You'd Prefer An Argonaute

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.

Allosteric regulation of Argonaute proteins by miRNAs

Sergej Djuranovic, Michelle Kim Zinchenko, Junho K Hur, Ali Nahvi, Julie L Brunelle, Elizabeth J Rogers & Rachel Green

Nature Structural & Molecular Biology 17, 144 – 150, February 2010.
doi:10.1038/nsmb.1736

This week’s summary and analysis by David Garcia:

It’s a good thing that one of the proteins central to RNAi, Argonaute, has a beefy, determined sounding name, because it’s everywhere these days. (Fortunately the wussy sounding “P-element induced wimpy testes,” or “PIWI” proteins are only a subclass of Argonautes, and not the other way around.) Argonautes owe their celebrity status to, of course, their relationship with widespread small RNAs. Numerous groups have studied Argonaute structure and function in vitro and in vivo. This week’s paper from Rachel Green’s group I think mostly convincingly shows Drosophila Ago1 operating allosterically, through a pleasing mix of structural bioinformatics, biochemistry, and cell culture experiments.

Focusing on the MID domain of eukaryotic Argonautes (Agos), the paper begins with bioinformatic analyses demonstrating sequence and structural similarities to the ligand binding domains of bacterial proteins that exhibit allosteric behavior, namely the coupling of metabolite binding in one site to active function in another distant site. Using the CLANS program they showed that MID domains themselves clustered into groups of sequence similarity that reflect their function: inhibition of translation (via miRNAs) or mRNA cleavage (via siRNAs). Agos that they classified as involved in translational repression, including DmAgo1, HsAgo1-4, and CeAlg1-2, formed a tight cluster separate from DmAgo2 and CeRde1, both which are involved in siRNA directed repression. They saw looser clustering for other domains in Ago, like PAZ or PIWI. Thus they argue that MID domain function, reflected in sequence, largely distinguishes various Ago family members. These conclusions might be tempered by the fact that some Agos, including HsAgo2 and DmAgo1, have dual-functionality: they can mediate mRNA degradation/translational repression and have slicer activity. (A growing body of evidence is showing that mRNA degradation is a substantial part of miRNA directed repression, although this doesn’t preclude their final model.)

The authors next searched for biochemical signatures that distinguish different Agos. Running tagged, purified MID domains over m7-GTP-Sepharose resins, they observed the miRNA Agos like DmAgo1 and CeAlg-1 bound more tightly than the siRNA Agos DmAgo2 and CeRde-1. Free nucleotides could compete with binding for DmAgo2 MID, suggesting a single site in this domain that binds the 5’ nucleotide of the sRNA was responsible for binding to the resin. In contrast, for the MID of DmAgo1, free nucleotides actually stimulated binding to the cap-like structure, suggesting a second allosteric site.

They next tested full-length Agos (DmAgo2 was slightly truncated) in the presence of miRNA, and saw strongly increased affinity for the cap-like structure for DmAgo1, but not for DmAgo2, indicating some crosstalk between two distinct sites that bind the sRNA and cap-like structure. The binding affinity of DmAgo1 for the cap-like structure increased with increasing [miRNA].

Filter binding assays were used to do the inverse experiment—whether binding of a miRNA can be stimulated by addition of free nucleotides/analogs with varying resemblance to the cap-like structure. They only saw stimulated miRNA binding with tri-phosphorylated nucleotides, which most closely resembled the cap. No such effect was seen for DmAgo2. Consistent with this result, addition of miRNA duplex stimulated binding of a labeled, capped mRNA ten-fold for DmAgo1, but no-fold for DmAgo2. Free nucleotides that resembled caps could compete away the labeled mRNA.

With these pleasant in vitro results in hand, demonstrating allostery for DmAgo1 but not DmAgo2, they moved in vivo into S2 cells. But here, while supporting their hypothesis, I find the results less satisfying due to their choice of reporter system. They employed a tethered Ago reporter system (reference) that examines Ago mediated inhibition of a luciferase reporter without need for a miRNA. The utility of the system comes from the ability to directly assess the effect of mutations in the tagged and tethered Ago on the reporter, without competition from endogenous Ago. But in my opinion the compromise is too great, ignoring the effect of a targeting miRNA on Ago structure and activity.

Anyways, they saw that DmAgo1 repressed the reporter strongly while DmAgo2 did not. Then they tested mutations in conserved residues in DmAgo1 predicted to be responsible for interacting with the 5’ nucleotide of the sRNA. These mutations actually caused substantial de-repression, which as they note, was very unexpected because the Ago protein is tethered to the mRNA, so it shouldn’t matter whether or not it can bind a 5’ nucleotide. They surmise that Ago needs to bind a miRNA for full activity, even if it’s tethered. (Another possibility is that even binding free nucleotides would be sufficient, and this was still perturbed in the mutants.)

Next they pursued an exposed region in the MID domain that could bind a cap. Mutation of a specific residue caused reduced binding to m7-GTP-Sepharose resin, and complete de-repression in the reporter assay. They declared this site the second allosteric site, which binds mRNA caps. In cell lysates, they observed that only the Ago variants that repressed in the reporter assays could effectively bind miRNA, cap structures, and GW182, a protein that has been implicated in miRNA directed repression.

So overall I found the bioinformatics and in vitro data very convincing, but for the in vivo data, despite the fact that it supports the allosteric model, I’m less enthusiastic because of the experimental system.

A burning question arising from their allosteric model is how the cap binds Ago. I wouldn’t naturally assume that the cap would be free and close to Agos moving along 3’ UTRs. Messenger RNAs are most efficiently translated when they’re circularized by protein-protein-RNA interactions between the PABPs and proteins that bind the cap. Could Ago or some other co-factor then destabilize these interactions to compete for the cap? Would this change be a prerequisite for Ago mediated repression? How reversible would the exchange be? What about mRNAs with multiple target sites–each message has only a single cap but may host multiple Agos? As the authors imply in the discussion section, Ago binding the cap may only be favorable when RISC is bound to an authentic target (potentially avoiding indiscriminate activation of Ago by the caps of other mRNAs floating nearby that aren’t necessarily themselves actively repressed), so there may be a kinetic argument to be made. Finally, has the allostery of DmAgo1, and potentially other Agos, co-evolved with its preference for miRNAs that usually target the 3’ UTR, a region perhaps physically closer to the cap of a circularized mRNA? Hopefully Rachel Green’s group and others are asking these questions. We crave answers.