You'd Prefer An Argonaute

RNA Journal Club 3/4/10

Posted in RNA Journal Club, RNAJC w/ review by YPAA on March 4, 2010

Regulation of Alternative Splicing by Histone Modifications

Reini F. Luco, Qun Pan, Kaoru Tominaga, Benjamin J. Blencowe, Olivia M. Pereira-Smith, Tom Misteli

Science 327: 996 – 1000, 19 February 2010.
DOI: 10.1126/science.1184208

This week’s substantial summary and analysis by Mohini Jangi:

This study is the latest in a string of papers within the last year that have sought to take a closer look at the link between chromatin modifications and splicing regulation.  It has now been analyzed and accepted in the field that splicing occurs co-transcriptionally, and that transcription can boost splicing efficiency and vice versa.  Focusing on transcription regulating splicing, there are thought to be two main mechanisms (although inter-related and not mutually exclusive) by which this occurs.  The first is that pre-mRNA processing machinery, including the spliceosome, can directly associate with the C-terminal domain of RNA Pol II as it is transcribing the gene.  This basically increases the chance that a splice site will get recognized because of the proximity of the splicing machinery to the nascent transcript.  The other is that nucleosome position can affect the speed of the elongating polymerase, which in turn will influence splice site recognition.  Interest in this second mechanism spurred this set of papers, which generally showed enrichment of specific chromatin marks, including H3K36me3, within exons compared to introns.  Here, the authors set out to address whether any specific chromatin marks were associated with alternative exon usage, specifically those events regulated by the splicing factor polypyrimidine tract binding protein (PTB).

They began by looking at a well-studied model in splicing regulation, fibroblast growth factor receptor 2 (FGFR2), which has a set of mutually exclusive exons IIIb and IIIc.  Exon IIIb is repressed by PTB in mesenchymal cell types (human mesenchymal stem cells in this study) and included in epithelial cell types (PNT2 cells).  ChIP assays on a number of histone marks showed H3K36me3 and H3K4me1 enrichment to correlate with PTB-dependent splicing of FGFR2.  When they extended this beyond FGFR2 to other PTB-dependent transcripts, this correlation also held, whereas PTB-independent transcripts did not show this enrichment.  Next they wanted to determine if these methylation marks are causal in the splicing regulation.  To this end, they overexpressed or depleted the H3K36 methyltransferases SET2/SETD2 or the H3K4 methyltransferase ASH2 and looked for repression of exon IIIb.  As expected, increased H3K36me3 and decreased H3K4me3 after modulation of methylation led to increased repression of IIIb.  To get at the molecular mechanism for this, they hypothesized that a component of the H3K4 demethylase complex that binds H3K36me3, MRG15, may also be able to drive this splicing switch.  Indeed, ChIP experiments showed MRG15 enrichment correlating with IIIb repression, and knockdown and overexpression experiments showed enhancement and repression of IIIb, respectively.  Co-IP’s also showed a small pool of PTB and MRG15 associating, and RNA-IP similarly showed both proteins associating with the nascent exon IIIb.  To bring this into a larger context, the authors performed high-throughput cDNA sequencing in hMSCs in the presence or absence of PTB, MRG15, or SETD2.  They saw that of 447 PTB-dependent and 186 MRG15-dependent splicing events, 65 were common, of which 61 changed in the same direction upon knockdown.  Furthermore, transcripts weakly regulated by PTB made up the largest fraction of co-dependent transcripts.  From their data, they suggested a model that MRG15 is serving as an adaptor between the splicing machinery, or more specifically PTB, and the chromatin, mediated by H3K36me3.

Not being a computational biologist myself, I glossed over the fact that their sequencing analysis was not very stringent nor did it go into much depth regarding the nature of the PTB and MRG15 co-dependent transcripts.  The discussion that came up during the journal club brought this to the forefront.  For example, they did not include basic controls, such as addressing what fraction of PTB-regulated transcripts are only weakly PTB-dependent, regardless of whether they are also MRG15-dependent. Another point of contention was how this might actually work in the cell – when you imagine PTB bound to chromatin at a specific locus, it is difficult to picture how this would be noticeably more efficient than PTB associated with the CTD of Pol II. The strength of the paper lay basically in the idea that methylation marks can actually drive changes in alternative splicing, and that there is a direct association between these marks and splicing factors involved in this regulation.  It further raises the questions of what other splicing factors and other histone marks might be working in this manner.  Overall, it was an interesting study that raised some good discussion, and there is most likely much more interesting work to come from it in the future.

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