You'd Prefer An Argonaute

Dicer-Independent Primal RNAs Trigger RNAi and Heterochromatin Formation

Mario Halic and Danesh Moazed

Cell 140 (4): 504-516, 19 February 2010.
doi: 10.1016/j.cell.2010.01.019

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 García:

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.

Noncoding RNA Gas5 Is a Growth Arrest– and Starvation-Associated Repressor of the Glucocorticoid Receptor

Tomoshige Kino, Darrell E. Hurt, Takamasa Ichijo, Nancy Nader, and George P. Chrousos

Science Signaling,
Vol. 3, Issue 107, 2 February 2010.
doi: 10.1126/scisignal.2000568

Journal club is seeing double this week, via an impartial presenter.

#1:

Genetic dissection of the miR-17~92 cluster of microRNAs in Myc-induced B-cell lymphomas

Ping Mu, Yoon-Chi Han, Doron Betel, Evelyn Yao, Massimo Squatrito, Paul Ogrodowski, Elisa de Stanchina, Aleco D’Andrea, Chris Sander, and Andrea Ventura

Genes & Development 23: 2806–2811, 15 December 2009.

#2:

miR-19 is a key oncogenic component of mir-17-92

Virginie Olive, Margaux J. Bennett, James C. Walker, Cong Ma, Iris Jiang, Carlos Cordon-Cardo, Qi-Jing Li, Scott W. Lowe, Gregory J. Hannon, and Lin He

Genes & Development 23: 2839–2849, 15 December 2009.

MicroRNA Function Is Globally Suppressed in Mouse Oocytes and Early Embryos

Nayoung Suh, Lauren Baehner, Felix Moltzahn, Collin Melton, Archana Shenoy, Jing Chen, Robert Blelloch

Current Biology, Online Ahead of Issue, 28 January 2010.
doi: 10.1016/j.cub.2009.12.044

Opposing microRNA families regulate self-renewal in mouse embryonic stem cells

Collin Melton, Robert L. Judson & Robert Blelloch

Nature advance online publication, 6 January 2010.
doi:10.1038/nature08725

Comprehensive discovery of endogenous Argonaute binding sites in Caenorhabditis elegans

Dimitrios G Zisoulis, Michael T Lovci, Melissa L Wilbert, Kasey R Hutt, Tiffany Y Liang, Amy E Pasquinelli & Gene W Yeo

Nature Structural & Molecular Biology, Advance online publication, 10 January 2010.
doi:10.1038/nsmb.1745

Transcriptional Control of Gene Expression by MicroRNAs

Basel Khraiwesh, M. Asif Arif, Gotelinde I. Seumel, Stephan Ossowski, Detlef Weigel, Ralf Reski, and Wolfgang Frank

Cell 140: 111–122, 8 January 2010.
DOI: 10.1016/j.cell.2009.12.023

HnRNP proteins controlled by c-Myc deregulate pyruvate kinase mRNA splicing in cancer

Charles J. David, Mo Chen, Marcela Assanah, Peter Canoll  &  James L. Manley

Nature AOP, 13 December 2009.
doi:10.1038/nature08697

Targeted 3′ Processing of Antisense Transcripts Triggers Arabidopsis FLC Chromatin Silencing

Fuquan Liu, Sebastian Marquardt, Clare Lister, Szymon Swiezewski, Caroline Dean

Science Express: 3 December 2009.
doi: 10.1126/science.1180278

This week’s punctilious summary and analysis by Igor Ulitsky:

The group of Caroline Dean in Norwich, as well as several other groups, has been extensively studying the regulation of the FLC gene in Arabidopsis. FLC is a key transcription factor in the flowering process, which is very tightly controlled in plants. Their studies have uncovered an extensive regulatory network that converges at the FLC locus, the mechanisms of which include transcriptional regulation, chromatin modification, small RNAs, and now also RNA processing. Two papers on FLC from Dean’s group were published last week – one in Science (Liu et al., the one I’ll focus on) and another in Nature (Swiezewski et al. Nature Vol 462). The Science paper focuses on the so-called “autonomous pathway” of FLC regulation, which promotes flowering by repressing FLC. Previous genetic analyses from the same group have uncovered a number of genes involved in this process, including two RNA-binding proteins, FCA and FPA, the cleavage and polyadenylation specificity factor FY, and an H3K4me2 demethylase, FLD. They have also previously shown (mostly in Liu et al., Molecular Cell 2007) that FCA is physically associated with the chromatin in an intron of FLC, between exons 6 and 7, but did not find clear evidence that FCA binding leads to processing of the FLC gene itself. Instead, it seemed that FCA played a role in regulating the transcription from the FLC locus, through chromatin modification by FLD. In addition, they characterized two anti-sense transcripts in the FLC locus, one short, ending around the physical location of FCA on the chromatin, and one long, ending at the FLC promoter. Interestingly, fca mutants had a higher long form/short form ratio than WT plants.

In this paper, Liu et al. first conducted a genetic screen for suppressors of FCA over-expression (which strongly repressed FLC). They used a fusion of FLC to LUC, and identified several genes that could activate FLC in presence of a strongly activated FCA. These included the known players FY and FLD, but also two additional genes, subunits of the CstF 3’ processing complex, which is highly conserved and essential in many species, including Arabidopsis. Through epistasis analysis, they could show that the CstF subunits indeed function in the FCA pathway. In addition, they show that these mutants have increased transcription of FLC, as evidenced by nascent RNA levels, Pol II binding, and H3K4me3 signatures. Their previous findings now pointed to a role of these subunits in regulating the long form/short form ratio of the antisense transcript. The paper doesn’t convincingly show that the sense FLC transcript is not affected, but it seems that the authors are convinced somehow that only the antisense is affected. Indeed, they find that the CstF mutants fail to process the 3’ of the antisense transcript, which leads in general to higher transcription of the anti-sense, which is similar to the elevation observed in fca mutants, and coincides with an increase in the FLC sense transcript. Overall, in different mutants in the FCA pathway, there is an increase in the abundance of the long form of the anti-sense transcript over the short form. The authors’ model for what happens in the WT strain, in which FCA represses FLC, is as follows: FCA/FY/CstF, through 3’ processing of the antisense transcript, causes shift in long-form/short-form ratio, which leads somehow to recruitment of FLD, which removes the H3K4me2 marks from the body of the FLC gene, leading eventually to down-regulation of both the sense and the anti-sense transcripts. They speculate on what may be the missing link between FCA/FY/CstF and FLD, but there is no clear evidence supporting it.

While the paper focuses on a single gene in Arabidopsis, there are several lines of evidence that this kind of regulation through 3’ processing of an antisense transcript occurs in other regulatory programs. In general, although the paper does not mention it, this kind of mechanism could explain why some regulatory proteins have conserved biding sites in the introns of their targets, as at least some of them may play roles in RNA processing of both the sense and the anti-sense transcipts.

To summarize, this paper, as well as another report on the FLC locus, show how amazingly complicated a relatively straight forward regulatory program (shutting down a target gene) can be. Another global implication that I found in this paper is how useful yet misleading genetic interactions can be. The authors found a genetic link between FCA and FLD, the chromatin modifier at the end of the pathway, in one of their previous studies. However, they could not find a direct physical link between them. As it appears now, FLD appears several pathway steps downstream of FCA, with complex machinery for 3’ processing of the antisense transcript separating them.

The paper was very interesting to read, and it gave a decent introduction to FLC pathway regulation. However, it wasn’t an easy feat to understand it thoroughly. The details of the unfolding of the pathway were not really clear in the first scan, and it was very difficult to understand which results were actually novel, and which have been reported previously. Part of the problem could be the extreme length limitations of Science publications, and lack of simple “textbook” figures in the paper.

RNA-Guided RNA Cleavage by a CRISPR RNA-Cas Protein Complex

Caryn R. Hale, Peng Zhao, Sara Olson, Michael O. Duff, Brenton R. Graveley, Lance Wells, Rebecca M. Terns and Michael P. Terns

Cell 139 (5): 945-56, 25 November 2009.
doi:10.1016/j.cell.2009.07.040

This week’s ace summary and analysis by Robin Friedman:

The CRISPR (clustered regularly interspaced short palindromic repeats) system is a set of DNA sequences and associated genes involved in prokaryotic immune defense. Since it was discovered that CRISPR loci generate small (usually 25-60 nt) RNAs that often match phage or plasmid sequence, it has been tempting to make an obvious analogy with eukaryotic RNA interference, which can also be used to protect against viruses. However, what little was known about CRISPR mechanism pointed to a DNA-dependent mechanism of invader recognition and defense. One complicating factor is that there are at least nine distinct subtypes of the CRISPR system based on different sets of Cas (CRISPR-associated) genes. In this paper Hale et al. examine a subtype, Cmr, that had not previously been studied.

The authors first showed that in P. furiosis, there are two main species of CRISPR RNA product (termed psiRNAs), 39 and 45 nucleotides long. They purified protein complexes containing these psiRNAs and subjected them to mass spectrometry, finding seven members of the CRISPR-associated Cmr family. Sequencing of the psiRNAs revealed that each species had an 8-nucleotide “psi-tag”, consisting of the 3’ end of the constant repeat sequence, followed by unique guide sequence.

To test the mechanism of the psiRNA-Cmr complex, the authors used several synthetic constructs with sequence similarity to P. furiosis psiRNAs. The complementary RNA sequence was specifically cleaved at two spots, but the sense RNA sequence, unrelated RNA sequences, and a complementary DNA sequence were not cleaved. Truncations of these synthetic complementary RNAs showed that cleavage occurs in the same location, suggesting that the Cmr complex cleaves 14 nucleotides from the 3’ end of the psiRNA. Finally, the authors reconstituted the Cmr complex in vitro with recombinant proteins and synthetic psiRNA and recapitulated the cleavage behavior of the native complex. Only one of the six included Cmr proteins were dispensable for cleavage.

This is one of the simplest Cell papers I have seen. However, its few results are thoroughly proven. There are many questions left to answer about CRISPR function both in this model system and in others, but the scope of this paper is merely to show that the CRISPR system can function through RNA cleavage. This paper finally provides evidence strengthening the appealing analogy between CRISPRs and eukaryotic RNAi, which is sure to stimulate more interest in the system.