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 because of technical limitations in purifying authentic nascent transcripts. We present a new approach to characterize 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 co-transcriptional splicing either occurs 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.
Highlights
Transcripts synthesized by eukaryotic RNA polymerase II (Pol II) are processed during transcription to gain functionality and control RNA stability
Since mNET-seq generates only short RNA sequence information, this limits analysis of cotranscriptional RNA processing, due to lack of information on the exact position where transcription starts, as well as on the continuity of the RNA molecule. To overcome these size limitations, we have developed a different procedure to isolate intact nascent transcript from the 5’cap site through to its 3’end within the Pol II active site; Polymerase Intact Nascent Transcript (POINT) technology (Figure 1A)
Following DNase digestion, intact nascent RNA ending within the Pol II active site is isolated by immunoprecipitation (IP) in NET-2 buffer with additional 3% Empigen to remove all steady state mRNA and rRNA from the Pol II IP fraction (Figure S1D)
Summary
Transcripts synthesized by eukaryotic RNA polymerase II (Pol II) are processed during transcription to gain functionality and control RNA stability. In higher eukaryotes where exons are generally much shorter than their adjacent introns, it is thought that splicing factors such as SR proteins initially bind to and define functional exons, known as the exon definition mechanism (Ule and Blencowe, 2019). These will in turn facilitate recruitment of U2 snRNP to the 3’SS and U1 snRNP to the 5’SS leading to spliceosome formation. Nascent transcript cleavage occurs in other transcript regions such as at pre-microRNA (miRNA)
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