Real-time imaging of cotranscriptional splicing reveals a kinetic model that reduces noise: implications for alternative splicing regulation

Abstract

A combination of several rate-limiting steps allows for efficient control of alternative splicing.

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

“…After 45 min, a strong reduction in the P-SF3b155 and poly(A) + RNA signals in speckles, was observed in the microinjected cells (Fig. 7e,f), consistent with a 0.4-7 min half time for splicing in mammalian cells 38 . These results support the idea that add-back of CDC5L to ∆CDC5L cells allows stalled spliceosomes to complete splicing, leading to a decrease in P-SF3b155, and that the resulting post-transcriptionally spliced mRNAs are subsequently released from speckles.…”

CRE promoter sites modulate alternative splicing via p300-mediated histone acetylation

“…[6][7][8] In vivo splicing is achieved within tens of seconds suggesting most introns are spliced out while RNA is still attached to Pol ll and the DNA template. [9][10][11] Consistent with this data, splicing factors have been shown to be recruited to the site of pre-mRNA synthesis and bind the nascent pre-mRNA [12][13][14] and 80% of active spliceosomes are attached to chromatin. 15 This coupling of premRNA splicing and Pol II transcription brings another layer of regulation.…”

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

“…We found that transcription and excision of short introns (1.3-1.4 kb) occurs in 20-30 s, which implies a splicing rate much faster than previously reported for the adenovirus-derived short intron based on FRAP experiments. 30 We believe the reason for this discrepancy derives from the higher resolution of our singlemolecule analysis: the rapid fluorescence fluctuations that we observed for a single pre-mRNA molecule are probably hidden in the bulk measurement of fluorescence recovery from a multitude of molecules at different stages of the splicing cycle. We then addressed the longstanding question of whether some introns are spliced faster than others.…”

“…Bertrand and colleagues combined such an approach with FRAP to measure co-transcriptional splicing kinetics. 30 They analyzed an ensemble population of pre-mRNAs synthesized from a gene cluster comprising ~20 copies of a reporter gene that contains MS2 binding sites in an artificial short intron derived from the adenovirus genome. Upon bleaching the MS2-GFP fluorescence associated with introns at the transcription site, they measured a half-life of 105 s for fluorescence recovery and estimated a mean splicing time of 162 s. This value was in good agreement with the splicing rates previously deduced from electron microscopy 6,7 and biochemical pulse-chase experiments.…”