RNA Journal Club 4/1/10
Akihisa Nakagawa, Yong Shi, Eriko Kage-Nakadai, Shohei Mitani, Ding Xue
Science Express, 11 March 2010.
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