A comprehensive analysis of 3' end sequencing data sets reveals novel polyadenylation signals and the repressive role of heterogeneous ribonucleoprotein C on cleavage and polyadenylation

Alternative polyadenylation (APA) is a general mechanism of transcript diversification in mammals, which has been recently linked to proliferative states and cancer. Different 3' untranslated region (3' UTR) isoforms interact with different RNA-binding proteins (RBPs), which modify the stability, translation, and subcellular localization of the corresponding transcripts. Although the heterogeneity of pre-mRNA 3' end processing has been established with high-throughput approaches, the mechanisms that underlie systematic changes in 3' UTR lengths remain to be characterized. Through a uniform analysis of a large number of 3' end sequencing data sets, we have uncovered 18 signals, six of which are novel, whose positioning with respect to pre-mRNA cleavage sites indicates a role in pre-mRNA 3' end processing in both mouse and human. With 3' end sequencing we have demonstrated that the heterogeneous ribonucleoprotein C (HNRNPC), which binds the poly(U) motif whose frequency also peaks in the vicinity of polyadenylation (poly(A)) sites, has a genome-wide effect on poly(A) site usage. HNRNPC-regulated 3' UTRs are enriched in ELAV-like RBP 1 (ELAVL1) binding sites and include those of the CD47 gene, which participate in the recently discovered mechanism of 3' UTR-dependent protein localization (UDPL). Our study thus establishes an up-to-date, high-confidence catalog of 3' end processing sites and poly(A) signals, and it uncovers an important role of HNRNPC in regulating 3' end processing. It further suggests that U-rich elements mediate interactions with multiple RBPs that regulate different stages in a transcript's life cycle

A comparative transcriptomic analysis of glucagon-like peptide-1 receptor- and glucose-dependent insulinotropic polypeptide-expressing cells in the hypothalamus

ObjectiveThe hypothalamus is a key region of the brain implicated in homeostatic regulation, and is an integral centre for the control of feeding behaviour. Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are incretin hormones with potent glucoregulatory function through engagement of their respective cognate receptors, GLP-1R and GIPR. Recent evidence indicates that there is a synergistic effect of combining GIP- and GLP-1-based pharmacology on appetite and body weight. The mechanisms underlying the enhanced weight loss exhibited by GIPR/GLP-1R co-agonism are unknown. Gipr and Glp1r are expressed in the hypothalamus in both rodents and humans. To better understand incretin receptor-expressing cell populations, we compared the cell types and expression profiles of Gipr- and Glp1r-expressing hypothalamic cells using single-cell RNA sequencing.MethodsUsing Glp1r-Cre or Gipr-Cre transgenic mouse lines, fluorescent reporters were introduced into either Glp1r- or Gipr-expressing cells, respectively, upon crossing with a ROSA26-EYFP reporter strain. From the hypothalami of these mice, fluorescent Glp1rEYFP+ or GiprEYFP+ cells were FACS-purified and sequenced using single-cell RNA sequencing. Transcriptomic analysis provided a survey of both non-neuronal and neuronal cells, and comparisons between Glp1rEYFP+ and GiprEYFP + populations were made.ResultsA total of 14,091 Glp1rEYFP+ and GiprEYFP+ cells were isolated, sequenced and taken forward for bioinformatic analysis. Both Glp1rEYFP+ and GiprEYFP+ hypothalamic populations were transcriptomically highly heterogeneous, representing vascular cell types, oligodendrocytes, astrocytes, microglia, and neurons. The majority of GiprEYFP+ cells were non-neuronal, whereas the Glp1rEYFP+ population was evenly split between neuronal and non-neuronal cell types. Both Glp1rEYFP+ and GiprEYFP+ oligodendrocytes express markers for mature, myelin-forming oligodendrocytes. While mural cells are represented in both Glp1rEYFP+ and GiprEYFP+ populations, Glp1rEYFP+ mural cells are largely smooth muscle cells, while the majority of GiprEYFP+ mural cells are pericytes. The co-expression of regional markers indicate that clusters of Glp1rEYFP+ and GiprEYFP+ neurons have been isolated from the arcuate, ventromedial, lateral, tuberal, suprachiasmatic, and premammillary nuclei of the hypothalamus.ConclusionsWe have provided a detailed comparison of Glp1r and Gipr cells of the hypothalamus with single-cell resolution. This resource will provide mechanistic insight into how engaging Gipr- and Glp1r-expressing cells of the hypothalamus may result in changes in feeding behaviour and energy balance

Pan-tissue analysis of APA regulation in hybrid mice

Alternative polyadenylation (APA), which is regulated by both cis-elements and trans-factors, is widespread across all eukaryotic species and is recognized as a major mechanism of gene regulation. It could change the 3'UTR of an mRNA transcript affecting its stability, translation efficiency, nuclear export and mRNA or translated protein localization, or, if an exonic/intronic polyadenylation site (PAS) upstream of the stop codon is used, it could affect a gene's coding region to produce different protein isoforms with distinct properties. Accumulating evidence suggests that global APA-mediated 3'UTR length change might play an important role in oncogenic transformation, pluripotency, lymphocyte activation, neuronal stimulation and in embryonic development and differentiation. However, recent studies found limited effects of 3'UTRs in most genes compared to other regulatory elements located in 5'UTRs or coding sequence. APA as a molecular trait is a low-level phenotype in the hierarchy of biological organization, and might only exert very limited effects on organismal fitness. Therefore, some researchers proposed the “error hypothesis”, stating that most observed APA is noise and that APA diversity within and between tissues is generally neutral or deleterious, and not functional. Similarly, it has been suggested that APA divergence between species is largely non-adaptive. This scenario would be consistent with the (nearly) neutral theory of molecular evolution, which predicts that genes under relaxed selective constraints accumulate neutral (or slightly deleterious) changes at a faster rate than those under stronger purifying selection. In order to clarify the general and tissue-dependent function and regulation of APA and its evolution in mammals, we applied 3'mRNA sequencing for multiple tissues of an F1 hybrid between the C57BL/6J (Mus musculus) and SPRET/EiJ (Mus spretus) mouse strains. We analyzed the factors regulating APA diversity and addressed the question whether APA is generally non-adaptive as proposed by the error hypothesis. In this study, we quantified all annotated PASs in nine tissues of the F1 hybrid mouse and comprehensively characterized different features of single-PAS genes and multi-PAS genes. Next, we checked the positional effects on PAS strength and discussed the functional difference between rank 1 and rank 2 PASs among distinct gene groups. By quantifying PAS usage in each allele, we studied the genes with divergent major PAS expression level and dN/dS ratio difference, and unveiled different evolutionary patterns between APA patterns and gene expression (mRNA levels). We found that in general APA of multi-PAS genes is consistent with the error hypothesis, and that most APA diversity within and between tissues appears to reflect noise, resulting from molecular error due to weak cis-regulation. However, we did not find different selective constraint in dN/dS between genes with high and with low APA diversity, but found strong correlation between mRNA abundance and APA accuracy. The minor and major relative PAS usage is also affected by PAS position. In addition to most major PAS, many minor PASs appear to have functional importance. They are highly conserved and can compete with the major PASs. Last, we found a small fraction of genes exhibits strongly tissue-regulated APA patterns. In these genes, PAS usage is under intensive trans-regulation between the C57BL/6J and SPRET/EiJ alleles in the F1 hybrid mouse. Whereas many divergent PASs exist between the two alleles in genes with low expression level and under relax selective constraints, comparing these with genes showing allelic mRNA transcript level differences, we unveiled different evolutionary patterns between APA and gene expression

A Multi-Omics Analysis of Transcription Control by BRD4

RNA polymerase II (Pol II) regulation during early elongation has emerged as a regulatory hub in the gene expression of multicellular organisms. Prior research links the BRD4 protein to this control point, regulating the release of paused Pol II into productive elongation. However, the exact roles and mechanisms by which BRD4 influences this and potentially other post-initiation regulatory processes remain unknown. This study combines rapid BRD4 protein degradation and multi-omics approaches, including nascent elongating transcript sequencing (NET-seq), to uncover BRD4’s direct protein functions. Applying NET-seq in comparative studies required experimental adaptations. First, analyses with spiked-in mouse cells proved essential for reliable normalization. Second, the study identified a disproportional enrichment of a chromatin-associated RNA class as NET-seq’s major limitation. Incorporating an additional enrichment step solved this problem and significantly increased Pol II coverage. The resulting high-sensitivity NET-seq method confirmed BRD4’s proposed role in early elongation by revealing a global defect in Pol II pause release upon BRD4 degradation. Observations from proteomics and chromatin immunoprecipitation followed by sequencing (ChIP-seq) experiments suggest that the failed recruitment of Pol II-associated factors (PAF) causes an assembly defect of a competent elongation complex. Interestingly, the elongation defect also affected transcribed enhancers. Pol II occupancy increased in a region proximal to the enhancer center, strikingly similar to the impaired Pol II pause release at genes. An integrated multi-omics analysis that included genome-wide 3D genome information revealed reduced interactions between these enhancers and other regulatory regions. Another unexpected result was the widespread Pol II readthrough transcription quantified by the developed readthrough index, revealing an apparent transcriptional termination defect. The implementation of long-read nascent RNA-sequencing (nascONT-seq) combined with a 3’-RNA cleavage efficiency test detected impaired 3’-RNA processing. Notably, those 3’-RNA cleavage defects correlated with the observed termination defects. A potential explanation is the BRD4-dependent recruitment of general 3’-RNA processing factors to the 5’-control region. These observations start to establish regulatory links between 5’ and 3’ control that require further validation. Overall, the results indicate a general BRD4-dependent 5’ elongation control point required for 3’-RNA processing and termination

To what end? - Computational tools to uncover regulators of pre-mRNA polyadenylation site selection

In eukaryotic cells, remarkably orchestrated regulatory steps ensure the availability of proteins and non-coding RNAs at the right spot, at the right time. While many of these steps, such as splicing, have been well studied since decades, the choice of the mRNA 3' end, which leads to expression of one of the many possible primary transcripts from a single locus has been recognized as key mechanism of post-transcriptional gene regulation only in recent years. Transitions between cell states have been found to be associated with specific patterns of change in poly(A) site usage, leading to coordinated changes in the length of 3' untranslated regions (3' UTRs). As 3' UTRs carry a plethora of cis-regulatory elements, their systematic shortening or lengthening has global effects on the responsivity of the transcriptome to regulation, which in turn affects essentially every aspect of RNA metabolism, including stability, transport and translation. However, the mechanisms underlying alternative polyadenylation (APA) under physiological or pathological conditions remain largely unknown. Likely, changes in poly(A) site choice are caused by changes in the availability of regulators that bind in the vicinity of poly(A) sites and impact their processing efficiency. The projects summarized in this thesis were devoted to a better understanding of the regulation of APA. Integrative analysis of a large number of data sets allowed us to establish a comprehensive annotation of poly(A) sites in the human genome. Tools developed for the projects described here could then exploit this resource to quantify and model the changes of poly(A) site usage in different contexts. In particular, the application of PAQR to quantify 3' end processing from RNA-seq data and of KAPAC to relate the abundance of individual sequence motifs to changes in poly(A) site usage led to intriguing insights into the regulation of APA in cancer. For glioblastoma, a CU-dinucleotide repeat motif was most significantly associated with the observed 3' UTR shortening, an effect that is likely to be explained by the binding of PTBP1, a factor previously known for its role in splicing regulation. Together with HNRNPC, another splicing factor that was implicated in the regulation of poly(A) site choice through analyses presented here, these results suggest an extensive coupling between splicing and 3' end processing. In particular, it appears that many regulators of both mRNA maturation steps exist and remain to be uncovered. Previous results from glioma cell lines indicated that PTBP1 levels directly affect proliferation and migration. Considering its role in splicing and 3' end processing, PTPB1 may emerge as an important regulator of gene expression with direct implications for tumor progression in glioblastoma. Potentially, PTBP1 can serve as therapeutic target or diagnostic marker in brain tumor. In summary, the work of this thesis illustrates how the deployment of computational tools can condense the information contained in large-scale data sets into biologically relevant results, shedding light on novel aspects of mRNA 3' end processing in physiological and pathological conditions. The uncovered regulators may be amenable to targeting by small molecules, thereby restoring the RNA processing patterns specific to the healthy states

Functional characterization of BRD4 in transcription elongation and termination

The eukaryotic transcription machinery consisting of RNA polymerase II (Pol II) and a small number of minimally required initiation, elongation and termination factors is well-characterized structurally and biochemically, while functional characterization traditionally has been hampered by the limitations of the available perturbation strategies and readouts. In addition, knowledge of accessory transcription regulators is likely incomplete. Proteins of the conserved bromodomain and extra-terminal domain (BET) family have been demonstrated to function in maintaining normal transcription genome-wide by promoting elongation. However, the underlying molecular mechanism and also the specific contribution of each of the protein family members remained elusive. Moreover, it was unclear if BET proteins serve additional direct or indirect functions in transcription or transcription-coupled processes. To address these aspects, this study combines targeted protein degradation, a new strategy to near-completely deplete a protein of interest within minutes to hours, with complementary transcriptome-, genome- and proteome-wide readouts of high temporal or spatial resolution. It provides evidence that specifically BRD4 is involved in transcription control, although BRD2 and BRD3 might have partially overlapping functions. Within 120 minutes, BRD4-selective degradation results in a global reduction of elongating Pol II from the gene body of most protein-coding and long non-coding RNA genes, as can be detected by native elongating transcript sequencing with spike-in normalization (SI-NET-seq). Concomitantly, Pol II accumulates in the promoter-proximal region, suggesting a defect in promoter-proximal pause release. Profiling elongation factors upon BRD4 depletion reveals a decrease in occupancy, suggesting an assembly defect of the active elongation complex. In particular, PAF1 binding is decreased throughout the transcribed region. Unexpectedly, BRD4-selective degradation also induces a severe transcription termination defect at a subset of genes. Particularly, Pol II continues transcribing on average three kilobases beyond its usual termination zone, which is accompanied by inefficient cleavage of the nascent transcript at the pA site. Chromatin immunoprecipitation with sequencing (ChIP-seq) reveals a significant reduction of RNA 3’ end processing factors of the CPSF and CstF modules near the 3’ gene end as well as in the promoter-proximal and the gene body region. The failure to recruit RNA 3’ end processing as well as PAF subunits, SPT5 and SPT6, but not CDK9, could be independently confirmed by quantitative mass spectrometry. Co-immunoprecipitation experiments indicate that the recruitment of 3’ processing factors either directly depends on BRD4 or could be mediated by elongation factors. Also, BRD4-selective degradation locally increases the binding of Pol II and SPT5 at transcribed enhancers, whereas enhancer accessibility appears to be not BRD4-dependent. Altogether, the data establish a role of BRD4 in coordinating the recruitment of elongation and RNA 3’ end processing factors during an early step of transcription.Die eukaryotische Transkriptionsmaschinerie — insbesondere RNA-Polymerase II (Pol II) und eine überschaubare Gruppe essentieller Initiation-, Elongations- und Terminationsfaktoren — ist strukturell als auch biochemisch gut charakterisiert, wohingegen die funktionelle Charakterisierung durch das Fehlen geeigneter Pertubationsmethoden und sensitiver experimenteller Analyseverfahren lange erschwert wurde. Auch sind viele nicht-essentielle Transkriptionsregulatoren wahrscheinlich noch unbekannt. Es konnte gezeigt werden, dass die durch zwei Bromodomänen und eine sog. extra-terminale Domäne gekennzeichneten Proteine der konservierten BET-Proteinfamilie nötig sind, die normale Transkriptionsaktivität der Zelle — vermutlich durch Regulation der Elongation — aufrecht zu erhalten. Unklar sind jedoch der molekulare Mechanismus als auch die Rolle der einzelnen Proteine der Proteinfamilie. Darüber hinaus ist denkbar, dass BET-Proteine direkt oder indirekt auch an weiteren transkriptionellen oder co-transkriptionellen Prozessen beteiligt sind. Die genannten Aspekte adressiert die vorliegende Studie einerseits durch die Verwendung eines relativ neuen Verfahrens zur gezielten und schnellen Degradierung eines Proteins — in diesem Fall BRD4 — und andererseits mit komplementären transkriptom-, genom- und proteomweiten Methoden, die eine hohe zeitliche oder räumliche Auflösung bieten. Auf diese Weise kann gezeigt werden, dass die Transkription in hohem Maße von BRD4 abhängt, obgleich nicht auszuschließen ist, dass BRD2 oder BRD3 eine ähnliche Funktion haben. So belegt SI-NET-seq, eine spike-in-gestützten Methode zur quantitativen Analyse naszenter RNA unter nativen Bedingungen, dass die gezielte Degradierung von BRD4 innerhalb von 120 Minuten zur globalen Verringerung transkribierender Pol II am Genkörper der meisten proteinkodierenden und langen nicht-kodierenden RNA-Gene führt. Gleichzeitig akkumuliert transkribierende Pol II in der promotor-proximalen Region, was auf einen Defekt am Übergang von der promotor-proximalen Pause zur produktiven Elongation hindeutet. Einen Hinweis auf eine Funktion von BRD4 bei der Assemblierung des Elongationskomplexes liefert die Beobachtung, dass die Chromatinbindung von Elongationsfaktoren nach BRD4-Degradierung verringert ist. Insbesondere PAF1 zeigt eine Abnahme in allen Genbereichen. Unerwarteterweise führt die gezielte Degradierung von BRD4 bei einer großen Gruppe von Genen auch zu einem Terminationsdefekt — im Durchschnitt erfolgt die Termination drei Kilobasen später als unter Kontrollbedingungen — und vermindert die Effizient, mit der die naszente RNA an der Polyadenylierungsstelle enzymatisch geschnitten wird. Tatsächlich binden CPSF- und CstF-Faktoren der 3'-RNA-Prozessierungsmaschinerie nach BRD4-Degradierung signifikant weniger am 3'-Ende der Gene, aber auch im promotor-proximalen Bereich und am Genkörper, wie mittels Chromatinimmunpräzipitation (ChIP) gezeigt werden kann. Eine signifikante Abnahme von 3'-Prozessierungsfaktoren sowie von PAF-Untereinheiten, SPT5 und SPT6, nicht aber von CDK9, ist auch durch quantitative Massenspektrometrie nachweisbar. Zudem legen Co-Immunpräzipitationsexperimente nahe, dass 3‘-RNA-Prozessierungsfaktoren entweder direkt durch BRD4 oder indirekt über Elongationsfaktoren rekrutiert werden können. Außerdem korreliert die BRD4-spezifische Degradierung mit der lokalen Zunahme von Pol II und SPT5 an transkribierten Enhancer-Regionen; die Zugänglichkeit des Chromatins gegenüber Tn5-Transposase erscheint hingegen nicht BRD4-abhänging zu sein. Zusammenfassend legen unsere Daten nahe, dass BRD4 zu einem relativ frühen Zeitpunkt des Transkriptionszyklus' die Rekrutierung von Elongations-, aber auch von RNA 3'-Prozessierungsfaktoren koordiniert