Modelling Reveals Kinetic Advantages of Co-Transcriptional Splicing

Messenger RNA splicing is an essential and complex process for the removal of intron sequences. Whereas the composition of the splicing machinery is mostly known, the kinetics of splicing, the catalytic activity of splicing factors and the interdependency of transcription, splicing and mRNA 3′ end formation are less well understood. We propose a stochastic model of splicing kinetics that explains data obtained from high-resolution kinetic analyses of transcription, splicing and 3′ end formation during induction of an intron-containing reporter gene in budding yeast. Modelling reveals co-transcriptional splicing to be the most probable and most efficient splicing pathway for the reporter transcripts, due in part to a positive feedback mechanism for co-transcriptional second step splicing. Model comparison is used to assess the alternative representations of reactions. Modelling also indicates the functional coupling of transcription and splicing, because both the rate of initiation of transcription and the probability that step one of splicing occurs co-transcriptionally are reduced, when the second step of splicing is abolished in a mutant reporter

RNA Polymerase II Pausing Downstream of Core Histone Genes Is Different from Genes Producing Polyadenylated Transcripts

Recent genome-wide chromatin immunoprecipitation coupled high throughput sequencing (ChIP-seq) analyses performed in various eukaryotic organisms, analysed RNA Polymerase II (Pol II) pausing around the transcription start sites of genes. In this study we have further investigated genome-wide binding of Pol II downstream of the 3′ end of the annotated genes (EAGs) by ChIP-seq in human cells. At almost all expressed genes we observed Pol II occupancy downstream of the EAGs suggesting that Pol II pausing 3′ from the transcription units is a rather common phenomenon. Downstream of EAGs Pol II transcripts can also be detected by global run-on and sequencing, suggesting the presence of functionally active Pol II. Based on Pol II occupancy downstream of EAGs we could distinguish distinct clusters of Pol II pause patterns. On core histone genes, coding for non-polyadenylated transcripts, Pol II occupancy is quickly dropping after the EAG. In contrast, on genes, whose transcripts undergo polyA tail addition [poly(A)+], Pol II occupancy downstream of the EAGs can be detected up to 4–6 kb. Inhibition of polyadenylation significantly increased Pol II occupancy downstream of EAGs at poly(A)+ genes, but not at the EAGs of core histone genes. The differential genome-wide Pol II occupancy profiles 3′ of the EAGs have also been confirmed in mouse embryonic stem (mES) cells, indicating that Pol II pauses genome-wide downstream of the EAGs in mammalian cells. Moreover, in mES cells the sharp drop of Pol II signal at the EAG of core histone genes seems to be independent of the phosphorylation status of the C-terminal domain of the large subunit of Pol II. Thus, our study uncovers a potential link between different mRNA 3′ end processing mechanisms and consequent Pol II transcription termination processes

RNA exosome depletion reveals transcription upstream of active human promoters.

Studies have shown that the bulk of eukaryotic genomes is transcribed. Transcriptome maps are frequently updated, but low-abundant transcripts have probably gone unnoticed. To eliminate RNA degradation, we depleted the exonucleolytic RNA exosome from human cells and then subjected the RNA to tiling microarray analysis. This revealed a class of short, polyadenylated and highly unstable RNAs. These promoter upstream transcripts (PROMPTs) are produced approximately 0.5 to 2.5 kilobases upstream of active transcription start sites. PROMPT transcription occurs in both sense and antisense directions with respect to the downstream gene. In addition, it requires the presence of the gene promoter and is positively correlated with gene activity. We propose that PROMPT transcription is a common characteristic of RNA polymerase II (RNAPII) transcribed genes with a possible regulatory potential

Expression of SNURF–SNRPN upstream transcripts and epigenetic regulatory genes during human spermatogenesis

The imprinted domain in human 15q11-q13 is controlled by a bipartite imprinting centre (IC), which overlaps the 5′ part of the paternally expressed SNURF–SNRPN gene. We have recently described two novel genes upstream of SNURF–SNRPN (PWRN1 and PWRN2), which are biallelically expressed in the testis. We have now found that PWRN1 represents an alternative 5′ part of SNURF–SNRPN, and that its expression in the brain is imprinted. To determine when the locus is activated during spermatogenesis and which factors are involved in this process, we have mined gene-expression data of testicular biopsies from men with different types of spermatogenic failure. Whereas PWRN1–SNURF–SNRPN and PWRN2 are expressed in post-meiotic germ cells only, a hitherto undetected SNURF–SNRPN upstream transcript is expressed already at meiosis. Several epigenetic factors (eg, MBD1 and MBD2 isoforms, MBD3L1, SUVH39H2, BRDT, and EZH2) are upregulated at specific stages of spermatogenesis, suggesting that they play an important role in the epigenetic reprogramming during spermatogenesis

A novel function of Tis11b/BRF1 as a regulator of Dll4 mRNA 3′-end processing

We report the characterization of Delta-like-4 (Dll4), an angiogenesis-related gene for which haploinsufficiency is lethal, as an additional target of Tis11b-mediated regulation. Unexpectedly, we show that Tis11b does not alter mRNA stability but rather seems to modulate 3′-processing of Dll4 mRNA in endothelial cells

Direct sequencing of Arabidopsis thaliana RNA reveals patterns of cleavage and polyadenylation

It has recently been shown that RNA 3 ′ end formation plays a more widespread role in controlling gene expression than previously thought. In order to examine the impact of regulated 3 ′ end formation genome-wide we applied direct RNA sequencing to A. thaliana. Here we show the authentic transcriptome in unprecedented detail and how 3 ′ end formation impacts genome organization. We reveal extreme heterogeneity in RNA 3 ′ ends, discover previously unrecognized non-coding RNAs and propose widespread re-annotation of the genome. We explain the origin of most poly(A)+ antisense RNAs and identify cis-elements that control 3 ′ end formation in different registers. These findings are essential to understand what the genome actually encodes, how it is organized and the impact of regulated 3 ′ end formation on these processes

Transcription Termination and Chimeric RNA Formation Controlled by <em>Arabidopsis thaliana</em> FPA

Alternative cleavage and polyadenylation influence the coding and regulatory potential of mRNAs and where transcription termination occurs. Although widespread, few regulators of this process are known. The Arabidopsis thaliana protein FPA is a rare example of a trans-acting regulator of poly(A) site choice. Analysing fpa mutants therefore provides an opportunity to reveal generic consequences of disrupting this process. We used direct RNA sequencing to quantify shifts in RNA 3' formation in fpa mutants. Here we show that specific chimeric RNAs formed between the exons of otherwise separate genes are a striking consequence of loss of FPA function. We define intergenic read-through transcripts resulting from defective RNA 3' end formation in fpa mutants and detail cryptic splicing and antisense transcription associated with these read-through RNAs. We identify alternative polyadenylation within introns that is sensitive to FPA and show FPA-dependent shifts in IBM1 poly(A) site selection that differ from those recently defined in mutants defective in intragenic heterochromatin and DNA methylation. Finally, we show that defective termination at specific loci in fpa mutants is shared with dicer-like 1 (dcl1) or dcl4 mutants, leading us to develop alternative explanations for some silencing roles of these proteins. We relate our findings to the impact that altered patterns of 3' end formation can have on gene and genome organisation