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Not collateral, as the grandson succeeds his grandsire in a d-ssrect line. 5. These mcns opinions are not the product of judgment, or the consequence of reason; but the ofa mind floating at all adventures, without choice, and without direction. of zeal for the government; want of ardour for the reigning prince. ln this age.
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These functions are mediated by transient interactions of the core complex with effector proteins In absence of the pre-EJC, many introns containing weak splice sites are retained.

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The pre-EJC facilitates removal of weak introns by a mechanism involving its prior deposition to adjacent exon junctions. In addition, the depletion of pre-EJC components results in frequent exon-skipping events, particularly at large intron-containing transcripts, although the mechanism is poorly understood 45 , 46 , In Drosophila , loss of Mago in the eye leads to several exon skipping in MAPK , resulting in photoreceptor differentiation defects.

Other large transcripts, often expressed from heterochromatic regions, show the same Mago-splicing dependency. Similarly, in human, exons flanked by longer introns are more dependent on the EJC for their splicing Here, we investigated the mechanism underlying the role of the pre-EJC in exon definition in Drosophila. We observed that depletion of pre-EJC components, but not of the EJC splicing subunit RnpS1, lead to a global decrease in promoter proximal pausing, altered Pol II phosphorylation state and premature entry into elongation.

These changes are concomitant with underlying changes in chromatin architecture and correlate strongly with exon skipping events. These effects are driven by non-canonical recruitment of pre-EJC components at promoters. Co-immunoprecipitation experiments indicated that Mago associates with Pol II but this association is largely dependent on nascent RNA. Altogether, our results demonstrate a direct role of the pre-EJC in exon definition via the control of promoter proximal pausing.

Thus, the pre-EJC is required for proper expression and splicing of large intron-containing genes. In contrast to intron definition, this exon definition activity only slightly required the EJC splicing subunit RnpS1, suggesting a distinct mechanism. Introns are spliced while nascent RNA is still tethered to Pol II, allowing coupling between splicing and transcription machineries 6 , 7 , 9 , 48 , 49 , To address whether the pre-EJC regulates splicing via modulation of transcription, we performed chromatin immunoprecipitation ChIP experiments for the different forms of Pol II.

Mago prevents premature release into transcription elongation. The tested regions for enrichment are shown in the scheme. Log2 fold changes against input control are shown on Y -axis, while X -axis depicts genomic coordinates. Log2 fold changes against input control are shown on Y -axis, while X -axis depicts scaled genomic coordinates. The quartiles were generated for genes that are Pol II bound in both control and Mago knockdown conditions.

Averaged read counts per million of mapped reads of two independent biological replicates from 4sU-Seq are shown on Y -axis while X -axis depicts scaled genomic coordinates. Averaged read counts per million of mapped reads of two independent biological replicates from 4sU-Seq are shown on Y -axis while X -axis depicts genomic coordinates. Next, we performed 4sU-seq to detect nascent transcripts a modified approach of 51 , see Material and methods , Fig.

Importantly, in contrast to the widespread change in promoter-proximal pausing, the average elongation rate was unaffected in Mago-depleted cells Supplementary Fig. Altogether, our data suggest that the pre-EJC controls Pol II pausing but does not significantly affect the elongation rate. To better dissect the role of pre-EJC in Pol II pausing we examined heat shock hsp genes, which possess a promoter-proximal Pol II that has been extensively characterized During HS, Pol II occupancy rose dramatically and the extent of this increase was similar in control versus Mago KD, suggesting that Mago has no impact on transcription initiation.

These results thus further suggest that the pre-EJC is specifically involved in the control of Pol II pausing rather than in transcription initiation. To test whether the pre-EJC might itself associate with chromatin, as suggested by previous immunostaining of pre-EJC components on polytene chromosomes of Drosophila salivary glands 38 , we performed ChIP-Seq experiments. Mago binding to promoter regions modulates Pol II pausing. Log2 fold changes against input control are shown on Y -axis while X -axis depicts scaled genomic coordinates. Log2 fold changes against input control are shown on Y -axis while X -axis depicts genomic coordinates.

Shown here is recruitment of pre-EJC components to an intronless Sry-delta and intron-containing T48 genes. The color labels to the right indicate the levels of enrichment. BoxB sequences blue rectangles were inserted upstream of the CDS of Firefly luciferase green rectangle. Bottom Quantification of the ChIP experiment.

Chromatin was prepared for the different conditions and followed by immunoprecipitation using antibody directed against total Pol II. The enrichment for three independent biological replicates is shown along with p -values for tested conditions. Accordingly, most of Mago binding to promoters was lost when the chromatin was treated with RNAse T1 prior to immunoprecipitation Fig. Consistently, the proportion of Mago or pre-EJC-bound genes was higher at highly expressed genes Fig.

Altogether these results suggest that pre-EJC binding to promoters might modulate promoter-proximal pausing. Transcription is tightly coupled to chromatin architecture Furthermore, the phasing of nucleosomes within the gene body was strongly altered upon Mago depletion Fig. Pre-EJC-bound promoters showed the most significant changes, consistent with a direct effect Fig.

We also detected a mild but significant negative correlation between changes in Ser2P enrichment and chromatin accessibility Fig. Therefore, Mago modulates histone marks and chromatin accessibility, likely via its promoter-proximal pausing activity. Mago controls chromatin accessibility.

The color labels to the right indicate the levels of nucleosomal occupancy. Composite metagene profiles are also shown, with nucleosomal occupancy level on Y -axis and genomic coordinates on X -axis. The example shown here is Dbp80 gene, a well-defined pre-EJC target. The changes were separated according to pre-EJC binding and a two-sample t -test was performed. Pre-EJC-bound promoters are highlighted by orange color. A mild negative correlation, as shown in the indicated pearson coefficient of correlation, between nucleosome and Ser2P occupancies was found.

We hypothesized that the underlying transcriptional changes upon Mago depletion might drive this size dependency. Indeed, Mago depletion led to an intron-size dependent increase in nucleosomal occupancy at promoters, and decrease in nucleosome occupancy along the gene body and at the TES Fig. In contrast, Ser2P enrichment displayed anti-correlative changes with respect to the nucleosomal occupancy Fig. Thus, Mago has a stronger impact on the transcriptional regulation of genes with longer introns than genes with shorter introns.

To determine whether these changes in nucleosomal and Ser2P occupancies result from pre-EJC binding, we calculated the percentage of genes bound by the pre-EJC in different classes relative to their representation in the total number of expressed genes. Interestingly, we found that pre-EJC binding was significantly over represented at genes containing longer introns Fig. Collectively, our results suggest that pre-EJC components preferentially bind and regulate the expression of large intron-containing genes via a direct transcriptional effect.

Mago controls chromatin accessibility and Pol II pausing in a gene size dependent manner. The proportion of pre-EJC-bound genes is increased for genes containing larger introns, relative to their abundance.

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The overlap between genes that are downregulated upon each pre-EJC component knockdown, and their respective target genes is significant. To determine whether pre-EJC components influence pausing through an interplay with NELF we re-analyzed the publicly dataset available from the ref. Given the similarity of NELF and Mago on pausing we tested whether their binding to promoters was dependent on each other.

To evaluate whether the change in Cdk9 occupancy was directly driven by Mago occupancy, we analyzed pre-EJC-bound and unbound genes. The increase in Cdk9 enrichment for pre-EJC-bound class was mild albeit significantly higher than the unbound class Fig. Rows indicate all the genes bound by Pol II and are sorted by decreasing Pol II occupancy, and the color labels to the right indicate the level of enrichment. To test this hypothesis, we attempted to rescue the splicing defects by decreasing the release of Pol II into gene bodies via simultaneously depleting Cdk9. These data suggest that Cdk9 and Mago antagonistically regulate transcription.

Altogether, these results strongly suggest that Mago regulates gene expression and exon definition via regulation of Pol II promoter-proximal pausing. Restoring pausing is sufficient to rescue Mago-associated exon skipping defects. Signal in the knockdown conditions was normalized to the control condition using Tubulin as loading control and quantification of the intensity was performed with ImageJ. The primers used for the analysis are indicated in the scheme above. Mago KD results in several exon skipping events, which are rescued upon simultaneous knockdown of Cdk9.

The level of exon skipping is compared to the control treatment, with RpL49 used for normalization. All photoreceptors are stained with anti-Elav antibody purple , and R8 the first class of photoreceptor to be specified with anti-Senseless green. Lower panel: Adult eyes of a control fly and flies with indicated KD. As shown previously, eye-specific depletion of Mago strongly impairs photoreceptor differentiation 45 , Strikingly, decreasing Cdk9 function in a similar background rescued eye development Fig.

Notably, the number of differentiated photoreceptors in larvae and adults was substantially increased. To determine if EJC-mediated promoter-proximal pausing is conserved in vertebrates, we investigated the function of Magoh, the human ortholog of Drosophila Mago. Thus, the function and mechanism of the pre-EJC in the control of promoter proximal pausing is conserved in human cells.

The role of Mago on promoter proximal pausing is conserved in human. There was no detectable interaction of Magoh with Ser2P. The specific bands for Pol II are highlighted by arrowheads in f. Western blot was performed with anti-Ser5P antibody.

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The quantification of the intensity was performed with ImageJ. This activity is required for proper recognition of exons. Our work uncovers an unexpected connection between the nuclear EJC and the transcription machinery via the regulation of Pol II pausing, which is conserved from flies to human. The premature release of Pol II into elongation in absence of the EJC results in splicing defects, highlighting the importance of this regulatory step in controlling downstream RNA processing events Fig. Promoter proximal pausing is a widespread transcriptional checkpoint, whose functions and mechanisms have been extensively studied.

Our study provides some answers to these questions. While it is not clear yet how the pre-EJC exerts this function, a simple mechanism would be by steric interference for Pol II binding, although more indirect mechanisms might also exist. However, we also found elevated Cdk9 binding and premature release of Pol II at Mago-unbound genes, albeit to a lesser extent compared to Mago-bound genes, suggesting that additional mechanisms must be involved. A recent study also supports these findings showing specific association of pre-EJC components on polytene chromosomes that depends on nascent transcription but is independent of splicing This is reminiscent to the binding of DSIF and NELF 63 , 64 , 65 , 66 , 67 , 68 , suggesting that interaction with Pol II and stabilization via nascent RNA is a general mechanism to ensure that pausing regulators exert their function at the right time and at the right location.

Nevertheless, since Cbp is also required to stabilize transcripts, the reduced Mago binding might result from this confounding effect. Moreover, other factors must be clearly involved as only a subset of genes is bound by the pre-EJC. This effect occurs via increased P-TEFb recruitment to gene promoters.

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Our previous work along with studies from other groups suggested that the EJC modulates splicing by two distinct mechanisms 43 , 44 , 45 , On one hand, the EJC facilitates the recognition and removal of weak introns after prior deposition to flanking exon-exon boundaries. We proposed that EJC deposition occurs in a splicing dependent manner after rapid removal of bona fide introns, which are present in the same transcript.

This function requires the activity of the EJC splicing subunits Acinus and RnpS1, which are likely involved in the subsequent recruitment of the splicing machinery near the weak introns. While this model is attractive, it does not however explain every EJC-regulated splicing event.

In particular, depletion of pre-EJC components results in a myriad of exon-skipping events, which occur frequently on large intron-containing transcripts this study and refs. In contrast to intron definition, this exon definition activity only slightly required the EJC splicing subunits, suggesting an additional mechanism.