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Angela Leong Feng Ping M13608 BL6403 Assignment 1: Critical Writing

Review: Nuclear RNAi maintains heritable gene silencing in Caenorhabditis elegans
Burton et al. (2011), Proceedings of the National Academy of Sciences, v. 108, p. 19683-19688.
Background Epigenetic changes are alterations in phenotype or gene expression arising not from modifications in nucleotide sequence, but extragenic events like DNA methylation [1]. In multicellular organisms, epigenetic modifications are generally mitotically stable in somatic cells, but not meiotically stable due to genetic reprogramming in germ and embryonic cells [2]. However, certain species exhibit epigenetic inheritance: in Linaria vulgaris, differences in floral shape arising from epigenetic mutations are inherited by offspring [3]. In RNA-interference (RNAi) in the nematode Caenorhabditis elegans, effector protein RDE-4 binds dsRNA and activates ribonuclease Dicer, which cleaves dsRNA into double-stranded siRNAs [4]. The guide strand integrates into RNA-induced-silencing-complex (RISC), base-pairs with

complementary sequences in mRNA, and initiates cleavage by Argonaute (Ago) proteins [5]. RNAdependent-RNA-polymerase (RdRP) produces secondary siRNAs using primary siRNAs as templates [6]. Nuclear-RNAi requires minimally nuclear-RNAi-defective 1–4 genes (nrde-1 to nrde-4). Ago NRDE3 guides secondary siRNAs into the nucleus, where NRDE-3 engages NRDE-1 and NRDE-2 to complementary sequences in emerging pre-mRNA transcripts. NRDE-4 enables NRDE-1 to bind chromatin, preventing RNA-polymerase-II elongation and controlling deposition of histone-3-lysine-9methylation (H3K9me) marks at RNAi-targeted genomic sequences [7, 8] RNAi in C. elegans is a multigenerational epigenetic change. dsRNAs introduced in parents induce the transmission of a RNAi-inheritance signal to progeny, regardless of whether the progeny possess the RNAi-targeted genes – indicating that a dominant extragenic factor controls RNAiinheritance. However, the compound acting as the RNAi-inheritance signal, the process through which the signal is passed down and preserved across generations, and the function of siRNA-controlled H3K9me in gene silencing are unknown.

Thus nuclear-RNAi. (H3K9me marks may be absent in embryo-stage because NRDE-3 is unexpressed. is necessary to preserve heritable siRNA expression. Offspring of nrde(-) strains exposed to gfp-dsRNA exhibited gfp-silencing at the embryonic-stage but not larval. as they matured.Major Findings and Conclusions Nuclear-RNAi preserves heritable silencing in offspring. Dpy-11-silencing in nrde-3(+/-) C. Progeny of wild-type animals exposed to dpy-11RNAi. elegans and were found in F1 offspring.) In nrde-1. elegans. . or associates weakly with dpy-11-gene which is expressed at low levels at embryo-stage. and nrde-4 mutants. failed to silence dpy-11. elegans was passed down to nrde-3(+/+) progeny but not nrde-3(-/-) progeny. in contrast to known multigenerational RNAi-silencing of germ-lineexpressed genes. to levels higher than in parent animals. Pos-1-RNAi caused embryonic arrest in F1 progeny of both nrde(+) and nrde(-) C. confirming presence of siRNAs in offspring and binding of NRDE-3 to siRNAs after RNAi. nrde-3. not parents. showing that NRDE-3 needs to both bind and guide siRNAs to the nucleus to preserve RNAi. which causes dumpy (Dpy) phenotype at larval-stage. while nrde(-) F1 offspring did not. Since silencing of dpy-11 and gfp-genes in somatic cells was relieved in F2 progeny. respectively. produced F1 progeny that displayed decreasing levels of NRDE-3-associated dpy-11-siRNAs and gfpsiRNAs. dpy-11-RNAi failed to induce heritable H3K9me. but not genes in embryonic-stage. suggesting that the extent to which RNAi activates H3K9me may increase with germ-line inheritance of silencing. nrde-3(-) offspring with NRDE-3(*NLS) and NRDE-3(PAZ*) mutant proteins. indicating that NRDE-3 functions in progeny. while unnecessary for inheritance of siRNA.and adult-stages. NRDE-3-associated dpy-11-siRNAs increased over 3000 times in parental C. RNAi-silencing of somatically-expressed genes lasts only one generation. and reappeared in F1 offspring after 48h. meaning that nuclear-RNAi is needed to establish and/or preserve heritable H3K9me. H3K9me chromatin immunoprecipitation revealed that H3K9me marks at dpy-11-locus increased after dpy-11-RNAi in wild-type animals. confirming that nuclear-RNAi is needed for heritable silencing of genes acting in larval-stage. showed Dpy phenotype. which cannot guide siRNAs to the nucleus and cannot bind siRNAs respectively. Nrde mutant parents exposed to dpy-11 and gfp-dsRNA respectively. After dpy-11-RNAi.

siRNA is likely the primary heritable agent. However. The results of RNAi. The results failed to distinguish if nuclear-RNAi mechanism was responsible for establishing or preserving H3K9me in progeny. Therefore. 12]. as C. Results in F2-progeny were reported sporadically. in particular nrde-2(-). In showing that pos-1-RNAi induced F1-embryonic arrest. elegans exhibits parental imprinting [16–18] which could result in silenced traits reappearing in subsequent generations. measurements of NRDE-3-bound dpy-11-siRNA and H3K9me were incomplete for certain genotypes. rde-1(-) and NRDE-3(*PAZ) strains – to confirm that their assay was selectively identifying dpy-11-siRNAs. The authors used three controls – an independent dpy-11-siRNA assay. Larval-stage genes daf-11 [9. Effects of RNAi should have been studied in further generations. and embryonic-stage genes ref-1 [14] and cdk-5 [15] used in similar studies could be utilised for verification. it is questionable if the findings can be generalized to all somatically-expressed larval/embryonic genes. This research is novel in its findings of siRNA as the RNAi-inheritance signal for somaticallyexpressed genes and the possibility that germ-line RNAi-inheritance promotes H3K9me. Given that the authors tested primarily only dpy-11-RNAi. nuclear-RNAi is required to maintain heritable siRNA expression and heritable RNAi-silencing of somatically-expressed genes. proving traits acquired by organisms in their . Comments and Critiques The evidence is strongly supported by controls. In conclusion. as the authors neglected measuring levels of other dpy-11-siRNAs. asna-1 [13]. 20] supporting Lamarckian inheritance. the inference that elevated H3K9me in inheriting offspring is not because offspring expressed more dpy-11-siRNAs is questionable. 10]. and RNAi-silencing is inherited even when the target gene is absent.Since H3K9me marks establish after siRNAs. and why nrde-1 and nrde-4 mutant progeny possessed higher levels of NRDE-3-bound dpy-11-siRNA than wild-type progeny. lin-4 [11. to ascertain that differences could only be attributed to mutated genes. heritable genesilencing mechanism involves NRDE-3 in progeny binding and guiding siRNAs to the nucleus. rde-4(-) strains exposed to pos-1-dsRNA acted as control to confirm that increased embryonic arrest was due to pos-1-RNAi. where siRNAs control H3K9me. It joins current research [19. RNAi in wild-type strains and their progeny provided a reference point for comparison against mutant progeny.The authors failed to account for different levels of H3K9me in different nrde-mutant progeny.

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