the ubiquitous presence of transcribing polymerases in the genome is a potential risk to the cell , as it can hamper the appropriate expression of canonical genes by interfering with their transcription ( jensen et  al . , 2013 ) .
pervasive transcription is controlled at the level of transcription termination and rna degradation , which can be coupled in s.  cerevisiae .
the main actors of this quality control pathway are the nrd1-nab3-sen1 ( nns ) transcription termination complex , the nuclear exosome , and the tramp complex ( for recent reviews , see porrua and libri , 2013a ; jensen et  al . , 2013 ) .
the nns complex is required for transcription termination of a large fraction of noncoding rnas ( ncrnas ) transcribed by rna polymerase ii ( rnapii ) , essentially cuts ( cryptic unstable transcripts ) , snrnas , and snornas ( steinmetz et  al .
cuts are short lived in wild - type yeast and are largely nonfunctional , although in a few cases their transcription has been shown to regulate gene expression ( kuehner and brow , 2008 ; thiebaut et  al . , 2008 ) .
transcripts terminated by the nns pathway are polyadenylated by the tramp complex and targeted by the nuclear exosome for degradation ( in the case of cuts ) or 3 end trimming ( in the case of snrnas and snornas ) .
the exosome is composed of a ring - shaped core to which two catalytic subunits , dis3p and rrp6p , associate .
the two enzymes are 3-5 exonucleases , and dis3p is also endowed with endonuclease activity ( chlebowski et  al . , 2013 ) .
because the central channel of the ring that drives the substrate toward the catalytic subunit dis3p is only wide enough to accommodate single - stranded rna , it has been proposed that the presence of an unstructured region of at least 30 residues is required for degradation ( chlebowski et  al . , 2013 ) .
rrp6p only associates with the nuclear form of the exosome and has overlapping and complementary roles to dis3p in rna degradation ( gudipati et  al . ,
the tramp complex is an important cofactor of the exosome that is required for the efficient processing and degradation of a variety of rnas produced by the three yeast rna polymerases ( wyers et  al . , 2005 ; san paolo et  al . , 2009 ;
tramp is composed of a poly(a ) polymerase ( trf4p or trf5p ) , a zinc knuckle rna - binding protein ( air1p or air2p ) , and the dexh - box rna helicase mtr4p ( lacava et  al . , 2005 ; vancov et  al . , 2005 ; wyers et  al . , 2005 ) .
polyadenylation of exosome substrates by tramp favors their degradation ( callahan and butler , 2010 ; kadaba et  al . , 2004 ;
lacava et  al . , 2005 ; rougemaille et  al . , 2007 ; vancov et  al .
, 2005 ; wyers et  al . , 2005 ) , and it has been proposed that poly(a ) tails added by tramp provide the unstructured extensions that allow threading of structured substrates through the central channel of the exosome ring .
it has also been shown that tramp stimulates exosome and rrp6p activity independently of polyadenylation ( wyers et  al . , 2005 ; callahan and butler , 2010 ; rougemaille et  al . , 2007 ) , although the mechanistic details of this stimulation are still unclear .
the nns complex is composed of the rna - binding proteins nrd1p and nab3p and the superfamily i helicase sen1p .
binding of the nrd1-nab3 complex to specific motifs on the nascent rna  constitutes the essential readout of transcription termination signals ( creamer et  al . , 2011 ; porrua et  al . , 2012 ; wlotzka et  al . ,
the actual termination step is most likely operated by sen1p that interacts with the nrd1-nab3 complex ( hazelbaker et  al .
, 2013 ; porrua and libri , 2013b ) . the nns complex has been shown to associate with tramp and the exosome , which is thought to favor degradation ( vasiljeva and buratowski , 2006 ) , although the molecular and mechanistic details of the interaction between the nns complex , the exosome , and the tramp are not well understood . nrd1p
interacts with the c - terminal domain ( ctd ) of the largest subunit of rnapii via a ctd interacting domain ( cid ) .
the cid recognizes heptapeptide ( ysptsps ) repeats in the ctd that are phosphorylated on the serine at position five ( ser5p ) ( kubicek et  al . , 2012 ; mayer et  al . , 2012 ; vasiljeva et  al . ,
2008 ) . because this modification mark predominates early in transcription , when nns - dependent termination preferentially occurs ( buratowski , 2009 ; gudipati et  al . , 2008 ; jenks et  al . , 2008
; steinmetz et  al . , 2006a ) , the cid - ctd interaction is believed to determine the regional specificity of termination ( gudipati et  al . , 2008 ; vasiljeva et  al . ,
. however , previous studies did not detect significant termination defects in a nrd1cid background ( vasiljeva et  al . , 2008 ) .
surprisingly , rnas produced by nns termination were found to be stabilized in nrd1cid cells , suggesting that the cid domain might favor degradation / processing by the exosome ( kubicek et  al . , 2012 ; vasiljeva et  al . ,
we analyze the role of the nrd1p cid in transcription termination and in promoting rna degradation / processing by the nuclear exosome .
we detected widespread termination defects at nns targets and defective recruitment of nrd1p to elongation complexes upon deletion of the cid .
surprisingly , we discovered that the cid also mediates the interaction between the nns complex and tramp by recognizing in trf4p a ctd mimic that we dubbed nim ( for nrd1 interaction motif ) .
we solved the solution structure of the interaction surface , and we show that the interactions of nrd1p with tramp and rnapii are mutually exclusive .
importantly , we demonstrate that the nrd1p - trf4p interaction stimulates the polyadenylation activity of tramp in  vitro , suggesting that the cid contributes to efficient degradation of exosome substrates by facilitating tramp recruitment and function .
our results demonstrate the existence of two alternative forms of the nns complex : one associated with rnapii and functioning in termination and the other associated with tramp and promoting rna degradation .
to assess the role of the nrd1p cid in the function of the nns complex , we reexamined whether this domain is required for efficient transcription termination by the nns pathway .
we compared the rnapii distribution by chromatin immunoprecipitation ( chip ) and tiling arrays in wild - type and nrd1cid cells . upon deletion of the cid , we observed persistent presence of  rnapii downstream of many genes , such as the cut nel025c , snr13 , snr47 , and cut065 ( figures 1a1c ) , indicating transcriptional readthrough .
these findings were confirmed by northern blot analyses ( figures 1d and s2a , available online ) and showed that in some cases ( e.g. , nel025c ) poor detection of the readthrough transcripts is due to the combined nuclear and cytoplasmic degradation of these species .
indeed , readthrough transcripts become prominent in cells in which both the nuclear exosome and the cytoplasmic nonsense - mediated decay ( nmd ) degradation pathways are defective ( i.e , in a rrp6p - depleted , upf1 mutant ; figure  1d ) .
metagene analyses suggested that readthrough occurs at the majority of nns targets , such as cuts and snornas ( figures 1e and 1f ; see also supplemental experimental procedures ) .
readthrough also occurs at small open reading frames ( orfs ; < 500  bp ; figure  s1a ) that have been shown to be partially nns dependent ( steinmetz et  al .
, 2006b ) and possibly at a subset of larger orfs ( figure  s1b ) , although in both cases it is not possible to clearly distinguish bona fide readthrough events from failure to terminate antisense transcription that is frequently observed at the 3 end of orfs ( neil et  al . , 2009 ; xu et  al . ,
consistent with this notion , genes with no detected antisense transcripts like rmd6 , dld3 , trs31 , ugo1 , and prp3 display no significant readthrough ( figures 1a and 1b ) .
we compared the genome - wide chromatin distribution of nrd1p in the presence and the absence of the cid by chip - chip analysis , normalizing to transcription levels as defined by rnapii occupancy in both strains ( figure  1 g ; for the non - normalized nrd1p occupancy , see figure  s1c ) .
metagene analyses for the four distinct classes of features showed that wild - type nrd1p is recruited to higher levels at cuts and snornas genes , but also at the 5 end of orfs as previously reported ( kim et  al .
2010 ) , although the 5 end recruitment peak was attenuated by rnapii normalization ( figures 1 g and s1d ) .
importantly , ablation of the cid domain affected recruitment in all instances , although higher nrd1p occupancy persisted at cuts relative to other features in nrd1cid cells , presumably because of rna - mediated recruitment .
as expected , no effects of cid deletion were observed at trna genes ( figure  s1e ) .
altogether , these results demonstrate that the cid is required for efficient termination at nns - dependent targets .
2008 ) , we observed that the levels of the nel025c cut , pre - snr13 , and pre - snr47 were increased upon deletion of nrd1 cid ( figure  s2a ) .
stabilization was stronger when the nuclear degradation machinery was compromised in rrp6 catalytic mutants ( figure  s2a ) , presumably because of partial redundancy in the degradation pathways .
interestingly , we also detected stabilization of some degradation intermediates derived from the u4 and u5 snrnas ( figure  s2b ) , suggesting a more general cid requirement for optimal activity of rrp6-exosome .
because the nns complex has previously been shown to copurify with the exosome and tramp complexes ( vasiljeva and buratowski , 2006 ) , we considered that the cid could be involved in mediating such interactions . to address this question ,
we first  performed coimmunoprecipitation assays with wild - type or cid tap - tagged nrd1p .
we consistently observed strong signals for the tramp components trf4p and air2p in nrd1p immunoprecipitates .
strikingly , however , tramp signals could not be detected in the absence of the cid in ribonuclease ( rnase)-treated extracts ( figure  2a ) , which was also reported by heo et  al .
as expected , deletion of the cid did not affect the interaction between nrd1p and nab3p ( figure  2a ) .
in contrast to the strong tramp signals , we only detected weak signals for rrp6p and dis3p in nrd1-tap immunoprecipitates upon rnase treatment ( figure  s3a ) .
this suggests that the tramp complex is a major partner of the nns complex relative to the exosome . in order to assess whether the interaction between nrd1p and the tramp is direct , we performed pull - down experiments using e.  coli extracts containing recombinant tramp components and nrd1 ( figure  2b ) .
we observed a robust , rna - independent interaction between rnrd1 and rtrf4 , even in the absence of rair2 and rmtr4 .
importantly , rnrd1cid failed to interact with rtramp ( figure  s3b ) , and recombinant isolated cid ( rcid - his6 ) alone was efficiently pulled down by rtrf4 ( figure  2c ) . taken together ,
these results demonstrate that nrd1p recognizes the tramp complex via a direct interaction with trf4p , the cid domain being necessary and sufficient for this interaction .
since the cid interacts with the rnapii ctd and trf4p , we considered that the same surface might be involved in the recognition of both targets .
consistent with this notion , two cid variants mutated at positions that are critical for binding to the ser5p ctd ( s25d and r28d ; kubicek et  al . , 2012 ) also failed to interact with rtrf4 ( figure  2c ) .
therefore , we surmised that the nrd1p cid might recognize a ctd mimic in trf4p . because the ctd is intrinsically disordered , we restricted our search to  the unstructured n- and c - terminal regions of trf4p .
we found a 9 aa motif at the very c - terminal end of trf4p , which contains several residues that are critical in the ctd for the interaction with the cid , including a glutamate that could mimic the ser5p in ctd repeats ( figures 2d and s3c ) . to assess the role of this motif , we immunoprecipitated nrd1-tap from strains expressing a mutant variant of trf4p lacking the last nine c - terminal amino acids and the two adjacent aspartic acid residues , which we surmised to be important for binding based on the cid structure ( kubicek et  al . , 2012 ) . despite identical steady - state levels of the wild - type and mutant trf4p ,
the interaction with nrd1p was abolished in the mutant , indicating that the c - terminal region is necessary ( figure  2e ) .
we set out to test whether the lack of interaction between nrd1p and trf4p contributes to the degradation / processing defects observed in the nrd1-cid mutant ( figure  s2 ) . to this end , we analyzed by northern blot the effect of the nim deletion on the levels of the nel025c cut and the snr13 precursor .
as shown in figure  2f , in trf4nim cells these nns targets were stabilized , although to levels slightly lower than those in an nrd1cid mutant . as for deletion of the cid , deletion of the nim
exacerbates the degradation / processing phenotype of exosome defective cells ( dis3-exo- ; figure  2f ) .
we did not observe any significant effect of the nim deletion on termination based on the detection of readthrough transcripts or rnapii chip ( figures 2f and s3e and data not shown ) , suggesting that the nrd1p - trf4p interaction is not required for transcription termination . taken together , these results demonstrate that nrd1p recognizes a ctd - like motif , nim , in the trf4p c - terminal region via  its cid domain and that this interaction contributes to the role of  the nns complex in promoting rna degradation by the rrp6-exosome .
previous studies have demonstrated that the cid of nrd1p binds to a fragment consisting of two canonical ctd repeats with the ser5p mark located in the upstream repeat ( kubicek et  al . , 2012 ) . this phospho - ctd fragment binds nrd1p cid with a kd of 130  m ( figure  3a ) . to assess the binding affinity of nrd1p cid to the nim
, we performed a quantitative solution - binding assay using fluorescence anisotropy ( fa ) measurements .
we found that nrd1p cid binds nim with an affinity roughly 100-fold stronger compared to the phospho - ctd peptide ( a kd of 1  m ; figure  3b ) . to assess
whether the interactions of the cid with ctd and trf4p are mutually exclusive , we first performed titration of fluorescently labeled nim peptides with nrd1p cid in the absence or presence of unlabeled ctd ( figure  3c ) .
the displacement of the binding isotherm in the presence of increasing ctd concentrations demonstrates that the ctd can outcompete nrd1p cid from binding to the nim , although the competition was only partially effective , as expected from the stronger affinity of the nim for the cid . importantly , increasing concentrations of unlabeled nim could effectively disassemble a preformed nrd1p cid - ctd complex in which the ctd was fluorescently labeled ( figure  3d ) .
the latter competition assay was used to calculate a kd of 120  m for nrd1p cid - ser5p ctd complex ( using a kd of 1.08  m for nrd1p cid - trf4p nim interaction ) , which is similar to the noncompetitive binding assay ( figure  3b ) . in both titration experiments we observed no additional increase of anisotropy , indicating that the nrd1p cid - ctd - nim ternary complex is not formed .
altogether , the fa data showed that the interactions of nrd1p cid with the ctd and the nim are mutually exclusive . to understand how trf4p is recognized by nrd1p
, we determined the solution structure of a reconstituted complex consisting of the cid ( residues 1153 ) of nrd1p and a 12 aa  nim peptide ( asp573-asp574-asp575-glu576-asp577-gly578-tyr579-asn580-pro581-tyr582-thr583-leu584 ) ( figures 4 and s4 ; table 1 ) .
the structure of nrd1p cid consists of  eight  helices in a right - handed superhelical arrangement ( figure  4 ) and is similar to the structure of nrd1p cid in the apo form ( vasiljeva et  al . , 2008 ) or bound to the phosphorylated ctd ( kubicek et  al . , 2012 ) .
the subtle differences originate from the extended interaction surface with trf4p nim , which involves loop 1-2 and helices 2 , 4 , and 7 of nrd1p cid ( figures 4a and s4a 
interestingly , [ h , n ] heteronuclear single quantum coherence ( hsqc ) titration experiments of nrd1p cid revealed that the protein amide resonances are in fast or slow exchange regimes between their free and bound forms relative to nmr timescale , when titrated with the phosphorylated ctd or nim , respectively ( figures s4c  s4e ) .
this observation is in agreement with the fact that the two substrates differ in their binding affinities to the cid by two orders of magnitude as evidenced by the fa data .
the structure of the nrd1p cid - trf4p nim complex shows that the specific recognition of the nim is facilitated by a hydrophobic  turn in the c - terminal region and negatively charged residues in the n - terminal region of the peptide ( figure  4b ) .
akin to the ctd , the nim peptide adopts the  turn conformation at asn580-pro581-tyr582-thr583 ( figure  4c ) .
pro581 and tyr582 of the nim  turn , along with the preceding tyr579 , dock into a hydrophobic pocket of the nrd1p cid that is formed by ile29 , tyr67 , met126 , leu127 , and ile130 ( figures 4a and s4 g ) .
tyr579 of the nim also forms intramolecular stacking with pro581 , and the hydroxyl group of tyr579 forms a hydrogen bond with a conserved asp70 of nrd1p cid ( figure  4a ) .
alanine substitution at position tyr579 of the nim strongly diminished the binding affinity for nrd1p cid ( figures 4e and s5b ) , confirming the importance of the intricate interaction network of this residue .
consistently , the asp70ala variant of cid displayed a significant drop in binding affinity for the nim peptide ( figures 4e and s5a ) .
tyr582 of the nim  turn stacks with the methyl groups of ile130 of the cid , and the perturbation of this interaction yields a 20-fold decrease in binding affinity ( ile130lys variant ) .
the n - terminal region of nim contains a stretch of negatively charged residues ( asp573-asp574-asp575-glu576-asp577 ) , which contains the putative ser5p mimic ( glu576 ) and interacts with a positively charged pocket formed by lys21 , ser25 , and arg28 in the 1-2 loop of cid ( figure  4b ) .
in particular , the carboxyl groups of asp577 and asp575 of nim form a hydrogen bond with ser25 and arg28 of the cid , respectively ( these hydrogen bonds are inferred from the final ensemble of structures , and they are indirectly defined by neighboring proton - proton nuclear overhauser effect [ noe ] ) . similarly , asp573 of the nim contacts lys21 of the cid via a hydrogen bond between the carboxyl group and the side - chain amino group of lys21 .
the importance of these contacts was further tested in a quantitative in  vitro binding assay using fa .
aspartate ( charge swapping ) or arginine substitutions at positions leu20 , lys21 , ser25 , and arg28 significantly decreased the binding affinity for the nim peptide ( figures 4a , 4e , and s5a ) .
the equivalent mutations in the nim also showed a decrease in the binding affinity for nrd1p cid , albeit with a lower magnitude .
notably , mutation of glu576 to alanine or to arginine partially affected binding , indicating that the putative phosphomimic is important , but not essential , for the interaction ( figures 4a , 4e , and s5b ) .
this suggests that the flanking aspartates of the asp - rich stretch may substitute one another in the single - point mutants .
altogether , the structural and binding data show that trf4p nim is recognized by nrd1p cid through two elements , hydrophobic  turn and asp - rich stretch , which is a recognition mechanism similar to that used for the recognition of phosphorylated ctd .
having established that nrd1p interacts directly with trf4p via the cid - nim interaction , we undertook the characterization of the interactions linking the nns complex and tramp with the nuclear exosome and rrp6p .
the strong and stable binding of  tramp to the nns complex ( figure  2a ) contrasts with the  weak , mostly rna - dependent signal that we observed for rrp6p and the core exosome in nrd1p immunoprecipitates ( figure  s3a ) .
we considered the possibility that rrp6p and the core exosome might be recruited to the nns complex via the nrd1p - tramp interaction .
however , the molecular details of the interaction between tramp and the nuclear exosome / rrp6p have not been elucidated .
therefore , we first performed pull - down assays using e.  coli extracts expressing recombinant tramp components and halo - tagged rrp6 .
these experiments revealed a clear and direct interaction between rtrf4 and rrrp6 irrespective of the presence of rmtr4 or rair2 ( figure  5a ) . to assess whether tramp also interacts with the nuclear exosome independently of rrp6p , we immunoprecipitated the core exosome using tap - tagged rrp41p from wild - type or rrp6 cells .
as shown in figure  5b , we detected significant signals for trf4p in purified core exosome from wild - type , but not from rrp6 cells , strongly suggesting that rrp6p bridges tramp and the core exosome .
the interaction we detected in  vivo between rrp6p and nrd1p is strongly dependent on the presence of rna ( figure  s3a ) .
however , we can not completely exclude the existence of direct but weak or transient contacts in  vivo that are not easily detected under our assay conditions .
therefore , we decided to assess whether a direct interaction could be detected between recombinant rrp6 and nrd1 . to address the rna dependency of the interaction
, we either treated our extracts with rnase a or used a variant of rnrd1 lacking the rna binding domain ( rnrd1-rrm ) .
as shown in figure  5c , we observed an interaction between rrrp6 and rnrd1 or rnrd1-rrm .
importantly , this interaction was not mediated by the cid since it was also detected with rnrd1cid . from these experiments , we conclude that rrp6p interacts directly with nrd1 in a cid - independent manner and with trf4p .
the latter interaction allows the association of tramp with the nuclear exosome ( figure  5d ) .
the trf4-air2 heterodimer possesses a distributive poly(a ) polymerase activity in  vitro ( lacava et  al . ,
2005 ; vancov et  al . , 2005 ; wyers et  al . ,
considering the high affinity of nrd1p and nab3p for their rna targets ( carroll et  al .
2011 ; porrua et  al . , 2012 ) , and the strong interaction of nrd1p with trf4p
, we surmised that the nrd1-nab3 heterodimer might stimulate polyadenylation of nns substrates by trf4p - air2p , for instance by improving recruitment or by stabilizing the trf4-air2 complex on the rna .
therefore , we assessed the effect of adding rnrd1 and rnab3 to in  vitro polyadenylation assays with recombinant trf4p - air2p ( figures 6 and s6a ) .
we first used a 40-mer rna substrate containing nrd1p and nab3p binding sites that we have previously shown to efficiently bind the nrd1-nab3 heterodimer in  vitro and elicit efficient nns - dependent transcription termination in  vivo ( porrua et  al . , 2012 ) .
as shown in figure  6a , using limiting concentrations of rtrf4-rair2 relative to the substrate , polyadenylation was markedly stimulated by the addition of purified recombinant nrd1p - nab3p , resulting in a longer length of the added poly(a ) tails .
we did not observe any substantial increase in the fraction of rna that is polyadenylated in response to the addition of rnrd1-nab3 ( compare the levels of nonadenylated substrate in figures 6a and 6b ) , suggesting that , at least in  vitro , stimulation preferentially occurs on molecules that have already undergone a polyadenylation cycle .
stimulation was dependent on the interaction between rnrd1 and rtrf4 because it was abolished by the use of rnrd1cid instead of wild - type rnrd1 ( figure  6b ) .
however , the addition of recombinant cid alone did not enhance the polyadenylation activity of rtrf4-rair2 , ruling out that stimulation could result from an allosteric activation of trf4p by the cid .
the whole rnrd1-rnab3 heterodimer was required because neither subunit alone could enhance polyadenylation ; rather , we observed a mild inhibition of rtrf4p - rair2 activity when adding only rnab3 to the reaction ( figure  6b ) .
finally , high - affinity binding of rnrd1-nab3 to the rna was required because no significant stimulation could be obtained when using a mutant substrate that binds the heterodimer with an affinity 20-fold lower than that of the wild - type version ( figures s6b and s6c ) .
taken together , our results strongly suggest that the simultaneous interaction of nrd1p - nab3p with trf4p - air2p and the rna enhances polyadenylation , most likely by favoring or stabilizing the association of trf4p - air2p with its substrate .
the nrd1-nab3-sen1 complex is of major biological relevance because of its central role in the biogenesis of snrnas and snornas and in the control of pervasive transcription in connection with the exosome and tramp complexes ( schulz et  al . ,
the cid domain of nrd1p has retained special attention because of its ability to bind the ctd of rnapii . however , despite a number of biochemical and structural studies ( vasiljeva et  al . , 2008 ; kubicek et  al . , 2012 ) , its actual function in  vivo has remained mysterious . in this work ,
we reexamine the role of the cid and demonstrate that it plays important roles in the efficiency of transcription termination and in rna degradation .
the genome - wide occupancy of rnapii upon deletion of the cid reported here clearly reveals a widespread role for this domain in  transcription termination of cuts , snornas , and to some extent , small orfs ( figures 1 and s1 ) , which is consistent with the known landscape of nns targets ( steinmetz et  al . , 2006b ) . while this work was in progress , heo et  al .
however , because deletion of the cid does not prevent nns termination , the interaction between nrd1p and the rnapii ctd only impacts the efficiency of the process .
deletion of the cid has a strong impact on nrd1p recruitment , even in the presence ( as in cuts ) of clusters of nrd1 and nab3 sites on the nascent rna , which have been shown to be required for recruitment ( gudipati et  al . , 2008 ) .
this can be best explained if recruitment depends synergistically ( and not redundantly ) on the interaction of the nrd1-nab3 complex with both the ctd and the nascent rna .
termination of mrna coding genes depends on the cleavage and polyadenylation factor / cleavage factor ( cpf / cf ) that also interacts with the nascent rna and the ctd .
we have previously shown that the cpf and the nns complex have partially overlapping sequence requirements and that the same rna sequence can be used as a terminator by either complex depending only on the distance from the transcriptional start ( gudipati et  al . , 2008 ; porrua et  al . , 2012 ) .
it is likely that early recruitment of the nns complex via the nrd1p cid - ctd interaction kinetically favors the appropriate recognition of rna binding sites that could otherwise be bound by the cpf complex , impairing termination by the nns pathway .
our results strongly suggest that the function of the cid in rna degradation relies on the interaction with a ctd - like motif in trf4p .
the strong association between the termination complex and the corresponding poly(a ) polymerase mirrors the association of pap1p with the cpf complex , suggesting that in both cases the appropriate poly(a ) polymerase is brought in the proximity of the 3-oh of the newly released ( or cleaved ) rna , thus preventing or limiting spurious cross - processing events .
the two alternative forms of tramp containing either trf4p or trf5p have partially redundant functions , but the former predominates in the degradation of cuts and the processing of snrnas and snornas ( san paolo et  al . ,
2009 ) . because trf5p does not contain a nim ( figure  s3d ) , this different target specificity can now also be explained by the interaction of the nns complex with trf4p .
in contrast to the robust interaction of the nns complex with tramp , we found the interaction of rrp6p and the exosome with the nns complex in  vivo to be strongly rna dependent ( figure  s3a ) , which is in apparent discrepancy with a previous report ( vasiljeva and buratowski , 2006 ) .
nevertheless , we could show a direct interaction between rrp6p and nrd1p in  vitro that is independent from the cid , suggesting that this interaction is too weak to withstand our immunoprecipitation conditions or that it forms only transiently in  vivo .
it is known that the rna quality control factors target a vast repertoire of defective molecules that are sorted because of kinetic competition between rna degradation and processing ( bousquet - antonelli et  al . , 2000 ; gudipati et  al . , 2012
) . however , when alternative routes to discarding are not desired , it is crucial to enforce degradation by the use of specific adaptors .
the nns complex fulfils such a function by recruiting the tramp to its targets after transcription termination via the cid - nim interaction .
this could stimulate degradation because of enhanced polyadenylation of the substrates , but it is also possible that degradation is stimulated by a poly(a)-independent mechanism ( callahan and butler , 2010 ; lacava et  al . , 2005 ; wyers et  al . , 2005 ) , maybe by recruiting the exosome by virtue of the direct trf4p - rrp6p interaction ( figure  5 ) .
we note that stimulation preferentially occurs on a fraction of already polyadenylated rnas rather than on the nonadenylated substrate .
it is possible that nrd1p - nab3p and trf4p - air2p compete for binding to the short rna substrates we used in our assays , which is also suggested by the inhibition of the polyadenylation reaction when rnab3 alone or rnrd1cid - nab3 are used ( figure  6b ) .
the emergence of a poly(a ) tail likely provides a binding platform in the vicinity of the substrate 3-oh that would be preferentially bound by trf4p - air2p .
it has previously been suggested that the rna helicase mtr4p modulates the activity of trf4p - air2p , restricting the addition of poly(a ) tails after 34 nt in  vitro ( jia et  al . , 2011 ) .
although very short poly(a ) tails ( 4 nt ) have also been observed in  vivo ( jia et  al . , 2011
2011 ) , it must be noticed that these represent average steady - state lengths , resulting from an equilibrium between synthesis and degradation .
when degradation is impaired , trf4p - dependent poly(a ) tails in  vivo are longer ( wyers et  al . , 2005 ; d.l . , unpublished data ) , most likely within the range required to allow threading of substrates through the exosome channel ( i.e. , roughly 30 nt ) .
the antagonistic impact of the nrd1-nab3 complex and mtr4p on trf4p activity might imply a temporal regulation of polyadenylation , restricted by mtr4p early after transcription to prevent the binding of pab1p ( jia et  al . , 2011 ) and stimulated later on by nrd1p - nab3p to favor degradation .
the ctd and nim share a sequence element that can form a  turn ( figure  4 ) .
the binding mode of the nim peptide at the  turn conformation resembles other previously determined structures of ctd bound to cids of pcf11p , scaf8 , rtt103p , and nrd1p ( figures 4c and s7 ) ( meinhart and cramer , 2004 ; becker et  al .
however , in contrast to these cid - ctd complexes , the  turn of the nim peptide has more extensive hydrophobic contacts with nrd1p due to the presence of tyr582 , the third residue of the  turn not present in any of the ctd repeats ( jasnovidova and stefl , 2013 ) .
the stacking of tyr582 with ile130 of nrd1p significantly contributes to the overall increase of binding affinity of nim to nrd1p in comparison to the phosphorylated ctd .
the other region of the nim peptide that contributes to the overall affinity is the aspartate - rich region located at the n terminus that intimately interacts with the electronegative pocket of nrd1p ( figure  4b ) .
to some extent , this mimics the recognition of phosphorylated ctd , but it involves more contacts , strengthening the overall binding affinity ( figure  4c ) .
it is also likely that other proteins contain these two elements with the same arrangement and therefore could interact with cid - containing proteins in a similar manner .
our structural data together with our competition assays demonstrate that the interactions of the cid with the ctd and the nim are mutually exclusive ( figures 4 and 5 ) .
this implies the existence of at least two distinct forms of the nns complex , one associated with the polymerase and the other associated with the tramp , which could represent pre- and posttermination forms of the nns complex .
although we show that the affinity of the cid for the nim is 100-fold higher than that for the ctd , the real balance between the two alternative complexes also depends on the number of rnapii and trf4p molecules available for interaction and , importantly , on the number of interaction targets , which is presumably higher for rnapii ( 25 possible diheptapeptides in the ctd ) .
we propose that by virtue of its alternative interactions , the cid controls the handover of the nns complex from rnapii to the tramp , which would temporally coordinate the two functions of the complex .
regulation of nns and tramp function might be reciprocal because interaction with the tramp might control the release of nrd1p from rnapii ( figure  7 ) .
this could be important for the downstream function of the nns complex in processing / degradation , but also for making the complex available for the interaction with new elongating rnapiis .
our results open up the interesting possibility that the dynamics of factors interacting with the ctd throughout the transcription cycle is regulated not only by the enzymes responsible for ctd modifications and proline isomerizations , but also by  competitive interactions with proteins containing ctd - like motifs .
chip and chip - chip experiments were performed as previously described ( mayer et  al . , 2010 ;
more complete details for these experimental procedures as well as construction of plasmids , yeast strains , rna analyses , and standard biochemical analyses can be found in the supplemental experimental procedures .
chip - chip data analysis was performed essentially as described ( mayer et  al . , 2010 ) .
briefly , we first performed quantile normalization between replicate measurements and averaged the signal for each probe over the replicate intensities .
the normalized chip signal at each nucleotide was calculated as the median signal for all probes overlapping this position .
profiles were smoothed using running median smoothing with a window half size of 75  bp . to average profiles over feature classes ,
features were aligned at their transcription start sites ( tsss ) and transcription termination sites ( ttss ) or poly(a ) addition sites for protein - coding genes , scaled to the same length ( i.e. , the median length of all transcripts in the class ) , and averaged by calculating the 5% trimmed mean at each genomic position . for better comparison of cuts and small - sized protein - coding genes , features of both classes with a length between 350 and 550  bp
note that annotation of cuts does not generally take into account the 3 end heterogeneity that is characteristic of these transcripts .
medium - sized protein - coding genes were selected by taking the 50% most highly expressed genes ( dengl et  al . , 2009 ) that were at least 200  bp away from neighboring genes , with an orf length of 1,238    300  bp .
note that because the size of cuts and snornas is generally similar to or lower than the resolution of the chip technique ( 200300 nt ) , the increased downstream rnapii signal also bleeds over the body of the metagene .
the apparent rnapii increase within cuts is also due to the fact that these genes have multiple termination sites ( neil et  al . , 2009 ;
wyers et  al . , 2005 ) that fall within the coordinates of each annotation ( and therefore of the metagene ) .
the possibility that deletion of the cid generally affects transcription initiation at cuts and snornas is very unlikely because ( i ) the rnapii increase in nrd1cid relative to the wt is always minimal at the 5 end and progressively increases toward the termination region ( figures 1a1c and 1e - f ) and ( ii ) we did not detect a significant increase in the levels of mature snrnas ( data not shown ) .
all nmr spectra for the backbone and side - chain assignments of 0.52.0  mm uniformly
n , c - labeled nrd1p cid in 50  mm sodium phosphate buffer ( ph  8.0 ) , 100  mm nacl ( 90% h2o/10% d2o ) were recorded on bruker avance 700 and 950 mhz spectrometers equipped with a cryoprobe at a sample temperature of 20c .
the spectra were processed using nmrpipe package ( delaglio et  al . , 1995 ) , and the protein resonances were assigned manually using sparky software ( t.g .
the h , c , and n chemical shifts of the bound form of nrd1p cid were assigned as described previously ( kubek et  al . , 2011 ;
all distance constraints were derived from the 3d n- and c - separated noesys and 2d h - h noesy ( with mixing time of 80  ms ) collected on a 950 mhz spectrometer .
intermolecular distance constraints were obtained from the 3d f1-c / n - filtered noesy-[c , h]-hsqc experiment ( peterson et  al . , 2004 ; zwahlen et  al . , 1997
) , with a mixing time of 150  ms on a 950 mhz spectrometer .
intramolecular distance constraints of the bound trf4p nim peptide ( unlabeled ) were derived from a 2d f1 , f2-c / n - filtered [ h , h]-noesy ( tm  = 150  ms ) ( peterson et  al . , 2004 ; zwahlen et  al . , 1997 ) .
the noes were semiquantitatively classified based on their intensities in the 2d and 3d noesy spectra .
the structure determination was performed as described previously ( kubicek et  al . , 2012 ) .
polyadenylation reactions were performed at 30c in a final volume of 20  l containing 2  nm 5 end - labeled rna substrate and 1  nm recombinant trf4-air2 in 20  mm tris - hcl ( ph 7.5 ) , 100  mm nacl , 0.5  mm mgcl2 , 10% glycerol , 0.01% nonidet p-40 , and 1  mm dithiothreitol in the presence of rnase inhibitors .
reactions were started upon addition of 2  l of an atp - mgcl2 mixture ( 20  mm each ) and stopped at different time points by collecting 4  l aliquots and mixing them with an equal volume of loading buffer ( 80% formamide , 0.05% w / v bromophenol blue , and 0.05% w / v xylen cyanol ) .
rnas were denatured for 5  min at 75c and separated by 10% ( w / v ) denaturing page .
after electrophoresis , gels were dried and analyzed using a phosphorimager scanner ( ge healthcare ) . to assess the effect of rnrd1 and rnab3 on the polyadenylation activity of rtrf4-air2 , individual proteins or the heterodimeric complex
were added to the reaction at a 3  nm final concentration and incubated for 10  min at 30c before starting the reaction .
a.t . and o.p . designed and performed molecular biology , genetic , and biochemistry experiments .
r.s . directed the research for all the studies performed at masaryk university , notably the determination of the solution structure and part of the biochemical analyses .
specifically , she directed the work of a.t . and designed experimental strategies relating to the discovery and functional characterization of the nim .
d.l . partially directed the work performed in the centre de gntique molculaire , contributing to the definition of the general experimental strategies and to the coordination of the research performed in the two main sites . d.l . also directed the work on the genome - wide analyses of nrd1cid and rnapii distribution . o.p .
 summarythe nrd1-nab3-sen1 ( nns ) complex is essential for controlling pervasive transcription and generating sn / snornas in s.  cerevisiae . 
 the nns complex terminates transcription of noncoding rna genes and promotes exosome - dependent processing / degradation of the released transcripts . 
 the trf4-air2-mtr4 ( tramp ) complex polyadenylates nns target rnas and favors their degradation . 
 nns - dependent termination and degradation are coupled , but the mechanism underlying this coupling remains enigmatic . 
 here we provide structural and functional evidence demonstrating that the same domain of nrd1p interacts with rna polymerase ii and trf4p in a mutually exclusive manner , thus defining two alternative forms of the nns complex , one involved in termination and the other in degradation . 
 we show that the nrd1-trf4 interaction is required for optimal exosome activity in  vivo and for the stimulation of polyadenylation of nns targets by tramp in  vitro . 
 we propose that transcription termination and rna degradation are coordinated by switching between two alternative partners of the nns complex . 
Introduction
Results
Discussion
Experimental Procedures
Author Contributions