Reciprocal intronic and exonic histone modification regions in humans.

While much attention has been focused on chromatin at promoters and exons, human genes are mostly composed of intronic sequences. Analyzing published surveys of nucleosomes and 41 chromatin marks in humans, we identified histone modifications specifically associated with 5' intronic sequences, distinguishable from promoter marks and bulk nucleosomes. These intronic marks were spatially reciprocal to trimethylated histone H3 Lys36 (H3K36me3), typically transitioning near internal exons. Several marks transitioned near bona fide exons, but not near nucleosomes at exon-like sequences. Therefore, we examined whether splicing affects histone marking. Even with considerable changes in regulated alternative splicing, histone marks were stable. Notably, these findings are consistent with exon definition influencing histone marks. In summary, we show that the location of many intragenic marks in humans can be distilled into a simple organizing principle: association with 5' intronic or 3' exonic regions

Bioinformatics analysis of multi-omics data elucidates U2 snRNP function in transcription

Transcription by RNA polymerase II (Pol II) is an important step in cell function and regulation. Pol II transcription has been shown to be coupled to pre-mRNA splicing, but the underlying mechanisms remain poorly understood. Co-transcriptional splicing requires the assembly of a functional spliceosome on nascent pre-mRNA, but whether and how this influences Pol II transcription remains unclear. To investigate this, we used a human erythroleukemic cell line and performed transient transcriptome sequencing (TT-seq) and mammalian native elongating transcript sequencing (mNET-seq) upon fast inhibition of U2 snRNP function. We further studied how the positive transcription elongation factor b (P-TEFb) recruitment is related to the Pol II pause duration, using chromatin immunoprecipitation and sequencing (ChIP-seq) of the PTEF-b kinase cyclin T1 (CycT1) upon U2 snRNP inhibition. I performed a bioinformatics analysis of the different datasets generated for this study and two additional published datasets. I also conducted a multiomics analysis combining TT-seq and mNET-seq data to calculate and quantify transcription kinetic parameters such as Pol II productive initiation frequency, pause duration and elongation velocity. Here we show that inhibition of pre-mRNA branch site recognition by the spliceosome component U2 snRNP leads to a widespread and strong decrease in new RNA synthesis from human genes. We further show that inhibition of U2 snRNP function increases the duration of Pol II pausing in the promoter-proximal region, impairs recruitment of the pause release factor P-TEFb, and reduces Pol II elongation velocity at the beginning of genes. Our results indicate that efficient release of paused Pol II into active transcription elongation requires the formation of functional spliceosomes and that eukaryotic mRNA biogenesis relies on positive feedback from the splicing machinery to the transcription machinery. We further show that the fast U2 snRNP inhibition affects the expression of genes related to RNA synthesis and it is not related to stress response genes. Our new multi-omics approach for the calculation of Pol II elongation velocity can be applied to further study the impact on Pol II kinetics regarding different splicing and transcription factors. This is of great importance to unravel the mechanisms behind Pol II transcription and splicing and understand how the disruption of this regulation leads to several pathological cell phenotypes and diseases.2021-08-2

Systematic Dissection of Roles for Chromatin Regulators in Dynamics of Transcriptional Response to Stress in Yeast: A Dissertation

The following work demonstrates that chromatin regulators play far more pronounced roles in dynamic gene expression than they do in steady-state. Histone modifications have been associated with transcription activity. However, previous analyses of gene expression in mutants affecting histone modifications show limited alteration. I systematically dissected the effects of 83 histone mutants and 119 gene deletion mutants on gene induction/repression in response to diamide stress in yeast. Importantly, I observed far more changes in gene induction/repression than changes in steady-state gene expression. The extensive dynamic gene expression profile of histone mutants and gene deletion mutants also allowed me to identify specific interactions between histone modifications and chromatin modifiers. Furthermore, by combining these functional results with genome-wide mapping of several histone modifications in the same time course, I was able to investigate the correspondence between histone modification occurrence and function. One such observation was the role of Set1-dependent H3K4 methylation in the repression of ribosomal protein genes (RPGs) during multiple stresses. I found that proper repression of RPGs in stress required the presence, but not the specific sequence, of an intron, an element which is almost unique to this gene class in Saccharomyces cerevisiae. This repression may be related to Set1’s role in antisense RNA-mediated gene silencing. Finally, I found a potential role for Set1 in producing or maintaining uncapped mRNAs in cells through a mechanism that does not involved nuclear exoribonucleases. Thus, deletion of Set1 in xrn1Δ suppresses the accumulation of uncapped transcripts observed in xrn1Δ. These findings reveal that Set1, along with other chromatin regulators, plays important roles in dynamic gene expression through diverse mechanisms and thus provides a coherent means of responding to environmental cues

Evidence for a DNA-Based Mechanism of Intron-Mediated Enhancement

Many introns significantly increase gene expression through a process termed intron-mediated enhancement (IME). Introns exist in the transcribed DNA and the nascent RNA, and could affect expression from either location. To determine which is more relevant to IME, hybrid introns were constructed that contain sequences from stimulating Arabidopsis thaliana introns either in their normal orientation or as the reverse complement. Both ends of each intron are from the non-stimulatory COR15a intron in their normal orientation to allow splicing. The inversions create major alterations to the sequence of the transcribed RNA with relatively minor changes to the DNA structure. Introns containing portions of either the UBQ10 or ATPK1 intron increased expression to a similar degree regardless of orientation. Also, computational predictions of IME improve when both intron strands are considered. These findings are more consistent with models of IME that act at the level of DNA rather than RNA

Origins and Impacts of New Mammalian Exons

Mammalian genes are composed of exons, but the evolutionary origins and functions of new internal exons are poorly understood. Here, we analyzed patterns of exon gain using deep cDNA sequencing data from five mammals and one bird, identifying thousands of species-and lineage-specific exons. Most new exons derived from unique rather than repetitive intronic sequence. Unlike exons conserved across mammals, species-specific internal exons were mostly located in 5' UTRs and alternatively spliced. They were associated with upstream intronic deletions, increased nucleosome occupancy, and RNA polymerase II pausing. Genes containing new internal exons had increased gene expression, but only in tissues in which the exon was included. Increased expression correlated with the level of exon inclusion, promoter proximity, and signatures of cotranscriptional splicing. Altogether, these findings suggest that increased splicing at the 5' ends of genes enhances expression and that changes in 5' end splicing alter gene expression between tissues and between species.Peer reviewe

Links between splicing, transcription and chromatin in Saccharomyces cerevisiae

There is increasing evidence from yeast to humans that splicing is mainly a co-transcriptional process, and it is becoming well established that splicing, transcription and chromatin are functionally coupled such that they influence one another. The present work explored the links between splicing and transcription and links between splicing and chromatin in the budding yeast Saccharomyces cerevisiae. Currently, there is little mechanistic insight into the contribution of the core transcription elongation machinery to co-transcriptional spliceosome assembly and splicing. To understand how members of the core transcription elongation machinery affect splicing, I used the auxin-inducible degron (AID) system to conditionally deplete essential and non-essential transcription elongation factors and I analysed the effects on RNA polymerase II, co-transcriptional spliceosome assembly and splicing. The transcription elongation factors that I analysed are all conserved from yeast to mammals and include: Spt5, Paf1, Ctk1, Bur1 and Bur2. Most notable were the effects of depletion of the transcription elongation factor Spt5, mutations in which were known to cause splicing defects. Here, Spt5 depletion resulted in reduced recruitment of the U5 snRNP to intron-containing genes, meaning proper co-transcriptional activation of the spliceosome was inhibited, explaining how loss or mutation of Spt5 results in splicing defects. This effect was not dependent on phosphorylation of Spt5, however, the unphosphorylated form of Spt5 enhanced co-transcriptional formation of the catalytically activated spliceosome. Together, these data indicate a two-part function for Spt5 in co-transcriptional spliceosome assembly in S. cerevisiae. Firstly, the physical presence of Spt5 is required for proper co-transcriptional recruitment or stable association of the U5 snRNP and B complex formation. Secondly, the loss of Bur1 kinase activity and at least the unphosphorylated form of Spt5 enhances co-transcriptional formation of the catalytically activated spliceosome and splicing. There is correlative and causative evidence that splicing affects chromatin structure and vice versa. Of particular interest to the present work are links between splicing and Histone 3 Lysine 4 trimethylation (H3K4me3), a chromatin mark associated with promoters of active genes. H3K4me3 has been shown to influence and be influenced by splicing in mammalian cells. However, the molecular basis of this is unknown. To further understand the links between splicing and H3K4me3, I used the AID system to conditionally deplete essential splicing factors that act at different stages of the splicing cycle and analysed the effects on H3K4me3. Whilst depletion of splicing factors that affect the first or second catalytic step of splicing reduces H3K4me3 on intron-containing genes, notably, depletion of the late-acting factor Prp22 reduces H3K4me3 in the absence of defects in splicing catalysis, suggesting a more direct role for Prp22. Prp22 is an RNA-dependent ATPase that proofreads to product of the second step of splicing and promotes mRNA release from the post-spliceosome. Interestingly, the effect of Prp22 on H3K4me3 is dependent on its ATPase activity. Furthermore, Prp22 and Set1 were found to interact in a pull-down assay and depletion of Prp22 results in reduced recruitment of Set1 to intron-containing genes. These data show a previously unknown link between Prp22, Set1 and H3K4me3 in S. cerevisiae. Collectively, these analyses provide new mechanistic insight into the links between splicing and transcription and links between splicing and chromatin in S. cerevisiae

The In Vivo Kinetics of RNA Polymerase II Elongation during Co-Transcriptional Splicing

Kinetic analysis shows that RNA polymerase elongation kinetics are not modulated by co-transcriptional splicing and that post-transcriptional splicing can proceed at the site of transcription without the presence of the polymerase

The In Vivo Kinetics of RNA Polymerase II Elongation during Co-Transcriptional Splicing

Kinetic analysis shows that RNA polymerase elongation kinetics are not modulated by co-transcriptional splicing and that post-transcriptional splicing can proceed at the site of transcription without the presence of the polymerase

Purifying selection on exonic splice enhancers in intronless genes

Exonic splice enhancers (ESEs) are short nucleotide motifs, enriched near exon ends, that enhance the recognition of the splice site and thus promote splicing. Are intronless genes under selection to avoid these motifs so as not to attract the splicing machinery to an mRNA that should not be spliced, thereby preventing the production of an aberrant transcript? Consistent with this possibility, we find that ESEs in putative recent retrocopies are at a higher density and evolving faster than those in other intronless genes, suggesting that they are being lost. Moreover, intronless genes are less dense in putative ESEs than intron-containing ones. However, this latter difference is likely due to the skewed base composition of intronless sequences, a skew that is in line with the general GC richness of few exon genes. Indeed, after controlling for such biases, we find that both intronless and intron-containing genes are denser in ESEs than expected by chance. Importantly, nucleotide-controlled analysis of evolutionary rates at synonymous sites in ESEs indicates that the ESEs in intronless genes are under purifying selection in both human and mouse. We conclude that on the loss of introns, some but not all, ESE motifs are lost, the remainder having functions beyond a role in splice promotion. These results have implications for the design of intronless transgenes and for understanding the causes of selection on synonymous sites