POINT Technology Illuminates the Processing of Polymerase-Associated Intact Nascent Transcripts

Abstract

SUMMARYMammalian chromatin is the site of both RNA polymerase II (Pol II) transcription and coupled RNA processing. However, molecular details of such co-transcriptional mechanisms remain obscure, partly due to technical limitations in purifying authentic nascent transcripts. We present a new approach to purify and profile nascent RNA, called Polymerase Intact Nascent Transcript (POINT) technology. This three-pronged methodology maps nascent RNA 5’ends (POINT-5), establishes the kinetics of co-transcriptional splicing patterns (POINT-nano) and profiles whole transcription units (POINT-seq). In particular we show by depletion of the nuclear exonuclease Xrn2 that this activity acts selectively on cleaved 5’P-RNA at polyadenylation sites. Furthermore POINT-nano reveals that splicing occurs either immediately after splice site transcription or is delayed until Pol II transcribes downstream sequences. Finally, we connect RNA cleavage and splicing with either premature or full-length transcript termination. We anticipate that POINT technology will afford full dissection of the complexity of co-transcriptional RNA processing.HIGHLIGHTSPOINT methodology dissects intact nascent RNA processingSpecificity of Xrn2 exonuclease in co-transcriptional RNA degradationSplicing suppresses Xrn2-dependent premature terminationDifferent kinetic classes of co-transcriptional splicing in human genes

Type I PRMTs and PRMT5 Independently Regulate Both snRNP Arginine Methylation and Post-Transcriptional Splicing

“…Notably, our SKaTER-seq analysis is limited to the number of genes the model was able to solve: roughly 50% of expressed genes that are typically longer genes with fewer isoforms. Nonetheless, our data is consistent with more recent studies highlighting that a considerable fraction of splicing remains to be completed after RNA pol II has transcribed at least a thousand nucleotides past the 3’ SS as well as after transcription has finished (Drexler et al 2020; Jia et al 2020; Sousa-Luis et al 2020).…”

FLEP-seq: simultaneous detection of RNA polymerase II position, splicing status, polyadenylation site and poly(A) tail length at genome-wide scale by single-molecule nascent RNA sequencing

“…The cotranscriptional processes, including mRNA capping, splicing, cleavage and polyadenylation, turn nascent RNAs into mature mRNAs that are eventually exported to the cytoplasm. Pre-mRNA splicing occurs mainly cotranscriptionally and is tightly coupled with Pol II elongation [1][2][3][4][5][6][7][8][9] . Many high-throughput sequencing methods have been developed to characterize nascent RNAs at the genome-wide scale quantitatively, and have revealed novel insights into transcriptional regulation by tracking Pol II position at nucleotide resolution as well as the status of splicing 10 .…”

Screening thousands of transcribed coding and non-coding regions reveals sequence determinants of RNA polymerase II elongation potential

“…On the other hand, the process of splicing has long been suggested to stimulate transcription, although the mechanisms underlying this process remain unclear. Various reports implicate splicing in stimulation of transcription initiation, pause-release, or through the suppression of premature termination [9][10][11][12][13][14] . Notably, a splicing-independent role for the 5' splice site (5'SS) has been suggested 6,8,10,[15][16][17] , and the U1 snRNP can bind to both a 5'SS and RNAPII without the full spliceosome present 18 .…”

“…Searches for sequence motifs that could convey these signals using MEME 36 and Homer 37 identified the same top hit enriched among the most abundant TSS-proximal mRNA inserts: a 5' splice site (5'SS) motif. The 5'SS and splicing generally have been suggested to stimulate RNA production [8][9][10][11][12][13][14][15][16][17] , and the mechanisms underlying this activity are currently a subject of great interest 18 . Thus, we used INSERT-seq to systematically assess the effect of intronic sequences on RNA production.…”

“…Notably, a splicing-independent role for the 5' splice site (5'SS) has been suggested 6,8,10,[15][16][17] , and the U1 snRNP can bind to both a 5'SS and RNAPII without the full spliceosome present 18 . However, prior experimental approaches to dissect the function(s) of 5' SSs have suffered several limitations, including reliance on a small number of introns under study [10][11][12]17 or the use of global splicing inhibitors 6,8,13,15,16 , which cause cellular toxicity and lead to indirect or off-target effects. Moreover, most analyses have focused narrowly on whether the presence of a 5'SS suppresses premature termination at cryptic PASs, in a process called 'telescripting' 15,16 , leaving open the question of a more general role in stimulating transcription.…”