Genome-wide Mapping of 5′-monophosphorylated Ends of Mammalian Nascent RNA Transcripts
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ABSTRACT:
SUBMITTER: Cortazar M
PROVIDER: S-EPMC10518774 | biostudies-literature | 2023 Sep
REPOSITORIES: biostudies-literature
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Project description:Transcription is a highly dynamic process. Consequently, we have developed native elongating transcript sequencing technology for mammalian chromatin (mNET-seq), which generates single-nucleotide resolution, nascent transcription profiles. Nascent RNA was detected in the active site of RNA polymerase II (Pol II) along with associated RNA processing intermediates. In particular, we detected 5'splice site cleavage by the spliceosome, showing that cleaved upstream exon transcripts are associated with Pol II CTD phosphorylated on the serine 5 position (S5P), which is accumulated over downstream exons. Also, depletion of termination factors substantially reduces Pol II pausing at gene ends, leading to termination defects. Notably, termination factors play an additional promoter role by restricting non-productive RNA synthesis in a Pol II CTD S2P-specific manner. Our results suggest that CTD phosphorylation patterns established for yeast transcription are significantly different in mammals. Taken together, mNET-seq provides dynamic and detailed snapshots of the complex events underlying transcription in mammals.
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Project description:The transcription cycle of RNA polymerase II (Pol II) correlates with changes to the phosphorylation state of its large subunit C-terminal domain (CTD). We recently developed Native Elongation Transcript sequencing using mammalian cells (mNET-seq), which generates single-nucleotide-resolution genome-wide profiles of nascent RNA and co-transcriptional RNA processing that are associated with different CTD phosphorylation states. Here we provide a detailed protocol for mNET-seq. First, Pol II elongation complexes are isolated with specific phospho-CTD antibodies from chromatin solubilized by micrococcal nuclease digestion. Next, RNA derived from within the Pol II complex is size fractionated and Illumina sequenced. Using mNET-seq, we have previously shown that Pol II pauses at both ends of protein-coding genes but with different CTD phosphorylation patterns, and we have also detected phosphorylation at serine 5 (Ser5-P) CTD-specific splicing intermediates and Pol II accumulation over co-transcriptionally spliced exons. With moderate biochemical and bioinformatic skills, mNET-seq can be completed in ∼6 d, not including sequencing and data analysis.
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Project description:BackgroundEukaryotic genomes undergo pervasive transcription, leading to the production of many types of stable and unstable RNAs. Transcription is not restricted to regions with annotated gene features but includes almost any genomic context. Currently, the source and function of most RNAs originating from intergenic regions in the human genome remain unclear.ResultsWe hypothesize that many intergenic RNAs can be ascribed to the presence of as-yet unannotated genes or the "fuzzy" transcription of known genes that extends beyond the annotated boundaries. To elucidate the contributions of these two sources, we assemble a dataset of more than 2.5 billion publicly available RNA-seq reads across 5 human cell lines and multiple cellular compartments to annotate transcriptional units in the human genome. About 80% of transcripts from unannotated intergenic regions can be attributed to the fuzzy transcription of existing genes; the remaining transcripts originate mainly from putative long non-coding RNA loci that are rarely spliced. We validate the transcriptional activity of these intergenic RNAs using independent measurements, including transcriptional start sites, chromatin signatures, and genomic occupancies of RNA polymerase II in various phosphorylation states. We also analyze the nuclear localization and sensitivities of intergenic transcripts to nucleases to illustrate that they tend to be rapidly degraded either on-chromatin by XRN2 or off-chromatin by the exosome.ConclusionsWe provide a curated atlas of intergenic RNAs that distinguishes between alternative processing of well-annotated genes from independent transcriptional units based on the combined analysis of chromatin signatures, nuclear RNA localization, and degradation pathways.
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Project description:Transcription termination in bacteria can occur either via Rho-dependent or independent (intrinsic) mechanisms. Intrinsic terminators are composed of a stem-loop RNA structure followed by a uridine stretch and are known to terminate in a precise manner. In contrast, Rho-dependent terminators have more loosely defined characteristics and are thought to terminate in a diffuse manner. While transcripts ending in an intrinsic terminator are protected from 3’-5’ exonuclease digestion due to the stem-loop structure of the terminator, it remains unclear what protects Rho-dependent transcripts from being degraded. In this study, we mapped the exact steady-state RNA 3’ ends of hundreds of E. coli genes terminated either by Rho-dependent or independent mechanisms. We found that transcripts generated from Rho-dependent termination have precise 3’-ends at steady state. These termini were localized immediately downstream of energetically stable stem-loop structures, which were not followed by uridine rich sequences. We provide evidence that these structures protect Rho-dependent transcripts from 3’-5’ exonucleases such as PNPase and RNase II, and present data localizing the Rho-utilization (rut) sites immediately downstream of these protective structures. This study represents the first extensive in-vivo map of exact RNA 3’-ends of Rho-dependent transcripts in E. coli.
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Project description:We have generated single-nucleotide resolution, nascent transcription profiles from HeLa cells by developing Native Elongation Transcript sequencing technology for mammalian chromatin (mNET-seq). Our extensive data sets provide a substantial resource to study mammalian nascent transcript profiles. We reveal unanticipated phosphorylation states for RNA polymerase II C-terminal domain (Pol II CTD) at both gene ends. We also observe that following 5’ splice site cleavage by the spliceosome, upstream exon transcripts are tethered to Pol II CTD phosphorylated on the serine 5 position (S5P) which is accumulated over downstream exons. We further show that depletion of termination factors substantially reduces Pol II pausing at gene ends leading to termination defects. Remarkably termination factors play an additional promoter role by restricting non-productive RNA synthesis and redistributing Pol II CTD S2P to promoters. These data demonstrate that CTD phosphorylation is more dynamic and variably distributed across mammalian transcription units than previously envisaged. To monitor nascent RNA within the mammalian Pol II complex, and its association with different CTD phosphorylation states, we employed mNET-seq methodology on HeLa cells, complemented with direct sequencing of chromatin-bound RNA (ChrRNA-seq). mNET-seq was preformed using the antibodies 8WG16, CMA602, CMA603 and CMA601, which are specific for unphosphorylated CTD, Ser2 phosphorylation, Ser5 phosphorylation and all CTD isoforms, respectively. In another experiment, to evaluate the effect of transcription termination factors in nascent RNA production by Pol II, mNET-seq and complemented with ChrRNA-seq was preformed on HeLa cells transfected with siRNA against PTBP1, CPSF73, CstF64+CstF64tau or Xrn2, and the gene profiles were compared with profiles from HeLa transfected with siRNA for Luciferase generated by the same protocol.
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