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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