Spliceosome assembly is coupled to RNA polymerase II dynamics at the 3′ end of human genes

Abstract

In the nucleus of higher eukaryotes, maturation of mRNA precursors involves an orderly sequence of transcription-coupled interdependent steps. Transcription is well known to influence splicing, but how splicing may affect transcription remains unclear. Here we show that a splicing mutation that prevents recruitment of spliceosomal snRNPs to nascent transcripts causes co-transcriptional retention of unprocessed RNAs that remain associated with polymerases stalled predominantly at the 3' end of the gene. In contrast, treatment with spliceostatin A, which allows early spliceosome formation but destabilizes subsequent assembly of the catalytic complex, abolishes 3' end pausing of polymerases and induces leakage of unspliced transcripts to the nucleoplasm. Taken together, the data suggest that recruitment of splicing factors and correct assembly of the spliceosome are coupled to transcription termination, and this might ensure a proofreading mechanism that slows down release of unprocessed transcripts from the transcription site.

Post-transcriptional spliceosomes are retained in nuclear speckles until splicing completion

“…2). Several previous reports suggested that nascent transcripts that are unable to be processed are retained at sites of transcription 39,43,44 . However, this retention was only observed when U1 and/or U2 snRNPs were prevented from associating with the newly transcribed pre-mRNA 39,43 , which is not the case when we block splicing either with SSA or by knockdown of CDC5L 39 .…”

“…Several previous reports suggested that nascent transcripts that are unable to be processed are retained at sites of transcription 39,43,44 . However, this retention was only observed when U1 and/or U2 snRNPs were prevented from associating with the newly transcribed pre-mRNA 39,43 , which is not the case when we block splicing either with SSA or by knockdown of CDC5L 39 . That there are at most only low levels of contaminating post-transcriptional spliceosomes in our chromatin fraction is supported by the fact that under conditions where we expect little or no co-transcriptional splicing, but rather post-transcriptional splicing (that is, after inhibiting transcription with DRB and splicing with SSA) there are only low levels (~15% of the amount found in control cells) of spliceosomes containing P-SF3b155 in the chromatin fraction (Fig.…”

The timing of pre-mRNA splicing visualized in real-time

“…To determine the fluorescence intensity emitted by a single pre-mRNA molecule, we treated cells with spliceostatin A (SSA), a potent splicing inhibitor that causes release of unspliced pre-mRNAs to the nucleoplasm. 33 After release from the transcription site, premRNAs diffused throughout the nucleus making it possible to resolve individual transcripts. Having determined the fluorescence intensity of a single premRNA, we then searched for cells that synthesized one reporter transcript at the time, i.e., cells with fluorescence fluctuations that started at background level, increased to a value in the range corresponding to a single pre-mRNA, and then returned again to background.…”

Establishment of a Monitoring System to Detect Inhibition of mRNA Processing

“…The effect of the SSA used in this study was comparable to previous studies. 8,11,12) In contrast, the addition of TG003 did not affect renilla luciferase activity at all (Fig. 1B).…”