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 14 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.

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

Since H3K9me marks establish after siRNAs, and RNAi-silencing is inherited even when the target gene is absent, siRNA is likely the primary heritable agent. In conclusion, heritable genesilencing mechanism involves NRDE-3 in progeny binding and guiding siRNAs to the nucleus, where siRNAs control H3K9me. Therefore, nuclear-RNAi is required to maintain heritable siRNA expression and heritable RNAi-silencing of somatically-expressed genes. Comments and Critiques The evidence is strongly supported by controls. RNAi in wild-type strains and their progeny provided a reference point for comparison against mutant progeny, to ascertain that differences could only be attributed to mutated genes. In showing that pos-1-RNAi induced F1-embryonic arrest, rde-4(-) strains exposed to pos-1-dsRNA acted as control to confirm that increased embryonic arrest was due to pos-1-RNAi. The authors used three controls an independent dpy-11-siRNA assay, rde-1(-) and NRDE-3(*PAZ) strains to confirm that their assay was selectively identifying dpy-11-siRNAs. However, the inference that elevated H3K9me in inheriting offspring is not because offspring expressed more dpy-11-siRNAs is questionable, as the authors neglected measuring levels of other dpy-11-siRNAs. The results failed to distinguish if nuclear-RNAi mechanism was responsible for establishing or preserving H3K9me in progeny. The results of RNAi, measurements of NRDE-3-bound dpy-11-siRNA and H3K9me were incomplete for certain genotypes, in particular nrde-2(-).The authors failed to account for different levels of H3K9me in different nrde-mutant progeny, and why nrde-1 and nrde-4 mutant progeny possessed higher levels of NRDE-3-bound dpy-11-siRNA than wild-type progeny. Given that the authors tested primarily only dpy-11-RNAi, it is questionable if the findings can be generalized to all somatically-expressed larval/embryonic genes. Larval-stage genes daf-11 [9, 10], lin-4 [11, 12], asna-1 [13], and embryonic-stage genes ref-1 [14] and cdk-5 [15] used in similar studies could be utilised for verification. Results in F2-progeny were reported sporadically. Effects of RNAi should have been studied in further generations, as C. elegans exhibits parental imprinting [1618] which could result in silenced traits reappearing in subsequent generations. 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. It joins current research [19, 20] supporting Lamarckian inheritance, proving traits acquired by organisms in their

lifetime can be passed to offspring. The authors believe dsRNAs, not naturally found in C. elegans, could be induced by environmental signals to activate RNAi, adapting offspring for unfavourable environmental changes a view supported by many contemporaries [21, 22]. By establishing a novel link between nuclear-RNAi mechanism and heritable gene silencing, the study has applications in advancing treatment of heritable diseases using RNAi [23], beyond silencing diseased genotype in patients to preventing its recurrence in future generations carrying the allele. References
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Word count: 1000 words (excluding references, in-line citations and headings)