Interplay between pre-mRNA splicing and microRNA biogenesis within the supraspliceosome

MicroRNAs (miRNAs) are central regulators of gene expression, and a large fraction of them are encoded in introns of RNA polymerase II transcripts. Thus, the biogenesis of intronic miRNAs by the microprocessor and the splicing of their host introns by the spliceosome require coordination between these processing events. This cross-talk is addressed here. We show that key microprocessor proteins Drosha and DGCR8 as well as pre-miRNAs cosediment with supraspliceosomes, where nuclear posttranscriptional processing is executed. We further show that inhibition of splicing increases miRNAs expression, whereas knock-down of Drosha increases splicing. We identified a novel splicing event in intron 13 of MCM7, where the miR-106b-25 cluster is located. The unique splice isoform includes a hosted pre-miRNA in the extended exon and excludes its processing. This indicates a possible mechanism of altering the levels of different miRNAs originating from the same transcript. Altogether, our study indicates interplay between the splicing and microprocessor machineries within a supraspliceosome context.


INTRODUCTION
MicroRNAs (miRNAs) are small $22-nt long molecules involved in the negative control of gene expression by binding mainly to the 3 0 UTR of target messenger RNA (mRNA) transcripts (1)(2)(3). A large fraction of miRNA genes are located in introns (4)(5)(6). The canonical biogenesis of intronic miRNAs from RNA polymerase II (Pol II) transcripts involves two main steps. The first takes place in the nucleus and is performed by the microprocessor. Key proteins of the microprocessor are DGCR8, which binds the RNA molecule, and Drosha, an RNase III type enzyme, which cleaves the primary (pri) miRNA transcript into a precursor (pre) miRNA stem-loop molecule of $70-80 bases (7)(8)(9)(10)(11). In the second step, which occurs after its export by exportin-5 to the cytoplasm (12,13), the pre-miRNA is cleaved by the RNase III Dicer yielding mature miRNA and its complementary miRNA* (14)(15)(16)(17)(18). The miRNA is then loaded on the RNA-induced silencing complex (RISC) (19)(20)(21), which directs its binding to its target gene.
Another cleavage pathway that takes place on introns is the pre-mRNA splicing process, where the introns are excised out of the pre-mRNA transcript and the exons are ligated. Splicing as well as other processing events of Pol II transcripts occur in the cell nucleus within a huge and highly dynamic ribonucleoprotein (RNP) machine-the supraspliceosome. The supraspliceosome is a 21 (±1.6)-MDa complex of RNA and proteins composed of four native spliceosomes connected by the pre-mRNA (22,23). The entire repertoire of nuclear pre-mRNAs, independent of their length and number of introns, is individually found assembled in supraspliceosomes [reviewed in (24)]. Components of the supraspliceosome include the spliceosomal U small nuclear RNPs (U snRNPs) and splicing factors, among which are Sm proteins; alternative splicing proteins such as SR proteins; the splicing regulatory factor heterogeneous RNP G (hnRNP G) hnRNP G (25); the alternative splicing factors RBM4 and WT1, which cointeract to influence alternative splicing (26); the alternative splicing regulator ZRANB2 (27); and other proteins that process the pre-mRNA, among which are the editing enzymes ADAR1 and ADAR2 (24). The supraspliceosome was shown to have both splicing and editing activities (28,29). Alternative splicing events were also shown to occur within the supraspliceosome (25,30,31). Splicing is a major event in the processing of Pol II transcripts. Therefore, the interplay between the processing of intronic pri-miRNAs and the processing of pre-mRNA is intriguing (32,33).
One way of coordination between intronic miRNAs processing and splicing occurs in short introns. In this case, the entire intron is a pre-miRNA, and the first step of miRNA biogenesis is the splicing of the intron (34,35). *To whom correspondence should be addressed. Tel: +972 2 6585162; Fax: +972 2 5617920; Email: sperling@cc.huji.ac.il The biogenesis pathway of these miRNAs, called 'mirtrons', does not involve the microprocessor. There are also mirtron-like splicing-independent miRNAs that require Drosha, but neither DGCR8 nor Dicer, for their processing and are called 'simtrons' (36).
However, most intronic miRNAs are processed by the microprocessor and, it seems, from the same pre-mRNA molecule as the mRNA (5,37,38). Several reports, with different conclusions, were published in recent years about the processing of the transcripts into mRNAs and miRNAs. Comparison of the level of pri-miRNA transcription expressed from either an intronic sequence or an intronic sequence flanked by exons, showed that the presence of the flanking exons increased the level of transcription, possibly due to prolonged time at the site of transcription and splicing (39). Microprocessing was shown to take place cotranscriptionally before splicing, and it was suggested that this processing enhanced splicing (40). Another study showed that pre-miRNA processing could occur in an intron before its splicing (5). Knock-down of Drosha did not reveal a strong effect on splicing, but introns without a pre-miRNA were spliced more rapidly than those with pre-miRNA. Supporting this, an in vitro system demonstrated that cleavage by the microprocessor can occur faster than splicing and that anti-Drosha antibodies can precipitate Yshaped splicing intermediates (41). In contrast, an artificial intron harboring an miRNA was shown to be spliced before the miRNA processing (42), similarly to what occurs with small nucleolar RNA (snoRNA) processing (43).
A recent study demonstrated a more complex interaction between splicing and microprocessing-it was shown that processing of miR-211, located in intron 6 of melastatin, promoted the splicing of that intron. An importance for the 5 0 splice site (SS) in miRNA biogenesis was also shown (44). In addition, a link between splicing and miRNA processing was found when splicing components were reported to associate or cosediment with the microprocessor and with pre-miRNA (41,45).
Because a large proportion of miRNA genes are located in introns, we searched for a cross-talk between their processing and pre-mRNA splicing. Here we show interplay between pre-mRNA splicing and intronic miRNA biogenesis. We demonstrate that inhibition of splicing increases miRNAs expression, whereas knock-down of Drosha increases splicing. We further show that the microprocessor components, Drosha and DGCR8, and the processed pri-miRNAs of the miR-106b-25 cluster of intron 13 of MCM7 (mini chromosome maintenance 7) are found in supraspliceosomes, together with pri-miRNAs. Importantly, we identified two novel alternative 3 0 splicing events, each between a miRNA pair of the miR-106 b-25 cluster, which extend exon 14 of MCM7 and change its coding capacity. In the novel splice isoform, miRNAs are included in the extended exon and are thus excluded from being processed into miRNA. Thus, changes in these splicing events were shown to differentially affect the levels of miRNAs originating from the same cluster. Overall, we introduce an extended interplay between the miRNA biogenesis and the pre-mRNA splicing within the supraspliceosome.

RNA extraction and real-time polymerase chain reaction
Total RNA from cell lines was prepared and treated with RNase-free DNase I as previously described (46). cDNA was synthesized by using 0.5-5 mg of RNA with dT 15 primer and Moloney murine leukemia virus reverse transcriptase (Promega). Polymerase chain reactions (PCRs) (20 ml) contained 10 pmol of each of the indicated primer pairs (see later in the text) and 1Â taq master mix, purple ( biotech). The amplified products were run on a 2% agarose gel. All experiments were performed with at least 2 or 3 biological repeats.

Drosha knock-down by small interfering RNA
Small interfering RNA (siRNA) targeted to Drosha 5 0 AACGAGUAGGCUUCGUGACUU 3 0 (7) and siGENOME Nontargeting siRNA (Dharmacon) were transfected into HeLa cells with DharmaFECT 1 (Dharmacon) according to the manufacturer's instructions with some modifications. Cells grown in 6-cm plates were transfected with 75-nM siRNA by using 0.176% DharmaFECT 1. After 72 h, total proteins and RNA were extracted and analyzed.

Preparation of supraspliceosomes, and analyses of protein and RNA
Supraspliceosomes were prepared from HeLa cells (CILBIOTECH) as previously described (47,48). Briefly, nuclear supernatant was prepared from clean cell nuclei by microsonication of the nuclei and precipitation of the chromatin in the presence of transfer RNA. The nuclear supernatant was fractionated on 10-45% (vol/vol) glycerol gradients. Centrifugations were carried out at 4 C in an SW41 rotor run at 41 krpm for 90 min (or an equivalent w 2 t). The gradients were calibrated with tobacco mosaic virus as a 200S sedimentation marker. Supraspliceosome peak fractions were confirmed by electron microscopy visualization. For a second fractionation, the 200S peak fractions were pooled, and the glycerol was removed by dialysis. The sample was then concentrated using Vivaspin 15R hydrosart 5 KDa (Sartorius, according to the instructions) and subsequently loaded on a second 10-45% glycerol gradient and centrifuged using the same parameters. For protein analysis, fractions were acetone-precipitated. For RNA extraction, fractions of the glycerol gradients (520 ml) were mixed with 150 ml of extraction buffer [50 mM Tris-HCl (pH 7.5), 300 mM NaCl] and 50 ml of 10% (w/ v) sodium dodecyl sulphate, and the RNA was recovered by extraction with phenol and precipitation in ethanol. Supraspliceosome fractions (sedimenting at 200S), were pooled, concentrated using vivaspin 15R 5000 MWCO HY (Sartorius) and analyzed.

Preparation and analysis of nuclear and cytoplasmic fractions
The purification was performed according to Qiagen's cytoplasmic RNA extraction using RNeasy protocol. Six-well plates were washed with phosphate-buffered saline followed by the addition of 175 ml of cold RLN buffer (50 mM Tris, pH 8, 140 mM NaCl, 1.5 mM MgCl 2 and 0.5% NP40). The cells were then scraped and moved to an Eppendorf tube on ice for 5 min. Centrifugation for 2 min at 300g at 4 C was then performed. The supernatant was transferred to a new tube, and both tubes (supernatant-cytoplasm and pelletnucleus) were centrifuged again for better purification. RNA was then extracted from both fractions using the TRI-reagent method.

Morpholino transfections
Antisense morpholino (Gene Tools) against the 3 0 SS between miR-93 and miR-25, 5 0 TCCTGTGAGGGAG ACCAGACCCTTT 3 0 , was transfected to HeLa cells with endo-porter (Gene Tools). One milliliter of medium without serum was added to 3.5-cm plates. Morpholino was added to a concentration of 10 mM followed by the addition of endo-porter, final concentration 0.6%. RNA was extracted 24 h later. The results represent two biological repeats and at least three repeats of each.

Spliceostatin A treatment
Spliceostatin A (SSA) (51), provided by Dr Minoru Yoshida (Chemical Genomics Research Group and Chemical Genetics Laboratory, RIKEN, The Institute of Physical and Chemical Research, Wako City, Saitama, Japan), was diluted in methanol and was transfected into HeLa cells. One microliter of 100 ng/ml SSA was added to a 3.5-cm plate with 1 ml of medium (final concentration 100 mM). As a control, 1 ml of methanol was added to 1 ml of medium. RNA was extracted 5 h later. The results represent two biological repeats and at least three repeats from each.

RESULTS
The microprocessor and pri-miRNAs are found within the supraspliceosome Given that a large proportion of miRNA genes are located within introns of genes transcribed by Pol II, and that RNA processing activities are performed within the supraspliceosome, we asked whether key microprocessor components are found in supraspliceosomes. To answer this question, we fractionated, on glycerol gradients, nuclear supernatants enriched with supraspliceosomes prepared from HeLa cells (24) and then re-fractionated the 200S fractions, where supraspliceosomes sediment, on a second glycerol gradient. WB analyses across the second gradient, with antibodies against the two microprocessor core proteins Drosha and DGCR8, show a similar distribution of the two proteins ( Figure 1A). Both peak at the 200S-supraspliceosome-region of the gradient, where Pol II transcripts as well as spliceosomal proteins and splicing regulatory factors (e.g. SR proteins, hnRNP G, previously shown to be associated with supraspliceosomes) sediment ( Figure 1A, bottom) (24,25,28,30,47,(52)(53)(54). In addition, both proteins appear as free proteins (or part of small complexes) at the top of the gradient, indicating dissociation from the large complexes during fractionation. The presence of microprocessor key components in supraspliceosomes fractions prompted us to further analyze the possible cross-talk between the microprocessor and the supraspliceosome by studying specific pri-miRNAs within the supraspliceosome. For this aim, we chose to study miR-25, a member of the miR-106b-25 cluster in intron 13 of MCM7, which harbors miRNAs 106 b, 93 and 25, and was already investigated with regard to splicing (5,40). This cluster is conserved in mammals, whereas the miR-25 and miR-93 are also conserved in Xenopous tropicalis and zebrafish (Supplementary Figure S1). The miR 106b-25 cluster has two paralogs-miR 17-92 and miR 106a-363. It is overexpressed in several cancers and was found to be prooncogenic, like miR 17-92 (55). To analyze how pri-miR-25 of this cluster is distributed across a glycerol gradient of fractionated nuclear supernatants enriched with supraspliceosomes prepared from HeLa cells nuclei, RNA was extracted from each gradient fraction, and RT-PCR was performed with primers flanking the pre-miRNA. It can be seen that pri-miRNA of miR-25 peaks at the 200S region where supraspliceosomes sediment ( Figure 1B). For comparison, we also analyzed the distribution across a glycerol gradient of a pri-miRNA of a single intronic miRNA. We chose pri-miRNA of miR-330 located in intron 4 of the echinoderm microtubule-associated protein like 2 (EML2) gene. RT-PCR analysis revealed that pri-miRNA of this miRNA also peaks in supraspliceosome fractions ( Figure 1B) indicating that more than miR-25 is present in these complexes.
Processed pri-miRNAs of the miR-106b-25 cluster are found in supraspliceosomes The finding of key microprocessor components and pri-miRNA sequences in supraspliceosomes fractions suggests interaction between the microprocessor and the splicing machine. To further substantiate this interaction, focusing on the miR-106b-25 cluster in intron 13 of MCM7, we searched for sequences of pre-miRNAs of the 106b-25 cluster within the deep sequencing data of small RNAs (<200 nt) present in supraspliceosome (24) fractions (8-10) isolated from HeLa cells nuclei (analysis of full sequencing data will be published elsewhere). Figure 2 shows that the supraspliceosome fractions harbor processed pri-miRNA-106b, pri-miRNA-93 and pri-miRNA-25. The data are in support of the previous results. Importantly, the analysis revealed that the sequences found in supraspliceosomes are not only of pri-miRNA (which might be part of an unprocessed intron) but of processed pri-miRNA-106b, pri-miRNA-93 and pri-miRNA-25, as manifested by the decline in the number of reads flanking each pre-miRNA of the 106 b-25 cluster (see also Supplementary Figure S2). Some are full-length pre-miRNAs and some are smaller that might be further cleavage products from the pre-miRNAs. The finding of both pri-miRNAs and processed pri-miRNAs of the 106b-25 cluster in supraspliceosome fractions indicates that the processing of these pri-miRNAs to pre-miRNAs likely occurs within the supraspliceosome.

Novel splicing events in the miR-106b-miR-25 cluster
To further explore the relationship between splicing and miRNA processing, we focused on the miR-106b-25 cluster in intron 13 of MCM7 (5,40), encouraged by the finding of both pri-miR 25 and pre-miR 25 sequences in supraspliceosome fractions. We next analyzed the expression of sequences of intron 13 of MCM7 using RT-PCR and different combinations of primer pairs ( Figure 3A). The analyses revealed two previously unknown alternative Figure 1. Microprocessor components and pri-miRNA sequences are found in supraspliceosomes. (A) Nuclear supernatants enriched for supraspliceosomes were prepared from HeLa cells and were fractionated in 10-45% glycerol gradients (28). Supraspliceosome peak fractions (8-10) were pooled and refractionated on a second glycerol gradient. Aliquots from gradient fractions were analyzed by WB using anti-Drosha and anti-DGCR8 antibodies. For comparison, distribution across glycerol gradients of regulatory splicing factors associated with supraspliceosomes, hnRNP G and SRSF5 SR proteins is shown (B) Nuclear supernatants enriched for supraspliceosomes were prepared from HeLa cells and were fractionated in 10-45% glycerol gradients (28). RT-PCR analyses of the distribution of pri-miR-330 and pri-miR-25 across the gradient using the indicated primer pairs that flank the respective pri-miRs. splicing events, alternative to the constitutive 3 0 SS of intron 13, that use two alternative 3 0 SSs, which are located within the cluster ( Figure 3A and B). One of the alternative 3 0 SSs is located between pre-miR-93 and pre-miR-25 (hence termed 93-25 3 0 SS, Figure 3A). The other is located between pre-miR-106b and pre-miR-93 (hence 106b-93 3 0 SS, Figure 3B). The identity of the bands corresponding to the two new splice variants was confirmed by DNA sequencing. The new splice isoform, the product of the 93-25 3 0 SS, seems to incorporate the sequence of pre-miR-25 into exon 14 of MCM7. We validated this result ( Figure 3E) by performing RT-PCR using primer a, (a sense primer from exon 13) and primer j (antisense primer, that spans the 3 0 splice junction of intron 13 and exon 14). The identity of this band was confirmed by DNA sequencing.
Both splicing events seem much less dominant than the constitutive splicing of intron 13 as RT-PCR with primers from exons 13 and 14 (primer pair a/c) gives only the constitutive product (see later in the text). The splicing from the 93-25 3 0 SS seems stronger than the 106b-93 3 0 SS one, again because the use of primer pair a/e gives this product predominantly ( Figure 3B). Also, RT-PCR with primer pair a/f gives more of the unspliced form than of the spliced one ( Figure 3B). The finding that the splicing from the 106b-93 3 0 SS occurs at a lower frequency than that from the 93-25 3 0 SS, might be due to the fact that this SS is newer in evolution ( Figure 3D). The finding of the novel 3 0 SS events introduces an additional aspect to the interplay between splicing and miRNA biogenesis. This is because transcripts that undergo splicing at the novel 3 0 SS include the relevant miRNA sequences within the extended exon, an uncommon feature of miRNAs (4). At the same time, the inclusion of the miRNA sequences in the extended exon changes the coding capacity of the MCM7 3 0 -end.

Conservation of the novel alternative splicing event
Because the miR 106b-25 cluster is conserved in mammals, we asked whether the sequences of the novel 3 0 SS, which might have implications on alterations in the levels of miR-25, miR-93 and of MCM7, are conserved in evolution. Comparison of the sequences of the miR-93-25 3 0 SS and its upstream poly-pyrimidine tract in different organisms shows a high level of conservation ( Figure 3C). The 93-25 3 0 SS is also found in other vertebrates besides mammals. Similar comparison of the sequences of the 106b-93 3 0 SS and its upstream poly-pyrimidine tract shows that they are less conserved ( Figure 3D). To test whether the 93-25 3 0 SS and 106b-93 3 0 SS are functioning in other organisms besides human, we tested the splicing pattern in rats. RT-PCR analyses of RNA prepared from the rat PC12 cell line showed that the 93-25 3 0 SS was also used in rat cells ( Figure 3A), but the splicing from the 106b-93 3 0 SS did not take place ( Figure 3B). The conservation of the novel 3 0 SS sequences indicates their biological relevance, and the use of the 93-25 3 0 SS suggests that this splicing isoform plays a functional role.
Because splicing at the 106b-93 3 0 SS is found in low levels, in the following sections, we focus our analyses on the more abundant 93-25 3 0 SS. We next asked whether the novel spliced isoform is found in supraspliceosomes. Interestingly, RT-PCR analysis of the distribution of the novel splice isoform (93-25 3 0 SS) across the glycerol gradients reveals that it cosediments with supraspliceosmes ( Figure 3F). This suggests that the constitutive, the novel alternative splicing events as well as the pri-miRNA processing events that MCM7 intron 13 undergoes, occur in supraspliceosomes.

The novel splice isoform is found in the nucleus and cytoplasm
The new splice isoform, the product of the 93-25 3 0 SS, incorporates the sequence of pre-miR-25 into exon 14 of MCM7. To investigate whether the new alternative splice isoform that changes the coding capacity of the MCM7 mRNA is exported to the cytoplasm, we analyzed the distribution of the spliced product between the nucleus and the cytoplasm (Figure 4). HeLa cells were fractionated into nuclear and cytoplasmic fractions, and the splicing of the MCM7 products were analyzed by RT-PCR (positive and negative controls for the fractionation are given in Supplementary Figure S3). Because the new spliced product harbors premature termination codons in all three reading frames, it is likely to be a substrate for the nonsense-mediated mRNA decay pathway (56,57). Thus, we performed the same experiment also in the presence of cycloheximide (CHX), which is known to inhibit nonsense-mediated mRNA decay by translational block (58). We observed that GAPDH and MCM7 exon Figure 2. Processing of pri-miRNA in supraspliceosomes. RNA was extracted from supraspliceosomes prepared from frozen HeLa cells, as previously described (28). Searching for sequences of pre-miRNAs of the 106b-25 cluster within the deep sequencing data of small RNAs (<200 nt), from this supraspliceosomal RNA (fractions 8-10), revealed that sequences of the pre-miRNA 106 b, 93 and 25 were found in supraspliceosomes. 13-exon 14 mRNAs are located mostly in the cytoplasm, and RNA from exon 13 of MCM7, which is expected to be in all the MCM7 RNA molecules, is similarly enriched in the cytoplasm. However, the product of splicing at the 93-25 3 0 SS is distributed evenly between the nucleus and the cytoplasm in the untreated cells. The addition of CHX elevated the level of this isoform in the cytoplasm (1.51 ± 0.14-fold compared with changes in mRNA level, measured using primers from exons 13 and 14). Yet, the distribution between nuclear and cytoplasmic fractions of this isoform remains relatively even compared with the distribution of the constitutive mRNA molecules, which is mainly cytoplasmic. First, these results show that the new 93-25 3 0 SS isoform is transported to the cytoplasm. Second, regardless of the CHX treatment, the 93-25 3 0 splice isoform is dispersed more evenly between the cytoplasm and the nucleus than the constitutive MCM7 mRNA, and the part of it that is found in the nucleus indicates that it might also have some function in the nucleus, presumably reflecting the competition between the novel splicing event and the production of the miRNA.

Inhibition of the splicing at the 93-25 3 0 SS results in elevated miRNA levels
Given that the 93-25 3 0 SS is located between the sequences of pre-miRNA 93 and 25, it is likely that changes in splicing at that site affect the level of the miRNAs of the cluster. To test this, we decided to inhibit this splicing event in a specific manner, and then test the effect on miRNA levels. For this purpose, we used antisense molecules in the form of morpholinos, antisense to the 93-25 3 0 SS and the polypyrimidine tract, 25 nt in length ( Figure 5A). Following incubation, RNA was extracted and analyzed by RT-PCR for mRNA and miRNA levels. The analyses confirmed the inhibition of splicing of the novel isoform and not of the canonical isoform ( Figure 5B).
To test the effect of the inhibition of splicing at the 93-25 3 0 SS on the level of miRNAs processed from this cluster, we performed quantitative RT-PCR using Taq-Man microRNA assay ( Figure 5C). For comparison, we  tested the effect of the morpholino on the level of let-7g (an intronic miRNA expressed in HeLa cells). Normalizing the effect of morpholino on the 106b-25 cluster to that on let-7g, it seems that the levels of miRs-106b and 93 were not so affected. However, the level of miR-25 was upregulated (although to a small extent, possibly due to the relatively small effect of the morpholino on the splicing reaction). Although other explanations cannot be ruled out, the results indicate that for the efficient processing of pre-miRNA-25, its inclusion in the intron, rather than in the exon, is favored. This is because inhibition of the splicing event that excludes it from the intron and leads to its inclusion in the alternatively spliced mRNA resulted in the increase of the level of this miRNA.

Inhibition of splicing by spliceostatin A upregulates the levels of the intronic miRNAs
After observing the effect of the specific inhibition of the novel splicing event on the interplay between splicing and miRNA biogenesis, we wanted to test the effect of general inhibition of splicing on this interplay. The inhibition was achieved using spliceostatin A (SSA), which inhibits splicing by binding to SF3b, a subcomplex of U2 snRNP, and keeping the splicing reaction in its primary state (51,59). As can be seen in Figure 6B, SSA inhibited both splicing reactions; namely, the constitutive splicing and the use of the 93-25 3 0 SS. The constitutive splicing seems less affected, perhaps because of its initial high level ( Figure 4). The splicing of exon 14-exon 15 is also inhibited ( Figure 6B, right panel). When we tested the effect of SSA on the level of the miRNAs, using quantitative RT-PCR (Taq-Man microRNA assay), we found that all three miRNAs of the cluster were upregulated ( Figure 6C). The level of the intronic let-7g was also upregulated ( Figure 6C). A number of possible interpretations comes to mind; for example, pri-miRNA processing from an intact intron is more efficient; retaining the intron by inhibiting splicing allows an extended period for processing of pri-miRNA. After splicing inhibition, RT-PCR with primer pair a/e did not amplify more of the unspliced intron 13 as expected ( Figure 6B), possibly because the pre-miRNAs were cleaved from the intron, thereby precluding it from being amplified. The amplification of the adjacent intron 14 (RT-PCR with primer pairs g/h and g/i), which lacks any miRNAs, was higher after splicing inhibition ( Figure 6B). Independent of the interpretation, this experiment shows interplay between splicing and pri-miRNA processing.

Drosha knock-down upregulates the novel splice isoform of MCM7
To test whether the microprocessing affects the splicing reaction-Drosha was knocked-down from cells by siRNA. Figure 7B shows WB with anti-Drosha antibodies verifying that the level of Drosha was reduced significantly following the addition of the siRNA. As cleavage of the pre-miRNAs out of the intron is downregulated after Drosha knock-down, it is expected that there would be more of the intact intron. This seems to be the case as on knock-down of Drosha, primer pair a/f gives significantly higher levels of the pri-miRNA 106b and 93 (5.25-fold increase, Figure 7C). The level of the pri-miRNA 25 (primer pair d/e, amplifying a part of intron 13 surrounding pri-miRNA 25), another substrate of Drosha, was also upregulated, but to a lesser extent when Drosha's levels decreased (1.62-fold increase, Figure 7C). This is not surprising, as knock-down of Drosha increased splicing from the novel alternative 93-25 3 0 SS, including pre-miRNA 25 in the exon of the novel splice isoform ( Figure 7C). These results demonstrate an effect of the microprocessor on splicing and further substantiate the cross-talk between these two pathways.

DISCUSSION
The finding that a large proportion of miRNAs are processed from pre-mRNA introns raises the question of how the two processing reactions, that of splicing and pri-miRNA processing, are coordinated. Our results show interplay between splicing and pri-miRNA processing. Coordination between splicing and pri-miRNA processing is likely facilitated by the presence of Drosha and DGCR8, the core of the microprocessor, in supraspliceosome fractions ( Figure 1A). Our finding of processed pri-miRNA in supraspliceosomes ( Figure 2) brings further support to the cross-talk between the splicing and miRNA processing machines and to the hypothesis that the processing of the tested intronic miRNAs occurs in supraspliceosomes. Earlier findings of association between certain splicing components and the microprocessor and pre-miRNAs (41,45) are in support of our findings.
To analyze the effect of splicing inhibition on miRNA biogenesis, we chose the splicing inhibitor SSA, which was shown to inhibit splicing by binding to SF3b and preventing the pre-spliced spliceosome from moving to an active state (51,59,60). A number of previous studies showed that intronic pre-miRNAs are processed by the microprocessor before splicing, but when assembled in spliceosomes (5,40,44). In the presence of SSA, introns are inhibited from being spliced out, as exemplified by intron 14 of MCM7, which does not harbor a pre-miRNA ( Figure 6). Importantly, the likelihood of cleavage of the pre-miRNAs from the looped-out intron increased upon SSA treatment, resulting in elevated levels of the miRNAs (miRs 106b, 93, 25 and let-7g, Figure 6). Accordingly, intron 13, harboring the 106b, 93 and 25 miRNAs is cleaved by the microprocessor and therefore is not detected by RT-PCR. These results show that splicing inhibition increases the levels of miRNAs, indicating competition between microprocessing and splicing.
Interestingly, the new splice isoform, the product of splicing at the 93-25 3 0 SS, incorporates the sequence of pre-miR-25 into exon 14 of MCM7 ( Figure 3B and E). miRNAs are usually not found in exons, and if so, they are predominantly in noncoding RNAs (4). We found that the inclusion of miR-25 in a coding mRNA affected its processing. The inhibition of splicing at the 93-25 3 0 SS by antisense morpholino ( Figure 5) led to an increase in the level of miR-25, in accordance with the model that the miRNAs are cleaved out of the intron and that the use of the 93-25 3 0 SS excludes pre-miR-25 from the intron. In addition, the processing of miR-25 was the least upregulated by the general inhibition of splicing by SSA that keeps the pre-spliced state ( Figure 6).
When we tested the effect of inhibition of the microprocessor on splicing, by knock-down of Drosha, we found that the level of the novel spliced isoform increased, likely because the SS was more available as less cleavage occurred by the microprocessor within the intron (Figure 7). These results add further support to the cross-talk between splicing and miRNA biogenesis.
Although both miRNA 25 and MCM7 mRNA can be generated from an MCM7 pre-mRNA spliced at the normal SSs, this is not the case when splicing occurs at the novel 3 0 SS. As shown in Figure 4, a detectable level of the new splice isoform (the product of splicing at the 93-25 3 0 SS), is found in the cytoplasm that might be translated into proteins. This alternative isoform encodes for a shortened protein that lacks the 3 0 carboxy terminus because the alternative exon harbors premature termination codons in all three reading frames. In view of our results, we suggest that expression of the novel splice isoform can lead to a modulation in the levels of the different miRNAs of the cluster and of the hosting mRNA isoforms originating from the same transcript.
A recent study of the effect of alternative splicing on miRNA levels in plants (61) showed that an alternative splicing event occurring on heat shock included intronic miR-400 in the alternative exon and excluded it out of the intron, leading to a reduction in miR-400 level. These results are consistent with our findings of the effect of the novel 3 0 SS on the level of miR-25. Both studies demonstrate the importance of alternative splicing in controlling miRNA levels.
The evolutionary conservation of the sequences of the pre-miR-106 b-25 cluster as well as the sequences of the novel 93-25 3 0 SS indicate the importance of these miRNAs and the biological relevance of the interplay between their processing and the splicing at the novel 3 0 SS. The 5 0 -end of intron 13 of MCM7 including pre-miRNA 106b up to pre-miRNA 93 is newer in evolution. Compatible with that, splicing from the 93-25 3 0 SS is more abundant than that from the 106b-93 3 0 SS and is also found in rat (Figure 3). One can speculate that early on, when there were only two miRNAs in that intron, an SS evolved to separate them. Now, a new SS is emerging separating the new miRNA from the older ones.
MCM7 plays a critical role in the G1/S phase transition, ensuring that the entire genome is replicated once and not more than once at each cell cycle (55). Because MCM7 pre-mRNA and pri-miR-106b-25 cluster are cotranscribed, they are both regulated by the transcription factor E2F1. Members of the miR-106b-25 cluster are key modulators of the TGFß tumor suppressor signaling pathway in tumors. While miR-106b and miR-93 inhibit cell cycle arrest through inhibition of p21, miR-25 inhibits apoptosis through affecting Bim (55). Being members of the same cluster, they are likely transcribed to a similar level along with MCM7 mRNA, and posttranscriptional processing events are likely responsible for the final levels of these three miRNAs and mRNA. The finding that both miR-106b and miR-93 inhibit cell cycle arrest, while miR-25 inhibits apoptosis, calls for different processing of miR-25 compared with the other two. The novel 3 0 splice isoform that we identified enables a differential control of the levels of miR-25 compared with miR-106b and miR-93. Although the miR-106b-25 cluster is coexpressed, in some cell lines such as human cholangiocarcinoma (H69, Mz-ChA-1 and KMCH), the miRNAs are expressed differentially, with miR-25 expressed at lower levels than miRs 106b and 93 (62,63). Furthermore, the novel 3 0 splice isoform, not only eliminates production of miR-25 from these transcripts, but also at the same time encodes for a defective MCM7 protein isoform with a shortened carboxy-terminal end. In view of the important role of MCM7 in replication, and in view of the fact that both MCM7 and miR-106b-25 cluster are pro-oncogenic, understanding the regulation of their processing is important for human health and disease. Importantly, it is likely that the level of the novel 3 0 splice isoform can be influenced by a number of cellular or environmental conditions that affect alternative splicing, thus changing the level of miR-25 compared with that of miR-106b and miR-93.
In conclusion, here we shed light on the interplay between intronic miRNA processing and splicing. We show an association between the two machineries-the splicing and the microprocessor, Drosha and DGCR8, within the supraspliceosome. We also suggest how miRNA processing can use alternative splicing to manipulate the levels of different clustered intronic miRNAs. Specifically, we show how miRNAs of the miR-106b-25 cluster, involved in the inhibition of several apoptotic pathways can be differentially expressed.