Nova Fong

Pre-mRNA splicing is a determinant of histone H3K36 methylation

A chromatin code appears to mark introns and exons with distinct patterns of nucleosome enrichment and histone methylation. We investigated whether a causal relationship exists between splicing and chromatin modification by asking whether splice-site mutations affect the methylation of histone H3K36. Deletions of the 3′ splice site in intron 2 or in both introns 1 and 2 of an integrated β-globin reporter gene caused a shift in relative distribution of H3K36 trimethylation away from 5′ ends and toward 3′ ends. The effects of splice-site mutations correlated with enhanced retention of a U5 snRNP subunit on transcription complexes downstream of the gene. In contrast, a poly(A) site mutation did not affect H3K36 methylation. Similarly, global inhibition of splicing by spliceostatin A caused a rapid repositioning of H3K36me3 away from 5′ ends in favor of 3′ ends. These results suggest that the cotranscriptional splicing apparatus influences establishment of normal patterns of histone modification.

RNA editing and alternative splicing: the importance of co‐transcriptional coordination

The carboxy‐terminal domain (CTD) of the large subunit of RNA polymerase II (pol II) is essential for several co‐transcriptional pre‐messenger RNA processing events, including capping, 3′‐end processing and splicing. We investigated the role of the CTD of RNA pol II in the coordination of A to I editing and splicing of the ADAR2 (ADAR: adenosine deaminases that act on RNA) pre‐mRNA. The auto‐editing of Adar2 intron 4 by the ADAR2 adenosine deaminase is tightly coupled to splicing, as the modification of the dinucleotide AA to AI creates a new 3′ splice site. Unlike other introns, the CTD is not required for efficient splicing of intron 4 at either the normal 3′ splice site or the alternative site created by editing. However, the CTD is required for efficient co‐transcriptional auto‐editing of ADAR2 intron 4. Our results implicate the CTD in site‐selective RNA editing by ADAR2 and in coordination of editing with alternative splicing.

Altered nucleosome occupancy and histone H3K4 methylation in response to `transcriptional stress'

We report that under ‘transcriptional stress’ in budding yeast, when most pol II activity is acutely inhibited, rapid deposition of nucleosomes occurs within genes, particularly at 3′ positions. Whereas histone H3K4 trimethylation normally marks 5′ ends of highly transcribed genes, under ‘transcriptional stress’ induced by 6‐azauracil (6‐AU) and inactivation of pol II, TFIIE or CTD kinases Kin28 and Ctk1, this mark shifted to the 3′ end of the TEF1 gene. H3K4Me3 at 3′ positions was dynamic and could be rapidly removed when transcription recovered. Set1 and Chd1 are required for H3K4 trimethylation at 3′ positions when transcription is inhibited by 6‐AU. Furthermore, Δchd1 suppressed the growth defect of Δset1. We suggest that a ‘transcriptional stress’ signal sensed through Set1, Chd1, and possibly other factors, causes H3K4 hypermethylation of newly deposited nucleosomes at downstream positions within a gene. This response identifies a new role for H3K4 trimethylation at the 3′ end of the gene, as a chromatin mark associated with impaired pol II transcription.

A 10 residue motif at the C‐terminus of the RNA pol II CTD is required for transcription, splicing and 3′ end processing

The RNA polymerase II C‐terminal heptad repeat domain (CTD) is essential for normal transcription and co‐transcriptional processing of mRNA precursors. The mammalian CTD comprises 52 heptads whose consensus, YSPTSPS, is conserved throughout eukaryotes, followed by a 10 amino acid C‐terminal sequence that is conserved only among vertebrates. Here we show that surprisingly, the heptad repeats are not sufficient to support efficient transcription, pre‐mRNA processing or full cell viability. In addition to the heptads, the 10 amino acid C‐terminal motif is essential for high level transcription, splicing and poly(A) site cleavage. Efficient mRNA synthesis from a transiently transfected reporter gene required the C‐terminal motif plus between 16 and 25 heptad repeats from either the N‐ or C‐terminal half of the CTD. Twenty‐seven consensus heptads plus the C‐terminal motif also supported efficient mRNA synthesis but not cell viability.

Fast ribozyme cleavage releases transcripts from RNA polymerase II and aborts co-transcriptional pre-mRNA processing

We investigated whether a continuous transcript is necessary for co-transcriptional pre-mRNA processing. Cutting an intron with the fast-cleaving hepatitis δ ribozyme, but not the slower hammerhead, inhibited splicing. Therefore, exon tethering to RNA polymerase II (Pol II) cannot rescue splicing of a transcript severed by a ribozyme that cleaves rapidly relative to the rate of splicing. Ribozyme cutting also released cap-binding complex (CBC) from the gene, suggesting that exon 1 is not tethered. Unexpectedly, cutting within exons inhibited splicing of distal introns, where exon definition is not affected, probably owing to disruption of the interactions with the CBC and the Pol II C-terminal domain that facilitate splicing. Ribozyme cutting within the mRNA also inhibited 3′ processing and transcription termination. We propose that damaging the nascent transcript aborts pre-mRNA processing and that this mechanism may help to prevent association of processing factors with Pol II that is not productively engaged in transcription.