Functional coupling of RNAP II transcription to spliceosome assembly

Abstract

The pathway of gene expression in higher eukaryotes involves a highly complex network of physical and functional interactions among the different machines involved in each step of the pathway. Here we established an efficient in vitro system to determine how RNA polymerase II (RNAP II) transcription is functionally coupled to pre-mRNA splicing. Strikingly, our data show that nascent premessenger RNA (pre-mRNA) synthesized by RNAP II is immediately and quantitatively directed into the spliceosome assembly pathway. In contrast, nascent pre-mRNA synthesized by T7 RNA polymerase is quantitatively assembled into the nonspecific H complex, which consists of heterogeneous nuclear ribonucleoprotein (hnRNP) proteins and is inhibitory for spliceosome assembly. Consequently, RNAP II transcription results in a dramatic increase in both the kinetics of splicing and overall yield of spliced mRNA relative to that observed for T7 transcription. We conclude that RNAP II mediates the functional coupling of transcription to splicing by directing the nascent pre-mRNA into spliceosome assembly, thereby bypassing interaction of the pre-mRNA with the inhibitory hnRNP proteins.

A wave of nascent transcription on activated human genes

“…Here, splicing and degradation of intronic RNA occur co-transcriptionally, with degradation of intronic RNA beginning even before the polymerase reaches the next exon, and well before it terminates. These results contrast with a genome-wide analysis that indicates that premRNA splicing in yeast is predominantly posttranscriptional (26); but the results are consistent with observations pointing to a functional coupling between transcription and splicing (27,28).…”

Concurrent splicing and transcription are not sufficient to enhance splicing efficiency

“…Functional coupling has also been inferred in experiments of Das et al (2006). These authors reported an apparent improvement in the kinetic parameters of splicing, of RNAP II-synthesized transcripts, and rapid formation of a splicing-and RNAP II-specific high MW complex.…”

“…In the past few years successful attempts were made to develop systems that coupled transcription and capping (Moteki and Price 2002), as well as transcription and polyadenylation (Adamson et al 2005;Rigo et al 2005). In vitro systems that combine transcription and splicing have also been described (Ghosh and GarciaBlanco 2000;Ibrahim et al 2005;Das et al 2006). However, while both transcription and splicing were shown to take place in these studies, it is unclear whether there was a functional coupling between the two processes, that is, whether the properties of the splicing reaction were affected in a significant way by transcription.…”

Hypoxia-inducible Expression of a Natural cis-Antisense Transcript Inhibits Endothelial Nitric-oxide Synthase

“…RNA was extracted from the supernatant (cytoplasmic fraction) and the nuclei pellet using guanidinium-based extraction protocols. Validation of the efficiency of nuclear/cytoplasmic partitioning was performed by quantifying levels of unspliced, pre-mRNAs (see below for primer sequences), which are known to be highly enriched in the nucleus due to their short half-life (20).…”