You'd Prefer An Argonaute

Caspase-Dependent Conversion of Dicer Ribonuclease into a Death-Promoting Deoxyribonuclease

Akihisa Nakagawa, Yong Shi, Eriko Kage-Nakadai, Shohei Mitani, Ding Xue

Science Express, 11 March 2010.
doi: 10.1126/science.1182374

This week’s methodische summary and analysis–impressively her third contribution to this blog–by Anna Drinnenberg:

In this paper Nakagawa and colleagues describe a new role for Dicer (DCR-1) in the apoptotic pathway of C. elegans. Briefly, the apoptotic pathway can be triggered by many different cellular stimuli, resulting in caspase activation and subsequent fragmentation of nuclear DNA. DNA fragmentation can be separated into two steps: (i) During the first phase a caspase-activated DNA endonuclease catalyzes formation of DNA nicks and breaks (ii) In mammals Caspase 3 activates the Caspase-activated deoxyribonuclease CAD generating 3’ hydroxylated (3’ OH) DNA breaks that can be detected by the TUNEL (TdT-mediated dUTP nick end Labeling) assay. Other endonucleases including EndoG, which translocates from the mitochondria to the nucleus, function at later stages to complete degradation of genomic DNA. Mutants of one or combinations of those endonucleases result in the accumulation of TUNEL-stained nuclei because the resolution of 3’ OH DNA breaks is impaired.

C. elegans has homologues of Caspase3 (CED-3) and EndoG (CPS-6), as well as other endonucleases involved in the second stage of DNA fragmentation, however, a homologue of CAD had not been found in the C. elegans genome. This group therefore aimed to identify the endonuclease that catalyzes the initial formation of 3’ hydroxylated DNA breaks. They performed an RNAi screen in the cps-6 deletion background and selected mutants that showed a decrease in TUNEL signal. DCR-1, a protein so far only known to be involved in RNAi pathways of C. elegans and other species was one hit from the screen.

They further confirmed the TUNEL results using DCR-1 deletion alleles, indicating that DCR-1 acts upstream of CPS-6 and other endonucleases. Moreover, they showed that DCR-1 has pro-apoptotic activity by counting the number of cell corpses during C. elegans embryogenesis in wild-type and dcr-1 deletion strains. Deletion strains of other factors involved in the C. elegans RNAi pathway did not show the same phenotypes indicating that the pro-apoptotic function of DCR-1 is independent of its role in the RNAi pathway. Furthermore, they found that DCR-1 is processed by CED-3, which cleaves its first RNAseIII domain. They named the c-terminal part of truncated form of DCR-1, “tDCR-1”, which lacks most of the full-length protein domains including the Helicase, the PAZ, and even one of the RNaseIII domains.

Studies in vitro incubating tDCR-1 with dsRNA or plasmid DNA showed loss of RNase activity, but a gain of DNase activity. The DNase activity of tDCR-1 appears to be weak, resulting in just a single cut of plasmid DNA instead of complete fragmentation. This would be consistent with a role for tDCR-1 in generating 3’OH DNA nicks, whereas completion of DNA fragmentation is carried out by other endonucleases like CPS-6. It would be therefore interesting to compare the enzymatic activity of tDCR-1 to CAD in the same in vitro assay. They also did some very interesting experiments that convincingly tied the function of the different forms of DCR-1 to either being involved in the apoptotic pathway or the RNAi pathway. For example, they rescued the mutant RNAi phenotype, but not the mutant apoptosis phenotype, of dcr-1 deletion animals by expression of an allele of DCR-1 resistant to CED-3 cleavage. In a complementary experiment, expression of tDCR-1 in the dcr-1 deletion background resulted in rescue of the apoptotic pathway but not the RNAi pathway. Finally, they showed the same acidic amino acids are important for both RNase and DNase activity, demonstrating the similarity of both enzymatic activities.

Nakagawa and collegues performed a very comprehensive study characterizing the role of DCR-1 acting as a functional analog of CAD, indicating that a conserved, caspase-mediated mechanism activates the apoptotic DNA degradation process in both C. elegans and mammals. Further studies could include visualizing the relative abundance of tDCR-1 versus full-length DCR-1 in the nucleus and the cytoplasm. Moreover, studies in other organisms, including those that have a homolog of CAD, could determine if the pro-apoptotic role of DCR-1 is specific to C. elegans or is conserved in other species as well. In conclusion, this study shows the participation of the same protein in two different unrelated pathways, an economical use of the genetic repertoire.

Citation for researchblogging.org:

Nakagawa A, Shi Y, Kage-Nakadai E, Mitani S, & Xue D (2010). Caspase-Dependent Conversion of Dicer Ribonuclease into a Death-Promoting Deoxyribonuclease. Science PMID: 20223951

Mammalian cell penetration, siRNA transfection, and DNA transfection by supercharged proteins

Brian R. McNaughton, James J. Cronican, David B. Thompson and David R. Liu

PNAS 106 (15): 6111-6116, 14 April 2009.
doi: 10.1073/pnas.0807883106

This week’s shrewd summary/analysis–impressively his third contribution to this blog–by David Weinberg:

In their 2009 PNAS paper, David Liu and colleagues demonstrate that a green fluorescent protein (GFP) variant that has been engineered to have a positively charged surface can penetrate mammalian cells and also chaperone nucleic acids into those same cells. The story began in 2007 when Liu’s lab published their initial characterization of so-called “supercharged” proteins. The motivation was to determine how changing the net charge of a protein can affect its stability. To do this, they began with an extra-stable GFP and changed as many surface-exposed residues as possible to positively charged residues (i.e., lysine and arginine). This resulted in a GFP variant with a net charge of +36 (herein referred to as +GFP) that folded and fluoresced similarly to the original GFP. Amazingly, however, +GFP was highly resistant to aggregation: boiling of the protein eliminated activity, but cooling the boiled protein restored most of the activity. This stabilizing effect of supercharging was not unique to GFP, as similarly supercharged variants of GST (a dimer) and streptavidin (a tetramer) showed similar properties.

So what does all of this have to do with RNA? In 2007, Liu’s lab also noted that +GFP could use its positively charge surface as “molecular Velcro” that can reversibly bind to RNA (tRNA) or DNA (plasmid dsDNA). Of course, +GFP-bound RNA is useless (to a first approximation) in a test tube. Where the PNAS paper begins is with the hypothesis that +GFP might be able to enter cells and thereby escort its bound nucleic acid cargo into the cell as well. The authors begin by conclusively showing that +GFP penetrate a variety of mammalian cells with an efficiency that varies with its charge. To satisfy the cell biologists in the audience, they provide an initial dissection of the mechanism of +GFP uptake using a variety of (mostly chemical) perturbations. From this, they conclude that the mechanism involves energy-dependent endocytosis that is dependent on actin polymerization and sulfated (positively charged) cell surface peptidoglycans but does not require caveolin or clathrin. Now focusing back on RNA, the authors demonstrate that +GFP can bind to siRNAs in vitro – not surprising given their previously published data showing that it can bind to tRNA. Moreover, +GFP-bound Cy3-labeled siRNAs can enter HeLa cells – their FACS data suggest that virtually all cells in the population take up the siRNAs to a similar degree, yielding a quite homogenous population of “transfected” cells. Although Lipofectamine (a standard transfection reagent) can also deliver Cy3-siRNA to HeLa cells, +GFP delivers ~100-fold more siRNA based on fluorescence. More impressively, in 4 other cell lines that are virtually resistant to Lipofectamine-mediated transfection, +GFP delivers huge amounts of Cy3-siRNA without any significant cytotoxicity. Not only do these siRNAs enter the cell, but they can interact with the RNA interference machinery and mediate gene silencing. Further characterization of +GFP-siRNA complexes reveals that +GFP enhances the stability of siRNA in serum and the protein is itself relatively stable in serum. Although less impressive in its efficiency, +GFP (with an HA tag) can also be used to transfect plasmid DNA that gets expressed in the nucleus.

The authors conclude that +GFP may provide an attractive alternative to nucleic acid delivery. Because it uses a general pathway (endocytosis) for delivery, it should in theory work in all cell types. In addition, it is relatively easy to use since it only requires mixing of recombinant +GFP (which can be readily made in E. coli) and the nucleic acid of interest, and adding this to cells. But beyond this immediate application as a transfection reagent, it seems that +GFP and supercharged proteins more generally may become a useful tool for all sorts of biology (which I have no doubt the Liu lab is already exploring).

Citation for researchblogging.org:

McNaughton BR, Cronican JJ, Thompson DB, & Liu DR (2009). Mammalian cell penetration, siRNA transfection, and DNA transfection by supercharged proteins. Proceedings of the National Academy of Sciences of the United States of America, 106 (15), 6111-6 PMID: 19307578

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.