Angela Leong Feng PingM13608BL6403 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]. RNA-

dependent-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 NRDE-

3 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-9-

methylation (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 RNAi-

inheritance. 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-11-

RNAi, 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-line-

expressed 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 gfp-

siRNAs, 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 gene-

silencing 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 [16–18]

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 somatically-

expressed 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.

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