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RESEARCH ARTICLE| 01 August 2008
A role for Q/N-rich aggregation-prone regions in P-body localization
Martin A. M. Reijns,
Martin A. M. Reijns *
Wellcome Trust Centre for Cell Biology, University of Edinburgh, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, UK
Ross D. Alexander,
Ross D. Alexander
Michael P. Spiller,
Michael P. Spiller ‡
Jean D. Beggs
Jean D. Beggs §
§ Author for correspondence (email: jbeggs@ed.ac.uk)
Present address: Medical Research Council, Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK
Present address: Faculty of Life Sciences, Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK
Accepted: 14 May 2008
Martin A. M. Reijns, Ross D. Alexander, Michael P. Spiller, Jean D. Beggs; A role for Q/N-rich aggregation-prone regions in P-body localization. J Cell Sci 1 August 2008; 121 (15): 2463–2472. doi: https://doi.org/10.1242/jcs.024976
P-bodies are cytoplasmic foci that are sites of mRNA degradation and translational repression. It is not known what causes the accumulation of RNA-degradation factors in P-bodies, although RNA is required. The yeast Lsm1-7p complex (comprising Lsm1p to Lsm7p) is recruited to P-bodies under certain stress conditions. It is required for efficient decapping and degradation of mRNAs, but not for the assembly of P-bodies. Here we show that the Lsm4p subunit and its asparagine-rich C-terminus are prone to aggregation, and that this tendency to aggregate promotes efficient accumulation of Lsm1-7p in P-bodies. The presence of glutamine- and/or asparagine-rich (Q/N-rich) regions in other P-body components suggests a more general role for aggregation-prone residues in P-body localization and assembly. This is supported by reduced P-body accumulation of Ccr4p, Pop2p and Dhh1p after deletion of these domains, and by the observed aggregation of the Q/N-rich region from Ccr4p.
P-body localization, Protein aggregation, Q/N-rich domains, Stress
Cytoplasmic mRNA-processing bodies (P-bodies) contain a variety of protein factors, some of which are involved directly in decapping (Dcp1p, Dcp2p), some in translational repression and/or activation of decapping (Pat1p, Dhh1p, Edc1p, Edc2p, Edc3p, Scd6p) and others are involved in deadenylation (Ccr4p, Pop2p, Not1p, Not2p, Not3p, Not4p, Not5p, Pan2p, Pan3p) or 5′ to 3′ degradation (Xrn1p). In addition, factors involved in nonsense-mediated decay and RNA interference (e.g. Ago1) are present in these foci (reviewed by Parker and Sheth, 2007). P-bodies in higher eukaryotes have also been called GW bodies after GW182 (also known as TNRC6A) (Eystathioy et al., 2003), a component that is required for their integrity (Liu et al., 2005), and which has a function in miRNA-mediated silencing (Jakymiw et al., 2005; Liu et al., 2005; Rehwinkel et al., 2005). In budding yeast, a cytoplasmic complex that consists of Lsm1p, Lsm2p, Lsm3p, Lsm4p, Lsm5p, Lsm6p and Lsm7p (hereafter referred to as Lsm1-7p), and which is involved in mRNA decapping and subsequent 5′ to 3′ decay (Bouveret et al., 2000; Tharun et al., 2000), localizes to P-bodies under certain stress conditions (Sheth and Parker, 2003; Teixeira et al., 2005). In higher eukaryotes, the equivalent of Lsm1-7p is present in similar foci, even under normal growth conditions (Ingelfinger et al., 2002; Eystathioy et al., 2003; Cougot et al., 2004). Lsm1-7p is thought to act as a chaperone, remodelling transcript-containing ribonucleoprotein particles (RNPs) at a step following deadenylation, thus promoting decapping (Tharun et al., 2000).
In yeast, no single protein component is responsible for P-body assembly but there is a level of interdependence in the recruitment of some of the components to these foci (Teixeira and Parker, 2007). By contrast, in human cells depletion of many components, with the notable exception of XRN1 and DCP2, affects localization of the others (reviewed by Jakymiw et al., 2007), suggesting that most components involved in early, but not late stages of mRNA decay are essential for P-body assembly. It is not known what makes any of these factors concentrate in cytoplasmic foci, although in yeast this seems to require RNA (Teixeira et al., 2005). More recently, various proteins in budding yeast have been implicated directly in P-body assembly, and the understanding of their physical and functional interactions is gathering pace. This includes Edc3p, Lsm4p (Decker et al., 2007), Pat1p (Pilkington and Parker, 2008), and Ded1p (Beckham et al., 2008).
LSM4 of Saccharomyces cerevisiae encodes an essential protein of 187 amino acids (aa). It is one of the seven subunits of the Lsm1-7p and Lsm2-8p (comprising Lsm2p to Lsm8p) complexes, the latter of which is needed for efficient pre-mRNA splicing through its role in U6 small nuclear RNA (snRNA) stability (Achsel et al., 1999; Mayes et al., 1999; Pannone et al., 1998; Salgado-Garrido et al., 1999) and localization (Spiller et al., 2007a) as well as U4/U6 di-snRNP formation (Verdone et al., 2004). The N-terminal 92 aa of Lsm4p include the Sm domain, which is involved in protein-protein and protein-RNA interactions within the Lsm complexes (Cooper et al., 1995; Hermann et al., 1995; Séraphin, 1995). This region is highly conserved between Saccharomyces species (Fig. 1A), and between budding yeast and humans (Fig. 1B). The C-teminal 95 aa are rich in asparagine (N; 36%) and serine (S; 17%), giving this region a highly hydrophylic character. It is less conserved than the N-terminus, however, homologues from various Saccharomyces species contain similar asparagine-rich stretches that vary in length and position. A notable exception is Lsm4p from S. kluyveri that has a glutamine (Q)-rich region (Fig. 1A). This N and/or Q-rich character of the Lsm4p C-terminus is conserved throughout the budding yeasts (supplementary material Fig. S1). By contrast, most Lsm4p homologues from higher organisms have an abundance of arginine and glycine residues in their C-termini, often in the form of RG repeats (Fig. 1B and supplementary material Fig. S2), that are important for interactions with the SMN complex. Symmetrical dimethylation of the arginine residues is thought to be important for regulation of snRNP assembly (Brahms et al., 2001; Paushkin et al., 2002). S. cerevisiae does not have a known SMN complex equivalent, providing a possible explanation for the absence of RG repeats in yeast Lsm4p. Experiments with Lsm4p of Kluyveromyces lactis suggest that the Lsm4p C-terminus is needed for efficient RNA degradation (Mazzoni et al., 2003a; Mazzoni et al., 2003b).
Here we describe the role of yeast Lsm4p and its C-terminus in Lsm protein aggregation. We show that the ability of Lsm4p to aggregate, although not essential, promotes efficient accumulation of Lsm1-7p in P-bodies. Many other P-body components contain Q/N-rich regions suggestive of a more general role for such aggregation-prone residues in efficient accumulation of these factors in P-bodies. In support of this hypothesis we show that the Q/N-rich region of Ccr4p is prone to aggregation under normal growth conditions and shows increased focal localization under stress conditions. Furthermore, we show that the Q/N-rich region of Ccr4p is essential for its accumulation in microscopically visible P-bodies, whereas those of Pop2p and Dhh1p, although not essential for P-body localization, promote their efficient accumulation in these cytoplasmic foci.
Lsm4p has an N-rich C-terminal region. (A) The N-terminus of Lsm4p (aa 1-92) contains the Sm domain; the C-terminus (aa 93-187; boxed) contains N-rich (or Q-rich) stretches of variable lengths. Scer, S. cerevisiae; Spar, S. paradoxus; Sbay, S. bayanus; Smik, S. mikatae; Scas, S. castellii; Skud, S. kudriavzevii; Sklu, S. kluyveri. (B) Lsm4 protein alignment of the budding yeast (yLsm4p) and the human (hLsm4p) protein. Sequences were aligned using ClustalW. Q residues are highlighted in grey, N residues in black and RG (arginine-glycine) repeats are underlined.
Overproduced Lsm4p or its C-terminus accumulate in foci
While investigating the localization of various GFP-Lsm4p fusions, we observed that GFP fused to full-length Lsm4p (GFP-Lsm4) or to its C-terminus (GFP-Lsm4C; aa 92-187) accumulates in cytoplasmic foci as well as in larger aggregates, in a variable percentage of cells (10-60%), whereas GFP fused to the N-terminal half of Lsm4p (GFP-Lsm4ΔC; aa 1-92), containing the Sm domain, does not (Fig. 2A). The number of GFP-Lsm4 foci increases after hypo-osmotic shock, indicating that aggregation can be triggered by stress, and that these newly formed foci are probably P-bodies. This is confirmed by colocalization of Dcp2-RFP with GFP-Lsm4p in foci formed after stress. By contrast, Dcp2-RFP is not particularly enriched in the larger Lsm4p aggregates during log-phase growth, suggesting that these are probably not P-bodies (Fig. 2B). In cells expressing GFP-Lsm4ΔC as the only copy of Lsm4p, Dcp2-RFP localizes to foci after osmotic shock, showing that P-bodies are formed (Fig. 2B). However, GFP-Lsm4ΔC localizes throughout the cell, indicating its failure to accumulate in P-bodies even under stress conditions. The virtual absence of GFP-Lsm4ΔC in microscopically visible P-bodies is not due to reduced levels of this truncated protein, as shown by western analysis (see below).
Other Lsm proteins aggregate upon Lsm4p overproduction
To investigate a potential link between these Lsm4p aggregates and P-bodies, the presence of other proteins was examined. We observed accumulation of all tested GFP-tagged Lsm proteins (Lsm1p, Lsm2p, Lsm6p, Lsm7p and Lsm8p) in similar cytoplasmic aggregates during log-phase growth of cells overproducing non-tagged Lsm4p (PGAL-LSM4 strain grown on galactose; Fig. 2C and supplementary material Fig. S3). This was even the case with the normally nuclear Lsm8p, indicating that not only Lsm1-7p but also Lsm2-8p aggregates when Lsm4p is present at high levels. Aggregates were observed mostly in the cytoplasm and occasionally in the nucleolus, judging from colocalization with the nucleolar protein Nop1p (data not shown). By contrast, normal localization was observed for each of the Lsm proteins in the absence of excess Lsm4p (Fig. 2C), although each of these GFP-tagged proteins was moderately overexpressed from the MET25 promoter. Lsm protein aggregates are therefore likely to be not physiologically significant, but simply the result of aggregation of Lsm4p-containing complexes when Lsm4p is present at higher than normal levels. As aggregation was observed with direct interaction partners of Lsm4p in the ring-shaped Lsm complex (Lsm1p, Lsm2p and Lsm8p) as well as with physically more distant subunits (Lsm6p and Lsm7p) it seems possible that overexpressed Lsm4p drives the aggregation of entire Lsm1-7p and Lsm2-8p complexes, probably via its C-terminus. This aggregation is specific to the Lsm proteins, as GFP alone localized throughout the cells regardless of Lsm4p levels (Fig. 2C), and the exclusively nuclear Lhp1p fused with GFP remained nuclear under these conditions and did not form aggregates, although many cells showed abnormal nuclear morphology, which is a phenotype associated with Lsm4p overproduction (supplementary material Fig. S3).
Overproduction of Lsm4p or its C-terminus leads to aggregation in cytoplasmic foci. (A) GFP-Lsm4 (pMPSLsm4), GFP-Lsm4C (pMPSlsm4D2) and GFP-Lsm4ΔC (pMPSLsm4D1) were overexpressed from the MET25 promoter in BY4741 cells grown in SD-Ura-Met. Localization was examined in cells during log-phase growth. (B) Colocalization of Lsm4p aggregates with Dcp2-RFP (pRP1155) was examined in BY4741 cells grown in SD-Ura-Leu-Met during log-phase growth or 20 minutes after hypo-osmotic shock. Dcp2-RFP (pMR171) localization was examined in PGAL-LSM4 cells expressing GFP-Lsm4ΔC grown in SD-Ura-His-Met (to prevent competition between GFP-Lsm4ΔC and endogenous Lsm4p for incorporation into Lsm1-7p), 20 minutes after hypo-osmotic shock. (C) Localization of GFP-Lsm1 (pGFP-N-Lsm1), GFP-Lsm6 (pMPSLsm6), Lsm8-GFP (pMR83) and GFP (pGFP-N-FUS) was examined in log-phase cells overproducing Lsm4p (PGAL-LSM4 cells grown in SDGal-Ura) and in cells with normal levels of Lsm4p (PGAL-LSM4 cells with pUSS1 grown in SD-Ura-Met). Nuclear DNA stained with DAPI is shown in blue.
A role for Lsm4p in Lsm1-7p P-body localization
The accumulation of GFP-Lsm4 and GFP-Lsm4C in foci when overexpressed, and failure of GFP-Lsm4ΔC to aggregate even under stress conditions, suggests a role for the Lsm4p C-terminus in targeting Lsm1-7p to P-bodies. As a complete Lsm1-7p complex is apparently needed for localization to P-bodies (Ingelfinger et al., 2002; Tharun et al., 2005) the C-terminal deletion is likely to affect localization of the entire Lsm1-7p complex. The localization of GFP-Lsm1 to P-bodies was therefore examined in cells producing either Lsm4p or Lsm4ΔCp (both non-tagged) from the native LSM4 promoter (i.e. not overproduced). In comparison with the accumulation of GFP-Lsm1 in P-bodies (for colocalization of GFP-Lsm1 with Dcp2-RFP see supplementary material Fig. S4) following hypo-osmotic stress of LSM4 cells, there was a reduction in the intensity of GFP-Lsm1 foci that formed in lsm4ΔC cells (Fig. 3A), as well as an apparent delay in their formation. To quantify this delay, the number of cells that displayed visible P-bodies 5 minutes and 1 hour after hypo-osmotic shock was counted in the LSM4 and lsm4ΔC strains. Whereas 85% of LSM4 cells displayed foci 5 minutes after hypo-osmotic shock, with only a small increase (to 90%) after 1 hour, only 4% of lsm4ΔC cells displayed foci after 5 minutes, increasing to 73% after 1 hour (Fig. 3B), with the majority of these foci still weaker than those observed in LSM4 cells. The localization of GFP-Lsm2 and GFP-Lsm6 to P-bodies after hypo-osmotic stress was similarly reduced in the lsm4ΔC strain compared with the LSM4 strain (Fig. 3C,D). Taken together, these data strongly suggest that the C-teminal domain of Lsm4p although not actually essential, is nevertheless important for efficient accumulation of Lsm1-7p in P-bodies under stress conditions. As the Lsm4p C-terminal domain seems to be important for efficient recruitment of Lsm1-7p to P-bodies, the C-terminal deletion might also have an effect on the accumulation of other proteins in P-bodies. However, no significant effect was seen on the localization of either Dcp1p or Dcp2p to P-bodies (Fig. 3E, and data not shown).
Detrimental effects of GFP-tagging Lsm4ΔCp
The complete absence of GFP-Lsm4ΔC from P-bodies after osmotic shock seems to contradict the mere reduction in P-body accumulation of GFP-Lsm1, Lsm2 and Lsm6 in the lsm4ΔC (non-tagged) strain. However, upon closer inspection, GFP-Lsm4ΔC was observed to localize weakly to P-bodies after hypo-osmotic shock in a small fraction of cells (<1%), and to accumulate in cytoplasmic foci in more than 90% of cells grown into late stationary phase (data not shown). Its reduced accumulation in P-bodies is likely to reflect negative effects of the GFP-tag in combination with the C-terminal deletion, possibly by reducing its incorporation into the Lsm1-7p complex. The negative effect of the GFP-tag is emphasized by a slow growth defect of the GFP-Lsm4ΔC strain at all temperatures compared with the lsm4ΔC strain (with non-tagged protein expressed from its native promoter), which shows slower growth only at 37°C (supplementary material Fig. S5). We cannot formally rule out the possibility that the difference between the non-tagged lsm4ΔC and the GFP-Lsm4ΔC strains is caused by their different levels of expression (native promoter vs MET25 promoter), although this is more likely to lead to the opposite of what we observe. While this manuscript was in preparation Mazzoni et al. reported that the asparagine-rich N-terminal region of the K. lactis Lsm4 protein, KlLsm4p, which is able to functionally replace its S. cerevisiae homologue, is essential for its own localization to P-bodies in budding yeast (Mazzoni et al., 2007). However, these authors only investigated the localization of a GFP-tagged version of this protein, and not the localization of other Lsm proteins in a strain expressing non-tagged klLsm4ΔCp. Thus the effect of the deletion may not have been distinguished from the additional, detrimental effect of the tag.
Absence of the Lsm4p C-terminus affects mRNA decay
To determine whether the Lsm4p C-terminal deletion affects mRNA decay, degradation of a PGK1pGmini reporter transcript (Mitchell and Tollervey, 2003) was investigated. This reporter is expressed from the GAL1 promoter, allowing its transcription to be switched off by growth on glucose. The rate of subsequent disappearance of the reporter transcript is used as a measure of its 5′ to 3′ degradation through the major mRNA decay pathway. A small effect was observed, as the mRNA half life increased from 3.4±0.9 minutes in wild type to 4.7±1.1 minutes in the lsm4ΔC strain, on the basis of the quantitative reverse-transcriptase PCR (qRT-PCR) data presented in Fig. 4C. Half lives calculated using data obtained from the northern blot were slightly higher compared with those determined by qRT-PCR, but the relative difference between the two strains was similar. In addition, the steady-state level of this transcript appears to be about 40% higher in the lsm4ΔC cells compared with the LSM4 cells (Fig. 4B). By contrast, no effect was observed on the splicing of pre-U3 RNA, compared with 12 hours of Lsm8p depletion (Fig. 4D), suggesting that Lsm4ΔCp does not detrimentally affect formation of Lsm2-8p or stability of U6 snRNA. It therefore seems unlikely that the stability or formation of Lsm1-7p is reduced because of this C-terminal deletion, unless the assembly requirements of these two complexes are significantly different. A similar effect on mRNA degradation was reported for klLsm4ΔC in K. lactis (Mazzoni et al., 2003a), whereas a seemingly stronger effect was observed for klLsm4ΔC in S. cerevisiae (Mazzoni et al., 2003b). The latter may reflect reduced incorporation of the mutant K. lactis Lsm4p into the S. cerevisiae Lsm1-7p complex. Decker et al. did not find a significant change in the half-lives of PGK1pG or MFA2pG reporter transcripts in the absence of the C-terminal 97 aa of Lsm4p, nor did they report on increased steady-state levels of these transcripts (Decker et al., 2007). The reason for this difference remains unclear; however, the strains used in these studies were constructed in different ways and in different genetic backgrounds. We cannot formally rule out that the effect we see on the PGK1pG half-life is caused by reduced expression and/or stability of Lsm4ΔCp, as we have no antibody to compare its level with that of full-length Lsm4p. However, the absence of an effect on splicing argues against this.
The Lsm4 C-terminus is required for efficient localization of Lsm1p to P-bodies. (A) GFP-Lsm1 (pGFP-N-Lsm1) localization 20 minutes after hypo-osmotic shock in LSM4 (MRY71) or lsm4ΔC (MRY73) cells. (B) Percentage of cells showing GFP-Lsm1 in foci 5 minutes or 1 hour after osmotic shock (n=100 cells per time point). (C,D) Localization of GFP-Lsm2 (pMPSLsm2) (C) and GFP-Lsm6 (pMPSLsm6) (D) in LSM4 or lsm4ΔC cells before and after hypo-osmotic shock (E) Dcp2-RFP (pMR159) localization in log phase LSM4 and lsm4ΔC cells grown in SD-His and 20 minutes after osmotic shock. All experiments in this figure were performed with strains expressing non-tagged Lsm4p or Lsm4ΔCp from the native LSM4 promoter.
The Lsm4 C-terminus is required for efficient mRNA degradation, but not splicing. (A) Degradation of PGK1pGmini reporter transcript in LSM4 (MRY71) or lsm4ΔC (MRY73) strains grown in SDGal-Ura after addition of glucose to 4% (w/v). scR1 RNA was used as a loading control. The asterisk indicates a stable degradation fragment. (B) PGK1pGmini transcript levels over time as a percentage of the level in LSM4 cells at t=0; averages of three northern blots with vertical bars indicating standard deviations. (C) Linearized degradation curves [–ln(PGK1t/PGK1t=0)] against time showing averages of qRT-PCR data of six RT repeats of two independent biological replicates; vertical bars indicate standard errors; half-lives indicated are based on the linearized qRT-PCR data (D) Northern blot detecting pre-U3 RNA and U3 RNA in LSM4 and lsm4ΔC strains grown in YPDA, and in a PGAL-LSM8 strain (MPS7) before and after 12 hours of growth on glucose.
Q/N-rich regions in other P-body components
Investigation of the aa sequences of all core components of P-bodies in yeast (Parker and Sheth, 2007) reveals Q and/or N-rich stretches of varying length in many of them, most of which are conserved between various Saccharomyces species (Fig. 5, Table 1 and supplementary material Fig. S6). Some (Lsm4p, Ccr4p, Pop2p and Not1p) were previously found in a genome-wide screen looking for yeast proteins with Q/N-rich domains (Michelitsch and Weissman, 2000). Michelitsch and Weissman used an algorithm to count these residues in consecutive aa 80-mers for each of the predicted open reading frames, finding an average Q/N-content of 7.7 per 80-mer in the yeast proteome (Michelitsch and Weissman, 2000). We counted Q, N and P residues in a similar fashion in each of the P-body core components. Our results (Table 1) show that all of the 20 proteins tested score above average for Q/N content (Graphic representations are shown in Fig. 5B,C and supplementary material Fig. S6). Interestingly, some of the Saccharomyces homologues show further extensions of Q repeats, e.g. Edc3p, Not3p, Not4p and Not5p (supplementary material Fig. S6). In addition, many of these polypeptides contain high numbers of proline residues in or just downstream of these Q/N-rich regions (Table 1). This is a feature that is also found in other aggregation-prone proteins, e.g. huntingtin, aggregation of which causes Huntington disease (Michelitsch and Weissman, 2000). Proline-rich regions often form extended and flexible regions, in many proteins apparently reaching out to facilitate interactions with other proteins, with phosphorylation having a potential regulatory role. Binding via these proline-rich domains is generally not very specific, but can be both very rapid and strong (Williamson, 1994; Kay et al., 2000). Furthermore, proteins with Q/N-rich domains have previously been shown to promote aggregation of heterologous proteins with similar domains (Derkatch et al., 2004). Indeed, Lsm4p was found as one of nine Q/N-rich proteins that, when overproduced, promote de novo appearance of [PSI+], the prion-form of the Q/N-rich Sup35 protein (Derkatch et al., 2001). On the basis of this behaviour as well as its structural similarities to Sup35p, these authors proposed that Lsm4p itself is a prion protein. Furthermore, Decker et al. showed that the prion-like Q/N-rich domain of the Rnq1 prion protein can, at least in part, functionally replace the C-terminal prion-like domain of Lsm4p (Decker et al., 2007).
Q, N and P residues counted per amino acid 80-mers in 20 different P-body components
Protein .
Q/N* .
Q .
P in Q/N-rich .
P (in best 80-mer)* .
Percentage of P (in best 80-mer) .
P-rich close to Q/N-rich .
Lsm4 38 5 33 1 2 2.5
Pop2 37 29 8 6 12 15.0 yes
Ccr4 34 11 23 4 10 12.5 yes
Not1 30 26 4 1 12 15.0 yes
Edc3 29 19 10 1 5 6.3
Dhh1 25 25 0 17 19 23.8 yes
Dcp2 24 13 11 8 20 25.0 yes
Pat1 23 19 4 18 23 28.8 yes
Scd6 22 8 14 2 15 18.8 (yes)†
Dcp1 22 4 18 2 4 5.0
Edc1 22 5 17 10 10 12.5 yes
Pan3 20 0 20 6 15 18.8 yes
Not3 19 9 10 2 13 16.3 (yes)†
Not5 15 8 7 3 12 15.0
Xrn1 15 5 10 1 24 30.0 (yes)†
Pan2 15 7 8 2 10 12.5
Lsm1 14 8 6 1 2 2.5
Not2 11 8 3 7 8 10.0
Edc2 8 5 3 6 9 11.3
Values indicate the number of residues in the highest scoring 80-mer for that protein
P-rich region near Q/N-rich region scoring <20
Other P-body components contain Q/N-rich regions. (A) Alignment of the N-terminal region of S. cerevisiae Ccr4p with homologues from closely related Saccharomyces species (see Fig. 1 legend for abbreviations). The Q/N-rich region is boxed, with Q residues highlighted in grey, N residues highlighted in black and P residues in red. (B) Schematic representation of P-body components with areas rich in Q and/or N residues indicated in white (approximately to scale; Not1p is broken to fit; lengths are indicated in numbers of aa). (C) Q, N and P residues were counted in aa 80-mers of Ccr4p, Pop2p, Not1p and Dhh1p starting at position 1, shifting ten aa at a time.
The Q/N-rich region from Ccr4p aggregates in cytoplasmic foci and responds to stress. (A) Localization of GFP-tagged Q/N-rich regions of Ccr4p (aa 1-229; pMR202), Pop2p (aa 1-156; pMR203) and Dhh1p (aa 427-506; pMR204) before and after hypo-osmotic shock (B) GFP-Ccr4(1-229) aggregates localize to the cytoplasm as shown in these fixed cells with DAPI stained nuclear DNA (C) The majority of GFP-Ccr4(1-229) aggregates does not colocalize with Dcp2-RFP (pRP1155) foci after osmotic shock.
Q/N-rich regions affect P-body localization
It is plausible that Q, N and/or P-rich regions have a role in the accumulation of proteins in P-bodies. We tested Q/N-rich regions from Ccr4p, Pop2p and Dhh1p for their ability to aggregate and/or accumulate in P-bodies when fused to GFP. The Q/N-rich N-terminal region of Ccr4p fused to GFP [Ccr4(1-229)] aggregates in cytoplasmic foci under normal growth conditions (Fig. 6A,B) in ∼20% of cells, and foci increase in numbers under stress conditions, with more than 50% of cells showing multiple foci per cell. Although the dynamics of increased focal accumulation resembled that of P-body formation, suggesting that the Q/N-rich N-terminus of Ccr4p is sufficient for P-body localization, we found that the majority did not colocalise with Dcp2-RFP (Fig. 6C). GFP-fusions of the Q/N-rich regions of Pop2p [Pop2(1-156)] and Dhh1p [Dhh1(427-506)], however, do not aggregate under normal growth conditions but show weak focal concentration in a low percentage of cells (<1%) when stressed, although the majority of cells do not show a change in localization (Fig. 6A). However, GFP-fusions of Pop2p and Dhh1p deleted for these domains [Pop2ΔN(147-433)] and [Dhh1ΔC(1-427)] do show decreased P-body localization compared to full-length Pop2p and Dhh1p (Fig. 7A), and Ccr4p deleted for 147 aa at its N-terminus [Ccr4ΔN(148-837)] completely fails to accumulate in cytoplasmic foci under stress conditions. We quantified the P-body localization of these proteins by counting the number of visible foci per cell at a set time after osmotic shock (Table 2). These numbers are an indication of the level of P-body localization, as a reduction in P-body accumulation will lead to a reduction in the number of visible P-bodies, which generally have variable sizes and/or intensities. Interestingly, deletion of a further 102 aa from the Ccr4p N-terminus [Ccr4ΔN2(250-837)] leads to exclusively nuclear localization (Fig. 7B). The latter suggests that Ccr4p normally shuttles between the nucleus and cytoplasm, and that its nuclear export depends on sequences within the N-terminal domain. The tendency for aggregation of these Q/N-rich regions is further emphasized by the fact that full-length Pop2p expressed from the MET25 promoter aggregates in bright nuclear foci when tagged at the C-terminus (Pop2-GFP, Fig. 7C), at a much lower rate when tagged at the (Q/N-rich) N-terminus (data not shown) and not at all in the absence of this N-terminus (Pop2ΔN-GFP, Fig. 7C). As these experiments were performed in the presence of natively expressed non-tagged proteins, which may contribute to the observed absence of GFP-Ccr4ΔN concentration in P-bodies, we investigated the localization of this protein in ccr4Δ as well as xrn1Δ strains. Whereas GFP-Ccr4ΔN still failed to concentrate in microscopically visible foci in the absence of native Ccr4p (Fig. 7D), some weak foci were observed in the absence of Xrn1p (supplementary material Fig. S7), which generally leads to larger and more abundant P-bodies by preventing 5′-to-3′ degradation of transcripts. For Ccr4ΔNp and Pop2ΔNp the reduced P-body localization is not due to reduced levels of these truncated proteins as western analysis showed no difference between levels of full-length and mutant proteins (Fig. 7E). As the level of Dhh1ΔCp was only 60% of that of full-length Dhh1p we cannot rule out that its reduced P-body localization is, in part, due to the lower protein level.
Quantification of P-body localization
Foci per cell .
Cells examined .
Ccr4 2.7±0.5 138
Ccr4ΔN (aa148-837) 0 >1000
Pop2 4.8±1.6 80
Pop2ΔN (aa147-433) 1.9±0.6 93
Dhh1 10.7±5.3 35
Dhh1ΔC (aa1-427) 3.5±1.1 83
In summary, although not absolutely essential, Q/N-rich sequences in Pop2p, Ccr4p and Dhh1p contribute to efficient accumulation of these proteins in P-bodies under stress conditions. This is most obvious for Ccr4p, which, in the absence of its N-terminal 147 aa is not microscopically detectable in P-bodies in otherwise normal cells. Increased focal accumulation under stress conditions of the N-terminal 229 aa fused to GFP, suggests that this region is capable of regulated aggregation in response to stress. The fact that the majority of these foci do not colocalize with Dcp2p, suggests that additional parts of Ccr4p are necessary for proper P-body localization, most probably through additional protein-protein interactions. It would therefore be interesting to further investigate the requirements of the Q/N-rich regions as well as other parts of these proteins for these interactions.
Is a mechanism for protein accumulation in P-bodies conserved?
As the C-terminal region of S. cerevisiae Lsm4p is semi-conserved between Saccharomyces species, at least in the high content of N and/or Q residues (supplementary material Fig. S1), the ability to promote Lsm1-7p accumulation in P-bodies is likely to be conserved in these yeasts as well as in other budding yeasts. In fact, this was shown to be true for the budding yeast K. lactis Lsm4p produced in S. cerevisiae (Mazzoni et al., 2007). The human homologue, however, does not show a significant enrichment in Q or N residues, apart from a short stretch of five glutamines. Indeed, full-length human LSM4 fused to GFP did not aggregate when overexpressed in wild-type yeast cells, nor did it accumulate in foci under stress conditions. Surprisingly, it mostly accumulated in the nucleus instead (data not shown). As it was not able to support viability in the absence of native Lsm4p expression (data not shown), it might be unable to form a functional complex with yeast Lsm proteins. It is possible that residues in other human LSM1-7 complex (comprising LSM1 to LSM7) members normally contribute to its accumulation in P-bodies. Notably, the short N or C-terminal extensions of LSM1, LSM2, LSM3 and LSM7 proteins contain relatively high levels of glutamine residues. However, if, as we propose, Q/N-rich sequences contribute to a rapid response to stimuli in yeast, this may not be needed in human cells, as the LSM1-7 complex accumulates in P-bodies even under normal growth conditions.
Q/N-rich regions from Ccr4p, Pop2p and Dhh1p contribute to efficient accumulation of these proteins in P-bodies. (B) Localization of GFP-tagged full-length Ccr4p (pMR212), Pop2p (pMR214), Dhh1p (pMR210) or truncated versions of these proteins (Ccr4ΔN(148-837) from pMR218, Pop2ΔN(147-433) from pMR215, Dhh1ΔC(1-427) from pMR211) before and after osmotic shock. All GFP-fusions were expressed in BY4741 cells and localization was examined in cells during normal growth (normal) or 20-40 minutes after osmotic shock (stress). (B) Localization of GFP-tagged Ccr4ΔN(148-837) and Ccr4ΔN2 (aa 250-837; pMR213) in fixed cells with DAPI-stained nuclear DNA (C) Localization of C-terminally GFP-tagged full-length Pop2p (pMR216) or Pop2ΔNp (pMR217); DAPI-stained nuclear DNA in blue. (D) Localization of GFP-Ccr4, GFP-Ccr4ΔN and Dcp2-RFP in ccr4Δ cells (Y10387) 30 minutes after hypo-osmotic shock (E) Anti-GFP western blot analysis of full-length and truncated Lsm4, Ccr4, Pop2 and Dhh1 proteins. Curiously, GFP-Ccr4 (122 kDa) migrates faster than GFP-Ccr4ΔN (106 kDa), but slower than GFP-Ccr4ΔN2 (95 kDa; Fig. 7E), and all three GFP-Ccr4 proteins migrate faster than their predicted molecular weights. The presence or absence of the highly polar N-terminal region of Ccr4p causes a change in the effective charge of the entire protein (predicted charges at pH 7 are –5.8, –4.0 and –5.5 respectively) and might affect protein conformation resulting in unusual migration during SDS-PAGE. Nop1p was used as a loading control.
Q/N-rich regions do not seem to be conserved in the human homologues of budding yeast Ccr4p, Pop2p and Dhh1p (CNOT6, CNOT8 and DDX6, respectively) proteins either; they are significantly shorter, lacking the N-terminal and C-terminal Q/N-rich regions respectively (supplementary material Fig. S8). Perhaps the function of these protein domains has been replaced by alternative domains, possibly in other polypeptides with which they interact. For example, GW182 contains an internal Q/P-rich region that is essential, but not sufficient, for its own P-body localization and that of Ago1 (Behm-Ansmant et al., 2006). Another P-body component specific to higher eukaryotes, Ge-1/Hedls, contains a C-terminal repetitive sequence rich in hydrophobic residues that is essential for P-body localization and parts of which aggregate in cytoplasmic foci that are not P-bodies (Yu et al., 2005). In addition EDC4 (also known as Ge-1, Hedls), DCP2 and TNRC6B from humans as well as other higher eukaryotes contain high levels of Q and/or N residues (Decker et al., 2007). Thus, alternative aggregation-prone regions might have replaced some of the yeast Q/N-rich domains in higher eukaryotes, at least some of which are likely to have a role in P-body assembly.
Aggregation of P-body components through their Q/N-rich regions could promote efficient P-body formation. Whether this is really the case and, if so, whether this occurs through prion-like aggregation or through specific interactions via putative modular `polar zipper' protein-protein interaction domains (Perutz et al., 1994; Michelitsch and Weissman, 2000) remains to be determined. The importance of the Q/N-rich protein Edc3p (also known as Lsm16p) in combination with Lsm4p in P-body assembly in yeast, which came to light while this manuscript was being revised (Decker et al., 2007), is in support of this hypothesis. An intriguing question is how Lsm4p aggregation, and that of other P-body components, is prevented under normal growth conditions. Post-translational modifications, e.g. phosphorylation of Lsm4p or other (Lsm) proteins, probably have a role. Such modifications could allow the cell to respond quickly and efficiently to changes in conditions, and might regulate the levels and intracellular localizations of Lsm1-7p and Lsm2-8p, in addition to promoting P-body localization. Such a mechanism could also regulate the competition between these two complexes that was observed by Spiller et al. (Spiller et al., 2007b). Similarly, post-translational modifications, e.g. of the N-terminal region of Ccr4p, could allow P-body localization of other proteins involved in RNA degradation. Q-rich regions in mouse TIA-1 and PUM2 have previously been shown to contribute to protein accumulation in stress granules (Gilks et al., 2004; Vessey et al., 2006). We now show that at least some of the Q/N-rich domains in P-body components have a role in the assembly of these RNA processing bodies. The presence of Q/N-rich regions in many other proteins that are involved in various aspects of RNA metabolism (Michelitsch and Weissman, 2000; Decker et al., 2007) hints at the possibility of a more general role for these prion-like domains in functional protein aggregation, in addition to stress-granule and P-body assembly.
Plasmids and strains
For a complete list of plasmids and strains used see supplementary material Tables S1 and S2.
Cells were grown at 30°C to mid-log phase in synthetic dropout (SD) medium. To stress cells, cultures were centrifuged and cells were resuspended in water. Live cells were placed on microscopy slides and examined by bright-field and/or fluorescence microscopy using a Leica FW4000 fluorescence microscope. Fixing of cells followed by DAPI staining was performed as previously described (Spiller et al., 2007b). Images were captured using LeicaFW4000 software (Scanalytics, Fairfax, VA) with a CH-250 16-bit, cooled CCD camera (Photometrics, Tucson, AZ).
RNA analyses
Cultures were grown at 30°C in synthetic dropout medium containing 2% (w/v) galactose. Transcription of the PGK1pGmini reporter gene was stopped by the addition of glucose to 4% (w/v) and 20 ml culture with an OD600 of 0.5 were snap-chilled at the indicated times after the addition of glucose. RNA extractions and northern blot analyses of 6% acrylamide/urea gels were as described (Mayes et al., 1999). The following oligonucleotide probes were used for northern hybridizations: to detect the PGK1pGmini reporter transcript 5′-AATTGATCTATCGAGGAATTCC-3′, to detect scR1 RNA 5′-ATCCCGGCCGCCTCCATCAC-3′ and to detect U3 RNA 5′-GGTTATGGGACTCATCA-3′. Northern blots were quantified using a STORM 860 PhosphorImager and ImageQuant software (Molecular Dynamics).
Quantitative reverse-transcriptase PCR
Ten μg of total RNA were treated with DNase1 (0.9 U RQ1, Promega) according to the manufacturer's instructions. cDNA was prepared from 5 μg of DNase-treated RNA in a 10 μl reaction: 1× first strand synthesis buffer, 2.5 mM DTT, 10 U RNase inhibitor (Roche), 0.75 mM dNTPs, 7.5 U ThermoScript RNaseH- (Invitrogen) and 500 nM of PGK1pGmini-specific primer (5′-AGCGTAAAGGATGGGGAAAGAGAA-3′), according to the manufacturer's instructions. A negative control reaction was performed in the absence of reverse transcriptase (RT). Any remaining RNA was hydrolysed by incubating reactions for 1 hour at 37°C after addition of 15 μl of 0.1 mg/ml RNaseA (Roche). Quantitative PCRs (qPCRs) were performed with SYBR Green JumpStart Taq ReadyMix (Sigma) in a Stratagene MX3005P real-time PCR machine in 10 μl reactions: 6 μl containing 5 μl 2× SYBR Green ReadyMix, 300 nM of each primer (F: 5′-ATTGAAATGAAATGAAATCGAAGGAATTTGG-3′; R: 5′-AGCGTAAAGGATGGGGAAAGAGAA-3′) and 0.5× ROX, plus 4 μl of cDNA template (diluted 1 in 20 after RT-PCR). Cycling parameters were as follows: 2 minutes at 94°C, then 50 cycles of 10 seconds at 94°C, 10 seconds at 63°C and 20 seconds at 72°C. Each qPCR reaction was performed in triplicate for each repeat RT reaction.
Western analysis
For crude protein extracts (Volland et al., 1994), yeast cells were lysed in 0.5 ml of 0.2 M NaOH on ice for 10 minutes, followed by TCA precipitation (final 5% w/v) for 10 minutes on ice. After centrifugation, the pellet was resuspended in 35 μl of dissociation buffer (0.1 M Tris-HCl pH 6.8, 4 mM EDTA, 4% SDS, 20% (v/v) glycerol, 2% (v/v) β-mercaptoethanol, 0.02% (w/v) BPB) and 15 μl of 1 M Tris base. Samples were heated at 95°C for 10 min before separation by SDS-PAGE. Proteins were transferred to PVDF membrane and detected with mouse anti-GFP (BD Bioscience) or anti-Nop1p antibodies, and sheep anti-mouse IgG-HRP (Amersham Bioscience).
Polypeptide alignments
Amino acid sequences of P-body components were obtained from the Saccharomyces Genome Database (http://www.yeastgenome.org/) or the NCBI Entrez Protein database (http://www.ncbi.nlm.nih.gov/sites/entrez). Alignments were made using the ClustalW Multiple Sequence Alignment tool (Thompson et al., 1994) inside Jalview 2.2 (Clamp et al., 2004).
We thank Roy Parker and David Tollervey for reagents, Jon Houseley for the critical reading of the manuscript, and David Barrass, Daniela Hahn and Olivier Cordin for assistance. This work was funded by a Wellcome Trust Prize Studentship 71448 and Royal Society support to MAMR, a studentship from The Darwin Trust of Edinburgh to MPS and Wellcome Trust Grant 067311. J.D.B. is the Royal Society Darwin Trust Research Professor. M.A.M.R. designed and performed all experiments under the supervision of J.D.B., except the qPCR experiment which was performed by R.D.A.; M.P.S. first suggested a potential role for the Lsm4p C-terminus in P-body localization; M.A.M.R. and J.D.B. wrote the manuscript.
Achsel, T., Brahms, H., Kastner, B., Bachi, A., Wilm, M. and Lührmann, R. (
). A doughnut-shaped heteromer of human Sm-like proteins binds to the 3′-end of U6 snRNA, thereby facilitating U4/U6 duplex formation in vitro.
Beckham, C., Hilliker, A., Cziko, A. M., Noueiry, A., Ramaswami, M. and Parker, R. (
). The DEAD-Box RNA Helicase Ded1p Affects and Accumulates in Saccharomyces cerevisiae P-Bodies.
Behm-Ansmant, I., Rehwinkel, J., Doerks, T., Stark, A., Bork, P. and Izaurralde, E. (
). mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes.
Bouveret, E., Rigaut, G., Shevchenko, A., Wilm, M. and Séraphin, B. (
). A Sm-like protein complex that participates in mRNA degradation.
Brachmann, C. B., Davies, A., Cost, G. J., Caputo, E., Li, J., Hieter, P. and Boeke, J. D. (
). Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications.
Brahms, H., Meheus, L., de Brabandere, V, Fischer, U. and Lührmann, R. (
). Symmetrical dimethylation of arginine residues in spliceosomal Sm protein B/B′ and the Sm-like protein LSm4, and their interaction with the SMN protein.
Clamp, M., Cuff, J., Searle, S. M. and Barton, G. J. (
). The Jalview Java alignment editor.
Cooper, M., Parkes, V., Johnston, L. H. and Beggs, J. D. (
). Identification and characterization of Uss1p (Sdb23p): a novel U6 snRNA-associated protein with significant similarity to core proteins of small nuclear ribonucleoproteins.
Cougot, N., Babajko, S. and Séraphin, B. (
). Cytoplasmic foci are sites of mRNA decay in human cells.
Decker, C. J., Teixeira, D. and Parker, R. (
). Edc3p and a glutamine/asparagine-rich domain of Lsm4p function in processing body assembly in Saccharomyces cerevisiae.
Derkatch, I. L., Bradley, M. E., Hong, J. Y. and Liebman, S. W. (
). Prions affect the appearance of other prions: the story of [PIN(+)].
Derkatch, I. L., Uptain, S. M., Outeiro, T. F., Krishnan, R., Lindquist, S. L. and Liebman, S. W. (
). Effects of Q/N-rich, polyQ, and non-polyQ amyloids on the de novo formation of the [PSI+] prion in yeast and aggregation of Sup35 in vitro.
Eystathioy, T., Jakymiw, A., Chan, E. K., Séraphin, B., Cougot, N. and Fritzler, M. J. (
). The GW182 protein colocalizes with mRNA degradation associated proteins hDcp1 and hLSm4 in cytoplasmic GW bodies.
Gilks, N., Kedersha, N., Ayodele, M., Shen, L., Stoecklin, G., Dember, L. M. and Anderson, P. (
). Stress granule assembly is mediated by prion-like aggregation of TIA-1.
Hermann, H., Fabrizio, P., Raker, V. A., Foulaki, K., Hornig, H., Brahms, H. and Lührmann, R. (
). snRNP Sm proteins share two evolutionarily conserved sequence motifs which are involved in Sm protein-protein interactions.
Ingelfinger, D., Arndt-Jovin, D. J., Lührmann, R. and Achsel, T. (
). The human LSm1-7 proteins colocalize with the mRNA-degrading enzymes Dcp1/2 and Xrnl in distinct cytoplasmic foci.
Jakymiw, A., Lian, S., Eystathioy, T., Li, S., Satoh, M., Hamel, J. C., Fritzler, M. J. and Chan, E. K. (
). Disruption of GW bodies impairs mammalian RNA interference.
Jakymiw, A., Pauley, K. M., Li, S., Ikeda, K., Lian, S., Eystathioy, T., Satoh, M., Fritzler, M. J. and Chan, E. K. (
). The role of GW/P-bodies in RNA processing and silencing.
Kay, B. K., Williamson, M. P. and Sudol, M. (
). The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains.
FASEB J.
Liu, J., Rivas, F. V., Wohlschlegel, J., Yates, J. R., III Parker, R. and Hannon, G. J. (
). A role for the P-body component GW182 in microRNA function.
Mayes, A. E., Verdone, L., Legrain, P. and Beggs, J. D. (
). Characterization of Sm-like proteins in yeast and their association with U6 snRNA.
Mazzoni, C., Mancini, P., Madeo, F., Palermo, V. and Falcone, C. (
). A Kluyveromyces lactis mutant in the essential gene KlLSM4 shows phenotypic markers of apoptosis.
FEMS Yeast Res.
Mazzoni, C., Mancini, P., Verdone, L., Madeo, F., Serafini, A., Herker, E. and Falcone, C. (
). A truncated form of KlLsm4p and the absence of factors involved in mRNA decapping trigger apoptosis in yeast.
Mazzoni, C., D'Addario, I. and Falcone, C. (
). The C-terminus of the yeast Lsm4p is required for the association to P-bodies.
FEBS Lett.
Michelitsch, M. D. and Weissman, J. S. (
). A census of glutamine/asparagine-rich regions: implications for their conserved function and the prediction of novel prions.
Mitchell, P. and Tollervey, D. (
). An NMD pathway in yeast involving accelerated deadenylation and exosome-mediated 3′ 5′ degradation.
Niedenthal, R. K., Riles, L., Johnston, M. and Hegemann, J. H. (
). Green fluorescent protein as a marker for gene expression and subcellular localization in budding yeast.
Pannone, B. K., Xue, D. and Wolin, S. L. (
). A role for the yeast La protein in U6 snRNP assembly: evidence that the La protein is a molecular chaperone for RNA polymerase III transcripts.
Parker, R. and Sheth, U. (
). P bodies and the control of mRNA translation and degradation.
Paushkin, S., Gubitz, A. K., Massenet, S. and Dreyfuss, G. (
). The SMN complex, an assemblyosome of ribonucleoproteins.
Perutz, M. F., Johnson, T., Suzuki, M. and Finch, J. T. (
). Glutamine repeats as polar zippers: their possible role in inherited neurodegenerative diseases.
Pilkington, G. R. and Parker, R. (
). Pat1 contains distinct functional domains that promote P-body assembly and activation of decapping.
Rehwinkel, J., Behm-Ansmant, I., Gatfield, D. and Izaurralde, E. (
). A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing.
Salgado-Garrido, J., Bragado-Nilsson, E., Kandels-Lewis, S. and Séraphin, B. (
). Sm and Sm-like proteins assemble in two related complexes of deep evolutionary origin.
Séraphin, B. (
). Sm and Sm-like proteins belong to a large family: identification of proteins of the U6 as well as the U1, U2, U4 and U5 snRNPs.
Sheth, U. and Parker, R. (
). Decapping and decay of messenger RNA occur in cytoplasmic processing bodies.
Spiller, M. P., Boon, K. L., Reijns, M. A. M. and Beggs, J. D. (
). The Lsm2-8 complex determines nuclear localization of the spliceosomal U6 snRNA.
Nucleic Acids Res.
Spiller, M. P., Reijns, M. A. and Beggs, J. D. (
). Requirements for nuclear localization of the Lsm2-8p complex and competition between nuclear and cytoplasmic Lsm complexes.
Teixeira, D. and Parker, R. (
). Analysis of P-body assembly in Saccharomyces cerevisiae.
Teixeira, D., Sheth, U., Valencia-Sanchez, M. A., Brengues, M. and Parker, R. (
). Processing bodies require RNA for assembly and contain nontranslating mRNAs.
Tharun, S., He, W., Mayes, A. E., Lennertz, P., Beggs, J. D. and Parker, R. (
). Yeast Sm-like proteins function in mRNA decapping and decay.
Tharun, S., Muhlrad, D., Chowdhury, A. and Parker, R. (
). Mutations in the Saccharomyces cerevisiae LSM1 gene that affect mRNA decapping and 3′ end protection.
Thompson, J. D., Higgins, D. G. and Gibson, T. J. (
). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.
Verdone, L., Galardi, S., Page, D. and Beggs, J. D. (
). Lsm proteins promote regeneration of pre-mRNA splicing activity.
Vessey, J. P., Vaccani, A., Xie, Y., Dahm, R., Karra, D., Kiebler, M. A. and Macchi, P. (
). Dendritic localization of the translational repressor Pumilio 2 and its contribution to dendritic stress granules.
J. Neurosci.
Volland, C., Urban-Grimal, D., Geraud, G. and Haguenauer-Tsapis, R. (
). Endocytosis and degradation of the yeast uracil permease under adverse conditions.
Williamson, M. P. (
). The structure and function of proline-rich regions in proteins.
Biochem. J.
(Pt 2),
Yu, J. H., Yang, W. H., Gulick, T., Bloch, K. D. and Bloch, D. B. (
). Ge-1 is a central component of the mammalian cytoplasmic mRNA processing body.


text:
Misologie (von griech.: misologia) bezeichnet allgemein die Ablehnung von vernünftig-logischem Denken. Als Problem findet sich dies bereits in Platons Phaidon, der darauf verweist, dass rechthaberische Argumente und logische Täuschungen, wie sie in der Eristik verwendet werden, den Getäuschten am Ende an der Vernunft zweifeln lassen.

Nach Immanuel Kant (vgl. Logik, Einleitung, Abschnitt III) ist die Misologie ein philosophischer Standpunkt, der sich zwar auf die Weisheit als oberstes Ziel der Philosophie beruft, die Wissenschaft aber ablehnt. Misologie – so Kant – entspringt einerseits der Leerheit wissenschaftlicher Erkenntnisse und einer daraus folgenden Eitelkeit, andererseits auch einer Unbefriedigung in Bezug auf die wissenschaftliche Tätigkeit. In der Grundlegung zur Metaphysik der Sitten übersetzt Kant "Misologie" selbst mit "Haß der Vernunft" ().

Kant beschreibt in der Grundlegung zur Metaphysik der Sitten, dass Misologie dann folgt, wenn "eine kultivierte Vernunft sich mit der Absicht auf den Genuß des Lebens und der Glückseligkeit abgibt". Diesen Schluss begründet er damit, dass je mehr sich die Vernunft mit der Glückseligkeit  befasst, das Bewusstsein entsteht, dass der Vorteil, den sie von den Künsten und der Wissenschaft erwartet, mehr Mühseligkeit als Glückseligkeit ist und die Glückseligkeit viel näher "unter der Leitung des bloßen Naturinstinkts" ist. Der Weg zur Glückseligkeit wird also viel weiter durch Vernunftgebrauch, als durch Instinktgebrauch. Diese Argumentation folgt nach der Festlegung, dass Glückseligkeit nicht der Zweck des Menschen, den die Natur demselben auferlegt hat, sein kann. Kant setzt die Gefahr der Misologie zur Unterstreichung dieser These ein, die Vernunft sei – im Gegensatz zum Instinkt – zu etwas viel würdigerem geschaffen, als zur Herbeiführung von Glückseligkeit.

Demgegenüber bestimmt Kant das Verhältnis von Weisheit und Wissenschaft positiv. "Denn Wissenschaft hat einen innern wahren Wert nur als Organ der Weisheit." Er fügt aber hinzu, dass auch die Weisheit ohne die Wissenschaft nicht auskommt, sondern ohne sie zu einem bloßen "Schattenriss einer Vollkommenheit" wird, "zu der wir nie gelangen werden."

Einzelnachweise

Literatur
 Gottlob Benjamin Jäsche (Hg.): Immanuel Kants Logik. Ein Handbuch zu Vorlesungen, Königsberg 1800.

Immanuel Kant

redpajama_set_name: RedPajamaWikipedia


Swimming pools constructed to the highly energy efficient Passive House Standard can help relieve the burden on municipalities significantly in the long term. This is demonstrated by two Passive House indoor swimming pools in Bamberg and Lünen (Germany), for which the Passive House Institute provided consultancy services. The Passive House Institute evaluated the pilot projects and has now published recommendations in a free handbook for planning of energy efficient pools. The handbook is also helpful for optimised operation of already existing pools.


text:
Illinois Gov. Bruce Rauner stays close to anti-union stance in annual address
Illinois Republican Gov. Bruce Rauner in his second state of the state address,on Wednesday, held to the same agenda from the playbook that he has used since his election to lead this solidly blue state. Focused on limits to collective bargaining, workmen's compensation reform, and a pro business environment, they have little chance of being passed by a General Assembly that is solidly controlled by Democrats.
Rauner faces intense criticism for not agreeing to a budget for this fiscal year, unless his measures are agreed upon, and as a result, has garnered stinging criticism. Two of which are glaring: the state's largest social services agency, Lutheran Social Services of Illinois, is ready to cut over 30 programs that serve 4,700 clients, due to lack of state funds, and 130,000 low-income college students who need tuition money awarded them through a state sponsored program, totalling $370 million.
These, and more unpaid bills, represent a $6 billion dollar deficit, a figure which is 12 percent of state spending, a significant increase from last year's amount of $4 billion. As CNBC has noted, "Without a formal budget, Illinois has been limping along by extending parts of last year's spending plan, settling on piecemeal appropriations and battling out the impasse in the courts," a pattern that is unsettling for residents, as well as politicians.
A long standing unfunded pensions deficit of $112 billion — one-quarter of the Illinois GDP, and nearly three times that of state revenue, also has to be addressed. But, in his thirty minute address Rauner struck the first salvo by saying, "If we don't offer a competitive environment for businesses, pretty soon the unions won't have any more jobs to unionize and the trial lawyers won't have any more businesses to sue."
In a move that some see as stagecraft, or as a way to drive a wedge between the State Senate president, John Cullerton, and the powerful Speaker of the House, Michael Madigan, he gave almost voluble support to the former without mentioning the latter. All part of what his staff says is an effort to help the pension deficit crisis.
The weeks leading up to the address provided a seeming solution to the crisis with he and Cullerton, hammering out a plan, only to have Rauner announce a version, that was later denounced by Cullerton as not being the plan they had agreed upon. According to the Chicago Tribune, "Cullerton has acknowledged that his pension plan faces likely union opposition, and Democrats are closely aligned with unions that are providing millions of dollars in campaign cash and ground troops for legislative races. There also are questions about whether the Cullerton-Rauner pension plan would pass legal muster."
In another dubious move, that some say is deliberately disingenuous, the governor touted education as a priority, especially K-12, but never said where the money would come from, or even addressed the Monetary Awards Program, the state's college assistance program, for low income university and community college students. "It's a little bit schizophrenic. It's one thing to say education's such a high priority and another to have these higher education institutions talk about closing their doors and laying off so many people," said Sen.Kwame Raoul, D-Chicago.
Rauner has also proposed a privatization of the state's economic arm, that seems less of a much heralded comprise, with madigan, but mostly an example of old-fashioned political horse trading. The Tribune reported that "The idea to create such an organization had been an early point of potential compromise between Rauner and Madigan last year. The two appeared to have brokered a deal to allow the governor to privatize a portion of the state's economic development efforts while giving the speaker his wish of a stand-alone agency to oversee the Abraham Lincoln Presidential Library and Museum, but talks broke down."
Using the power of a not-for-profit entity "to dole out state grants and negotiate tax incentives for private businesses on behalf of the Department of Commerce and Economic Opportunity," is, they said, "likely to stir controversy because privatization of state functions typically raises concerns about transparency and accountability. It also sent a signal to Madigan and his Democratic allies that Rauner is willing to work around the legislature to get things."
In his closing remarks, he said: "Our job in this Capital is to improve the lives of all the people of Illinois, through more economic opportunity, better educational opportunity, and more value for their hard-earned tax dollars. To achieve a grand compromise, we must cast partisanship and ideology aside. We must break from the politics of the past and do what is right for the long term future of our state. I'm ready – and it's my genuine hope that you are too. Let's continue this journey together. Illinois can't wait any longer."
Reaction from the other side the aisle was predictable, and Senate Democrat candidate Bob Fioretti said in a statement, "Rauner insists on blaming unions for our financial mess, but this old saw isn't the problem now, and it hasn't been the problem for the decades anti-union business interests have claimed as much. More and more economic research shows unions are good for workers and good for the surrounding economy by raising wages."
Fioretti also noted while they agree on some things: reducing the prison population, the importance of education, vocational training and addiction treatment, he said they do not agree on the solutions, "The solution in Illinois, in Chicago and in the 5th State Senate District isn't to break unions, it's to invest in our schools. Fix the disparities that deny impoverished neighborhoods a fair share of funding and require Chicago teachers to contribute more to their pensions than teachers anywhere else in the state," he said.
Another Democrat, State Sen. Melinda Bush, of suburban Grayslake, in her statement, using blunter terms, said, "The problem this past year has not been with the governor's initiatives, but with his actions," and that "After a year that pulled the rug out from under seniors, children and the needy, I hope his call for cooperation is real. It is time for Illinois to move past partisan bickering."
Turning up the volume even higher was SEIU Illinois Healthcare Vice President Greg Kelley, who in the statement released by his office, noted, "The governor has caused massive cuts to the social safety net in Illinois as part of a scheme to enact his "Turnaround Agenda" to lower wages and worker protections. And it has been people of color and women who have disproportionately borne the brunt of his scorched-earth strategy."
With the voices of his critics ringing in his ears, and the shouts of demonstrators outside the capitol building screaming, "Budget first," what many seem to be asking is this: "Is he listening?"
Illinois Gov. Bruce Rauner wants state control of Chicago Public Schools
Illinois Gov. Bruce Rauner says state will take control of Chicago schools
Anti-Beyonce rally sparks Anti-Anti-Beyoncé protest rally: Black Girl Rising
Obama's swan song: Final State of the Union address
State of the Union Address 2016 live stream online info, TV start time & channel

redpajama_set_name: RedPajamaCommonCrawl


If you are searching for water filtration, here is the list of businesses and websites related to 'water filtration' on Directory.ac. Add your company to this list.
Gold Coin Car Title Loans : The Most Worthy Title Loans. Looking for a low rate car title loans in Napa California? Gold Coin Title Loans will offer you the most flexible and hassle-free loan.
Independent Group Agency Insurance : California Insurance Specialists since 1987. IGA is an insurance in Los Angeles providing auto insurance, home insurance, condo insurance, renters insurance, motorcycle insurance, and more.


WASHINGTON (Oct. 31, 2017) — The National Transportation Safety Board determined Tuesday the probable cause of a fatal 2016 collision between a motorcoach and tractor-trailer truck near Palm Springs, California, was the California Department of Transportation's inadequate transportation management plan for stopping highway traffic near utility work, coupled with fatigue related to undiagnosed obstructive sleep apnea and inaction by a driver due to fatigue.
In the early-morning darkness of Oct. 23, 2016, on Interstate 10, the motorcoach was traveling at highway speed when it crashed into a stopped truck, resulting in the death of the motorcoach driver and 12 motorcoach passengers. The truck and other traffic had been stopped on the highway by police for utility work. When traffic resumed, the truck did not move. The motorcoach struck the rear of the truck two minutes later, intruding about 13 feet into the truck trailer and pushing it 71 feet forward.
The NTSB determined the probable cause of the crash was Caltrans' inadequate transportation management plan for stopping traffic, which resulted in a hazardous situation in which law enforcement did not detect the truck's lack of movement following the traffic break and did not provide any advance warning to the bus driver of the potential for stopped traffic ahead.
The board also determined the truck driver did not resume driving after the traffic stoppage because he most likely fell asleep due to fatigue related to his undiagnosed, moderate-to-severe obstructive sleep apnea. Despite the fact the truck driver was severely obese and at a very high risk for obstructive sleep apnea, he had not been tested for the condition. And although the Federal Motor Carrier Safety Administration Medical Review Board has developed guidance for screening for obstructive sleep apnea, the FMCSA has not disseminated this guidance to the medical examiners it certifies to perform commercial driver's license medical examinations.
The NTSB's investigation also revealed the bus driver had untreated diabetes, but the FMCSA-certified medical examiner did not diagnose the bus driver's condition or refer the driver for further testing despite a positive glucose urine test during the driver's medical certificate examination. The NTSB also found that the bus driver did not take actions to avoid the crash because he too was likely fatigued and did not expect to encounter stopped traffic.
The board issued eight safety recommendations based upon the findings of the investigation, making two recommendations to the FMCSA, three to the Federal Highway Administration, one to the trucking company, one to three national trucking associations and one to the International Association of Chiefs of Police and the National Sheriffs' Association.
The abstract of the NTSB's final report, which includes the findings, probable cause and safety recommendations is available online at https://go.usa.gov/xn22F. The final report will be publicly released in the next several days.


THE CODE PLUG AND PROGRAMMING SOFTWARE FOR DOWNLOAD IS FOR THE CS750 DMR RADIO. THIS CODE PLUG WAS MADE FOR USE WITH THE W1EDH CLUB AND ARES SYSTEM FOR THE STATE OF CT.
THE CODE PLUG AND PROGRAMMING SOFTWARE FOR DOWNLOAD IS FOR THE CS700 DMR RADIO. THIS CODE PLUG WAS MADE FOR USE WITH THE W1EDH CLUB AND ARES SYSTEM FOR THE STATE OF CT.


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PORT ELIZABETH COLLEGE
Institution/Brands
Port Elizabeth College is an institution for tomorrow with a rich illustrious past. We focus on developing people today to enable them to face the challenges of tomorrow by equipping them with relevant practical skills to enable them to attain their dreams and aspirations.
Welcome to Port Elizabeth College!
Port Elizabeth College is an institution for tomorrow with a rich illustrious past. We focus on developing people today to enable them to face the challenges of tomorrow by equipping them with relevant practical skills to enable them to attain their dreams and aspirations. This underpins our vision of being the preferred provider of Further Education and Training (FET) Programmes, which complement the human resource and skills development needs of a competitive, developing nation.
Our three main campuses; Russell Road Campus, Dower Campus and Iqhayiya Campus are reflected in our logo which signifies unity and strength in order to empower students with quality market-related skills in order to contribute to the socio-economic development of the country.
Our core values are expressed on a day-by-day basis by
Respecting the rights and dignity of each individual
Personal integrity and ethical behaviour
Cultural diversity, tolerance and equality
Professionalism and excellence
Quality service provisioning
Accountability between all stakeholders
Open communication, transparency and shared decision making at all levels
Innovative and constructive change in line with the human resource development strategy and skills development strategy of the country
Why PE College?
Quite simply, Port Elizabeth College performs with excellence as a priority. Our approach to education is largely driven by the will to exceed expectations. Complacency has not followed our accolades and achievements; instead we always strive to improve our performance. Port Elizabeth College caters for driven individuals with the knowledge that education is the fundamental entity of our prospering nation. We cater for individuals who want to get ahead, our slogan "Get Ahead, Get an Education" translates our stance.
For general enquiries contact the switchboard.
Contact number: +27 41 509 6000
Contact email: N/A
Website: http://www.pecollege.edu.za/


This Bible verse wall decal from James 1:5 reminds us to pray always with an eye of faith. We need to take time to pray and remember that the Lord is with us and in control. Put this Bible verse wall decal anywhere in your home, at work if you can, or even as a wall decoration for church classrooms.


Made to Dance in Burning Buildings is a fusion of poetry, theatre, and violent and visceral contemporary dance, which poses the question: how do we heal from trauma?
It's about Ava, a young black woman, who is raped by her boyfriend's friends while he watches; she develops PTSD as a result. Metaphorically, she fractures into five different women who each represent a different facet of her personality (Romance, Despair, Madness, Perseverance, and Missing You). Poetically, these women narrate her journey through PTSD while she, her first love, and her abusers explore through dance the trauma and the events leading up to it.
For the play, Anya is the inaugural winner of the $10,000 Voice is a Muscle Grant from the Corporeal Voices Foundation which is run by best-selling author Lidia Yuknavitch. The grant recognizes a writer of color from Oregon who demonstrates exemplary merit and a passion for social justice.
The play will be performed at Joe's Pub at The Public Theater on April 24, 2018 at 7pm. Tickets are $20. Purchase tickets here.
It will receive its World Premiere at Shaking the Tree Theatre in Portland, OR in Winter 2019.


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FUNDS AND GRANTS
FILMS > FINISHED >
THE LAST DAY OF SUMMER
POLISH TITLE: KONIEC LATA
REPRESENTED BY KFF
The film is a poetic, emotional journey to the times of our childhood and a story about difficulties of entering the world of adults. We want to take a closer look at the relations between peers, between children and adults (teachers, parents) to see the stages of growing up. We will observe the process from 7 year old kid who learns how to read to a 17 year old graduate who has to decide about his future. In 50 minutes we will see 10 years from the period in which so many changes occur and in which our personality develops.
FILM REPRESENTED BY THE KRAKOW FILM FOUNDATION
Piotr Stasik
Anna Dymek
Piotr Stasik »
Graduate of the Andrzej Wajda Master School of Film Directing and the Institute of Applied Social Sciences at the University of Warsaw. He is a co-initiator of the "ę" Society of Creative Initiatives and worked as a lecturer in the Film Kindergarten of the Wajda School as well as a chairman of the Film 1,2 Association. He made a film series which includes How to Make a Movie (2010) and Muzykoteka szkolna (2011). His documentaries "Diary of Journey" (2013), "The Last Day of the Summer" (2010), "7 x Moscow" (2005) were screened and awarded at many festivals. In 2016 he was nominated for the European Film Award for his film "21xNew York".
AT FESTIVALS:
54th International Leipzig Festival for Documentary and Animated Film - DOK Leipzig, 2011 (screening)
28th Kassel Documentary Film and Video Festival, Germany, 2011 (screening)
59th Belgrade Documentary and Short Film Festival, Serbia, 2012 (screening)
VERZIO International Human Rights Documentary Film Festival, Hungary, 2012 (screening)
17th Guanajuato International Film Festival, Mexico, 2014 (screening)
PLUS CAMERIMAGE 2010 - International Film Festival Of The Art Of Cinematography, Poland, 2010 (Grand Prix Golden Frog Award)
goEast Film Festival, Germany, 2011 (Documentary Award "Remembrance and Future")
International Documentary Film Festival "Flahertiana", Russia, 2011 ("Silver Nanook" Award for the best short film)
Vilnius Film Shorts, Lithuania, 2011 (competition)
Doclisboa International Film Festival, Portugal, 2011 (competition)
Cortopotere International Short Film Festival, Italy, 2011 (competition)
Minsk International Film Festival Listapad, Belarus, 2011 (competition)
Festival Tous Courts, Aix-en-Provence, France, 2011 (Prix RTP2 Onda Curta)
MEDIAWAVE International Film and Music Festival, Hungary, 2012 (competition)
Pärnu Film Festival, Estonia, 2012 (competition)
NEWSLETTER I express my consent to the processing of my contact data by the Krakow Film Foundation with its registered office in Krakow (30-364), u. Pychowicka 7, in order to receive its newsletter. The data will be processed for the period until your consent to the delivery of the newsletter is withdrawn or the newsletter is discontinued by the controller. I confirm at the same time that I was instructed about my right to: demand access to my data, correct it, remove it or limit its processing, file a complaint, transfer data, withdraw my consent and file a complaint to the supervisory authority. My consent is voluntary, but its refusal will make it impossible for me to receive the newsletter.
LIKE US Ministerstwo Kultury, Dziedzictwa Narodowego i Sportu Polski Instytut Sztuki Filmowej The project is co-financed by the Ministry of Culture, National Heritage and Sport of the Republic of Poland from the Culture Promotion Fund and the Polish Film Institute Krakowskie Biuro Festiwalowe Krakow Film Commission
Copyright © 2009–2023 Polish Docs Developed by: Agencja Interaktywna Y0 Design: terenprywatny.com hosting: Kei.pl Privacy policy


The teacher of my Me'ah class this semester is terrific. She's managed to break down the complex topic of medieval Jewish history into bite sizes neither too small and frothy nor massive and overwhelming. And she--unlike one of the good, but not great, instructors we had last year--is not the star of each lesson. I usually don't mind when a teacher injects herself into the subject matter, and enjoy hearing the personal perspectives which can bring a topic to life. But there's a line beyond which I become uncomfortable. I want to learn the thing, not the teacher's reactions to the thing. I want the teacher to be a little anonymous and distant so I can concentrate on something else besides the teacher.
My first reaction to Girl Meets God, a book I'm reading thanks to a deep and insightful review by The Velveteen Rabbi, was that its author had crossed this line. I kept getting distracted by the author's voice, kind of whiny and self-centered, despite the great story of a spiritual journey. I must admit that the subject matter, Orthodox Jewish woman converts to Christianity, also made me a little uncomfortable. I tried to wipe all biases from my mind as I began Chapter 1, but found myself casting judgments at every page: what's wrong with you, to do such a thing? I also remembered that I once contemplated--in secret, quietly, but the thought existed nevertheless--this same act, leaving a Judaism that seemed completely irrelevant. Maybe I bristled because parts of her story hit too close to home. But, really, I just wanted to shout at the author: why couldn't you find another kind of Judaism, like I did? Why did you leave us?
But, really, I just wanted to shout at the author: why couldn't you find another kind of Judaism, like I did? Why did you leave us?
YES. I can really relate to this. I had conflicted feelings about that -- part of me wanted to ask why she left us, while another part of me felt sad that I apparently couldn't escape my old binarisms -- but it was definitely a strong undercurrent of my reading experience.


text:
Manish Dabas
Air Force Brat | Foodie | Cricketer | Realist | Meditator | Humanitarian | MUNer | Computer Engineer
Faizal Januar Ramdhoni
Student of International Relations, ambitious, hard worker, active, open-minded
Marko Kovacevic
Founding Chief Editor at MUNPlanet. Reader, writer, researcher - IR and UN.
Chairing MUN
TEIMUN, a model UN based in the Hague is one of the oldest in Europe (founded in 1987). On Monday, July 21 TEIMUN 2014 kicks-off for a week of quality MUNing, a perfect opportunity to talk to Dyonne Niehof, chair of the Board of Directors. She answered questions about idealism and real-world agenda, as well as how MUNs play into world politics as a possible agent of change, and more.
MUNPlanet: TEIMUN is one of the MUNs in Europe with the greatest tradition. How is it like to chair the Board of Directors?
Dyonne: Well, as you can probably imagine, so far it has been a great experience. I was given the opportunity to be the chairman of an entire foundation, and to lead a group six ambitious students. During my student life I have been an active part in several committees and organizations, but this was a whole new level. TEIMUN is an organization run by students, but in the Hague, Brussels and the international community the foundation is being perceived as a professional international organization. The difference between the student life in Groningen and the working hours at our office was sometimes huge, but also very interesting.
During the past year, we experienced the craziest things. We were invited to network drinks and ceremonies where we were, without a doubt, the youngest attendants. The previous year, we had the opportunity to meet ministers and politicians, and we even went to an event where the Dutch royal family was present. You can imagine that for 21-year-old students these situations are sometimes overwhelming; but because we were encouraged to actively network and represent our organization, we learned a lot.
MUNPlanet: The theme of this year's TEIMUN Conference is 'Pursuing Perpetual Peace: Ideals and Interests at War'. How does this topic resonate with the present and future of International Relations?
Dyonne: This year's topic does not only resonate with International Relations, but with decisions that you make every day. If you would take note of it, you would probably find an example every day of a struggle between your ideals and your interests. A very simple example is the following: going to your lecture on Monday morning at 9 AM. Your ideal will probably be to pass your courses at the university with good grades, but your interest on a Monday morning around 8.30 AM might be to stay in your bed. And this is only a conflict between your own ideal and interest, imagine what the situation would be like if there would be something much larger at stake, or if other parties are involved as well.
A very clear example that resonates with International Relations is the issue of human rights and immigration. Almost every world leader advocates human rights and equality, but not all policies are directed towards these ideals. Immigration in the European Union has been a hot topic for years, since countries are reluctant to bear the costs of immigration in light of the current European crisis. The immigration issue within the EU is also a topic during this TEIMUN conference, and I am very much looking forward to see whether the delegates will be able to let their ideals prevail over their (national) interests .
"I believe that MUNs do not necessarily change the way you perceive the world, but they broaden your world. During MUN conferences we will not be able to bring everlasting peace, but we can try taking baby steps by creating awareness and facilitating an international dialogue."
"The immigration issue within the EU is also a topic during this TEIMUN conference, and I am very much looking forward to see whether the delegates will be able to let their ideals prevail over their (national) interests."
MUNPlanet: In what ways do you think MUNs influence the way people think of the world – I cannot help but notice that Immanuel Kant's famous work is in the foundation of this year's conference topic.
Dyonne: A very special feature of MUNs, and I believe TEIMUN in particular, is that you meet people from all over the world. Last year, my council discussed the issue of opium trade in Afghanistan. During the second day, we found out that one of the delegates in my council lives in Afghanistan, and that he knows people who trade opium. Not because those people are criminals, but because they need a way to survive. Suddenly this problem was not 'just' a global issue and something that happens far away, but it became really personal.
Representing another country encourages you to see international problems from a different perspective. This increasing mutual understanding between the delegates, since you perceive the world from a different view. For example, if you are a Dutch national, you read more or less the same newspaper every day, you look at the same (Dutch) news every day, and you talk to your (Dutch) friends about the news and about problems in the world. It is fairly ease to represent and protect these interests, because you were raised in a certain environment and with certain beliefs. But when you are encouraged to step into someone else's shoes, you have to step out of your comfort zone and suddenly take someone else's preferences and ideas into account. From my own experience I know that this can be quite a challenge, but if you succeed this increases your understanding of the world and will bring you closer to your fellow delegates. Therefore , I believe that MUNs do not necessarily change the way you perceive the world, but they broaden your world. During MUN conferences we will not be able to bring everlasting peace, but we can try taking baby steps by creating awareness and facilitating an international dialogue.
MUNPlanet: TEIMUN is a conference in which both first time MUNers and experienced MUNers can participate. What is your number one hint for first timers?
Dyonne: The most important thing that first time MUNers have to do, is to stand up and speak right away. I can fully understand why an MUN conference can be really overwhelming the first time. The first time I attended an MUN conference, I did not understand the Rules of Procedure, and I was really afraid that I was going to make a lot of stupid mistakes, so I understand the feelings of the first timers. However, our Chairs have been delegates at TEIMUN before, and they have been informed about the experience of their delegates, so they will make sure that everyone will be at ease and feel comfortable. And it is an open door, but the best way to learn a skill is by making mistakes and being corrected. After all, TEIMUN is a conference you attend in order to learn something new and exciting.
"The most important thing that first time MUNers have to do, is to stand up and speak right away… the best way to learn a skill is by making mistakes and being corrected."
MUNPlanet: What part of the Conference are you looking forward to most?
Dyonne: Personally, and I know, my fellow Board members are going to make fun of me because of this answer: I am mostly looking forward to Saturday July 26, since it will be my birthday. During this day, TEIMUN has the annual Football Match, in which this year all councils have the opportunity to find out who has the best football players, and we will have a BBQ at the Beach of Scheveningen. The Beach party afterwards is always amazing, since by that time you have already made a lot of friends, and it is the last official party of the TEIMUN conference. You can chill at the beach, tan in the sun, have good food at the beach, and even go swimming in the sea if you like, since it is usually really warm in the Netherlands during that time. The fact that it will be my birthday, and that I have the chance to celebrate that with over 250 students from all over the world makes it even more special.
MUNPlanet: How does the Hague, being a UN city, influence the spirit of MUNing?
Dyonne: The Hague is a fantastic city to host an MUN conference. The city hosts one of the most influential international organizations, and it the political centre of the Netherlands. Part of the TEIMUN program are excursions to e.g. the Peace Palace, the Organisation for the Prohibition of Chemical Weapons, the Humanity House, the Red Cross, etc. We try to give our participants an excursion that a regular tourist would not be able to do. The aim with these excursions is that we try to find the link between the MUN and the future careers of our participants. We also try to create this by organizing our network drinks, for which we have invited several companies and organizations that will have an informal talk with our delegates about their work and their careers. The Hague gives us the opportunity to give our delegates an extra experience, on top of our main activity: MUNing.
MUNPlanet: The past decade has seen an increased dynamics and globalization of MUN as a concept. How do you see MUNs at the moment, and in the years ahead?
Dyonne: In the future, I hope that MUNs will become increasingly cultural, diverse and globalized. One of the most interesting parts about MUNing for me is meeting all these people from all over the world, who have different ideas and beliefs and are very willing to share them with you. We will never be able to fully agree with each other, or to fully understand someone else who's opinions are the opposite of yours, but MUNs are tools that bring students together, and I hope that in the future they will continue to do so.
MUNPlanet: One of the goals of MUNPlanet is to get the brightest members of the community together to take part in a meaningful dialogue and interaction. Do you believe that MUNers can step forward and bring about the change as a transnational leadership force?
Dyonne: Well, as I mentioned before, I believe that key to international peace is mutual understanding and the facilitation of dialogue. I think that MUNPlanet is a great platform for MUNers to get in contact with each other. Since young people are ambitious, able to think out of the box, and have a drive to make a difference. At this stage, I think that MUNs can function as a training ground for future diplomats and can contribute to our understanding of the world.
If MUNers would like to step forward and bring about the change as a transnational leadership force, MUNers should try to find ways to actively influence world politics. The Nuclear Summit in The Hague for example, could have had a MUN-like activity the week before, where exactly the same topics were being discussed. I think it would be very interesting to see whether students would be able to come up with alternative answers to the issues that were discussed by the world leaders a week later. These specific activities would, in theory, be able to exert influence on world politics, but for now I believe that the benefits of MUNs are mostly visible in the long run.
"The Hague gives us the opportunity to give our delegates an extra experience, on top of our main activity: MUNing."
"If MUNers would like to step forward and bring about the change as a transnational leadership force, MUNers should try to find ways to actively influence world politics."
MUNPlanet: There must be something you would like to add in the end…
Dyonne: What I would like to share with all MUNers is a new project that I am going to work on from September onward. I will be part of the International Interdisciplinary Conference about the Legacy of the First World War (http://www.iclww1.org/), which will take place in June 2015 in The Hague. My task is to organize a Historical Council, on the peace treaties that made an end to the First World War. It is a very ambitious plan, since Historical Councils are usually very technical and very hard to simulate, but I think that this topic is very interesting and with the help of experienced chairs it will be a very interesting event. Once applications for chair positions or delegates are open, I will make sure to keep all MUNPlanet members updated on this unique event, since I think that MUNPlanet hosts the crème de la crème of the MUNers out there. But before this new adventure starts, the TEIMUN 2014 conference will start in a few days, and I am sure that together with my Board and with the staff, we are going to have an amazing week. I hope to welcome some of you in The Hague in a few days, or perhaps we will see each other somewhere else in the future.
MUNPlanet: Dyonne, thank you for talking to MUNPlanet. We wish you a successful TEIMUN conference, and are looking forward to hearing from you soon.
Are you ready to be part of Model UN history and organize your first conference? DownloadOrganizing Successful Model UN Conference eBookand get started!
Also, see:
TEIMUN 2014
Bret Stephens About Great War, Great Journalism and Great Mindset
The First World War Centenary: A Special Interview with Miguel Angel Moratinos
The First World War Centenary: A Special Interview with Prof. Christopher Clark
Introducing Fridays With MUNPlanet
"MUNs are the beginning of a long journey to making a positive contribution to the world"
54 conferences later: Diego Salama on leadership and key MUN lessons
MUNers Are Great OSCE Youth Ambassadors - A View From Denmark
Living In the Golden Age of MUNs
Einstein, Music and Leadership: Look How Tobias Henz Works to Unite MUNers in Germany
"Bring Your Thesis to the World!" - Lamprini Basdeki and Her Way From MUNs to UN
Juliane Jokinen on MUNs and Entrepreneurship - The Estonian Way
Learning By Doing - In "the Stronghold of MUNing" with Finn Carlson
Are You Ready to Take the Challenge? Milena Milicevic on MUNs, Leadership and Entrepreneurship
"Pelé Can Teach Us About How to Succeed In the World" [Special World Cup Interview With Moises Costa]
"MUNs Are About Making a Change and Empowering People to Make a Change", A View From Egypt
Founding Chief Editor at MUNPlanet. Reader, writer, researcher - IR...
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Posted by ESA on May 29, 2007
CLIMATE: BILL FORCING U.S. PARTICIPATION IN CLIMATE CHANGE TALKS CLEARS HOUSE PANEL
The Bush Administration would be required to participate this year in international global climate change negotiations aimed at setting binding targets to limit heat-trapping emissions under legislation approved May 23 by a near party-line 29-16 majority in the House Foreign Affairs Committee.
Chairman Tom Lantos' (D-CA) bill, H.R. 2420, calls on Bush to work "more actively and constructively" through the United Nations on a future climate treaty that can go into effect upon the 2012 expiration of the Kyoto Protocol.
It orders the U.S. to send a high level delegation — headed by the Secretary of State — to the next major U.N. global climate change conference scheduled this December in Bali, Indonesia.
The bill also orders Bush to work on an international climate agreement that requires "binding mitigation commitments from all major emitting countries based on their level of development."
The legislation does not give a specific numerical target for emission cuts but instead urges U.S. diplomats "to seek international consensus" on a target stabilization rate for greenhouse gases in the Earth's atmosphere that reflect the recent recommendations of the Intergovernmental Panel on Climate Change.
Lantos' bill also sets up a State Department Office on Global Climate Change headed by a presidential-appointed ambassador who must be Senate confirmed. And it puts an emphasis on exporting new U.S. energy technologies to emerging economies, namely China and India, through trade missions and several new offices.
The White House quickly signaled opposition to the measure and defended its efforts on climate change.
Visit this site to view the bill: www.eenews.net/features/documents/2007/05/23/document_gw_01.pdf
CLIMATE: U.S. ACCUSED OF STALLING PROGRESS ON RAINFOREST PLAN
U.S. officials are blocking progress on a proposal from rainforested countries seeking the start of a new program to combat global climate change, according to environmentalists tracking United Nations talks in Bonn, Germany.
Papua New Guinea, Costa Rica, and Brazil, along with the European Union, suggested the launch of a "national baseline" measurement system to allow countries with rainforests to determine whether deforestation is speeding up or slowing down. The measurements could later be used to start up a payment program for countries that do not clear carbon-absorbing trees.
But Bush Administration officials attending the two-week conference resisted the measurement plan, saying further study would be needed before it could gain U.S. approval.
Steve Schwartzman, Director of International Programs at Environmental Defense commented, "It's scandalous that the United States has said we're not going to be a party to the Kyoto Protocol because it doesn't do anything to reduce emissions from big developing countries, and then when Brazil comes forward with a proposal, the U.S. says, 'No, I think we need another workshop.'"
The top U.S. climate negotiator, Harlan Watson, said that the rainforested countries are free to advance their proposal, but they should do it under the existing Kyoto Protocol system and not during the debate on a follow-up treaty that would go into effect once Kyoto expires in 2012.
AGRICULTURE: HARKIN TARGETS BIG CHANGES TO CONSERVATION PROGRAMS
Senate Agriculture Committee Chairman Tom Harkin (D-IA) is planning a dramatic reorganization of conservation programs in the next Farm Bill and wants to pump $6 billion more into conservation spending.
Harkin's Farm Bill proposal would consolidate the Agriculture Department's largest working lands programs into one new initiative called the Conservation Stewardship Incentives Program. Its annual budget would be twice that of the entire Fish and Wildlife Service. The Agriculture Chairman would put another $3 billion into the other conservation programs. The money would come from the "reserve fund" given to the Farm Bill in the 2008 budget resolution, which requires offsets.
Harkin does not have those offsets in hand yet and is basically going to "buy now, pay later," with the hopes that his colleagues will be so impressed with the conservation spending, they will help him come up with the offsets.
Meanwhile, House Agriculture Committee Chairman Collin Peterson (D-MN) said he would restore controversial cuts he made to the Conservation Security Program in his Farm Bill proposal, bringing it up to baseline levels.
Peterson appears to have backed away from some of the hard budget lines he drew before the conservation and energy markups earlier this week, when he had told members they could not tap any more of the reserve funds to try to boost funding for favorite programs.
But after a meeting with House leadership, Peterson said that he is feeling more confident about being able to find offsets for funding and would let subcommittees tap more money in the reserve.
AGRICULTURE: POLLINATOR PROTECTION ACT INTRODUCED IN SENATE
A group of 29 senators introduced a bill May 24 that would open all Farm Bill conservation programs to habitat protection for bees and other pollinators.
The "Pollinator Protection Act" does not create new Farm Bill programs or change their funding. Rather, it directs the USDA offices that dole out conservation funds to help producers develop habitat or farming practices that could benefit pollinators.
Senate Agriculture Committee Chairman Tom Harkin (D-IA) and roughly half of the Agriculture Committee members have signed on as cosponsors.
Note: The Ecological Society of America signed a group statement of support for the Pollinator Protection Act, along with many other organizations, including The Nature Conservancy, National Wildlife Federation, and National Audubon Society.
ENDANGERED SPECIES: HOUSE LAWMAKERS ASK INTERIOR TO HALT ENDANGERED SPECIES ACT REWRITE
A group of House lawmakers asked the Bush Administration to stop its rewrite of Endangered Species Act (ESA) regulations.
Seventy-six members, most of them Democrats, told Interior Secretary Dirk Kempthorne in a letter to "reconsider any attempts" to change the law until he seeks the advice of congressional committees with ESA jurisdiction.
The Administration can move forward with its regulations without congressional approval, but Congress could put future work on the regulations on hold by blocking funding.
Interior has been under fire since draft regulatory changes became public in March. That draft would scale back federal power to list species or prevent disruptive activities in their habitat.
Fish and Wildlife Service Chief Dale Hall said the Agency has thrown out those proposals and is drafting new regulations. Reps. Maurice Hinchey (D-NY), Jim Moran (D-VA), and Christopher Shays (R-CT) were the lead authors of the letter, which is signed by half the Democrats on the Interior Appropriations panel.
Hinchey also sent a letter to Interior Appropriations Committee Chairman Norm Dicks (D-WA) asking him to include language in the spending bill that would limit the Administration's ability to overhaul the ESA.
Visit this site to view the letter to Secretary Kempthorne: www.eenews.net/features/documents/2007/05/16/document_gw_01.pdf
ENDANGERED SPECIES: FWS BUREAUCRATS ORDERED SCIENTISTS TO BACK DELISTING OF ARIZONA EAGLE
High-ranking Fish and Wildlife Service officials in Washington, D.C. ordered the agency's field biologists to overlook some scientific information and back the removal of Arizona's bald eagles from the endangered species list, according to Interior Department documents obtained and released May 18 by the Center for Biological Diversity, an advocacy group.
Biologists argued against delisting the eagle but were told by agency supervisors to seek only information that would back removing the bird from the list, according to department meeting notes. The group, which filed a lawsuit to maintain the eagle's listing, obtained the documents through a Freedom of Information Act request.
Agency notes suggest the FWS decision came as a "policy call" from higher-ranking officials in the regional office and Washington. Some biologists' initial reactions were to keep the eagles on the list as a distinct population segment (DPS). The delisting is expected to occur next month.
Sources: Energy and Environment Daily and Greenwire


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This was Brainerd - Oct. 31
OCTOBER 31 20 years ago (1991) Golly, what a gully! Crow Wing County's new landfill - a $3.46 million, five-acre hole in the ground - opens for business in four days. The old landfill received its last load of debris today. The old landfill, buil...
By Matt Erickson
20 years ago (1991)
Golly, what a gully! Crow Wing County's new landfill - a $3.46 million, five-acre hole in the ground - opens for business in four days. The old landfill received its last load of debris today. The old landfill, built 18 years ago for $60,000, will be closed.
(Photo) The Jaycees will have about 20 members wearing fluorescent vests on Halloween night, helping law enforcement handle safety. Pictured are Pat Krueger, Jim Nelson and Greg Oyen of the Jaycees, with Bruce Burke, Brainerd Police, and Deputy Frank Ball.
An apparent misunderstanding in the Village of Baxter has left Tuesday's election with a wide open race for mayor. The name of Darrell Powers had been placed on the ballot, but he said it was without his knowledge. Incumbent Don Sande has declined to run for re-election.
Masked youngsters prowling through Brainerd streets tonight in search of "trick or treats," will wear overcoats, mittens and overshoes. The young ghosts will plow through knee-deep snow. The annual Halloween parade this afternoon was abandoned.
Federal prohibition agents last night captured a truck loaded with 900 gallons of moonshine whiskey five miles northwest of Little Falls. Agents, acting on a tip, had been searching for it for two days. It had a flat and went into the ditch. The driver disappeared.
100 years ago (1911)
On his 200 acre farm on Gull Lake, Alderman James Elder is devoting time and attention to the propagation of pine trees, clearing brush as needed. It is historic land, for one section near the lake was site of the old Episcopal St. Columba mission 60 years ago.
-Compiled by Terry McCollough
Related Topics: THIS WAS BRAINERD
Matt Erickson joined the Brainerd Dispatch in 2000 as a reporter, covering crime and courts and the city of Brainerd. In 2012 he was promoted to night editor and in 2014 was promoted to editor of the newspaper.
This Was Brainerd - Jan. 31
A look through the Brainerd Dispatch archives with Terry McCollough combing the microfilm for tidbits of history through the decades going back to 1923.
By Terry McCollough
Review: 'Living' comes alive in bittersweet drama
MOLLETTE: Classified document stink piles


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