RNA silencing and genome regulation

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Closely related RNA silencing phenomena such as posttranscriptional and transcriptional gene silencing (PTGS and TGS), quelling and RNA interference (RNAi) represent different forms of a conserved ancestral process. The biological relevance of these RNA-directed mechanisms of silencing in gene regulation, genome defence and chromosomal structure is rapidly being unravelled. Here, we review the recent developments in the field of RNA silencing in relation to other epigenetic phenomena and discuss the significance of this process and its targets in the regulation of modern eukaryotic genomes.

Introduction

RNA silencing is a general term for a particular collection of phenomena in which short RNA molecules trigger repression of homologous sequences. It is a highly conserved pathway, found in a large variety of eukaryotic organisms, and its main characteristic is the use of small RNA molecules of 21–28 nucleotides that confer high specificity to the target sequence. Originally, it was described as part of a ‘co-suppression’ phenomenon in plants 1, 2, 3 or ‘quelling’ in Neurospora crassa [4] and was later attributed to a posttranscriptional gene silencing process (PTGS; see Glossary) occurring in the presence of complementary RNA molecules that would bind and form double-stranded RNA [5]. A closely related effect described in Caenorhabditis elegans as ‘RNA interference’ (RNAi) 6, 7 also requires long double-stranded precursor RNAs to induce and sustain efficient posttranscriptional repression of homologous sequences.

In RNA silencing, double-stranded RNA (produced by various mechanisms) enters the ‘canonical pathway’ after cleavage into small (21–28 nt) RNA duplexes by the helicase/RNase-like III Dicer [8]. Following unwinding, a single-stranded small RNA (small interfering RNA: siRNA) becomes part of protein complexes in which PAZ/PIWI domain proteins (PPD or Argonaute) are central players 9, 10 (Figure 1a,b). These RNA-induced silencing complexes (RISC) then target homologous mRNAs and exert silencing either by inducing cleavage (‘slicing’) or, as in the case of micro-RNA-loaded RISC (see below), by also eliciting a block to translation (Figure 1c,e). RNA-dependent RNA polymerase (RdRP) also plays a role in nematodes [11], plants 12, 13 and fungi 14, 15 but is apparently not required or detectable in the genomes of flies and vertebrates. RdRP amplifies the RNAi/PTGS response by generating more double-stranded RNA from single-stranded targets that can then enter and continue to stimulate the RNA silencing pathway (Figure 1a). This positive-feedback system is crucial in plants and worms to amplify the siRNA signal transmitted from cell to cell and to mount a systemic form of silencing 16, 17.

It is now evident that the core machinery required for RNA silencing plays crucial roles in cellular processes as diverse as regulation of gene expression, protection against the proliferation of transposable elements and viruses and modifying chromatin structure. While it appears that the basic pathway has been conserved, specialization has adapted the common RNA silencing machinery for these different purposes. This is implied both by the diversity of Argonaute proteins found in different species, such as C. elegans (more than 20), Arabidopsis thaliana (10) [18] and humans (8) [19] and also by the distinct phenotypic effects that arise from disrupting different Argonaute genes 20, 21. This specialization is most obvious in plants, which also encode multiple RdRP and Dicer-like proteins that are relevant for distinct small RNA pathways [22]. Here, we discuss these different pathways and the various levels through which small RNAs can influence the activity of the genome.

Section snippets

Regulation of gene expression – microRNAs

MicroRNA regulation is a clearly specialized branch of the RNA silencing pathway that evolved towards gene regulation, diverging from conventional RNAi/PTGS. MicroRNAs are a specific class of small RNAs that are encoded in gene-like elements organized in a characteristic inverted repeat. When transcribed, microRNA genes give rise to stem–looped precursor RNAs from which the microRNAs are subsequently processed 23, 24, 25. The released miRNAs are incorporated into RISC-like complexes containing

Defence – transposable elements and viruses

RNA silencing was first recognized by its effect on the expression of multicopy transgenes. This curious phenomenon was then interpreted as a process of genome defence against foreign ‘invading’ sequences. In fact, it was observed in the early 1990s that, in plants, co-suppression or PTGS could play a role in defending against viral invasion [29]. Known core components of the RNAi pathway were found to be required for repressing transposable elements (TEs) in several eukaryotes: C. elegans 30,

Transposable elements and heterochromatin

In general, TEs and related DNA sequences are often found in chromatin domains that are transcriptionally silent and structurally distinct from the open euchromatic regions [41]. These heterochromatic regions have conspicuous features, which can include dense methylation of DNA (5-methylcytosine; 5-Me-C), hypo-acetylation of lysine residues in the N-terminal tails of histones H3 and H4 and methylation of specific lysine residues such as lysine 9 on histone H3 (H3K9me2/3). Some of these

RNA silencing reaches chromatin

The same pathway that acts to repress genes posttranscriptionally can enforce modification of homologous chromatin in a way that alters its structure and consequently its function. Transcriptional gene silencing (TGS) (Figure 1d) was initially observed in plants and was associated with repression of exogenously introduced transgenes and viral suppression [44]. Remarkably, the presence of dsRNAs homologous to the promoter or the coding region in the DNA result in robust silencing that persists

Chromosomal function – the fission yeast centromere

In fission yeast, silent chromatin assembled over the outer repeat arrays at the centromeres is required for proper chromosome segregation during mitosis. The high density of cohesin complexes associated with this silent chromatin ensures that sister chromatids are held tightly together at centromeres after DNA replication and up until the onset of anaphase 52, 53. RNA silencing must play a direct role in this process in fission yeast as deletion of any gene encoding key RNAi components leads

Repeats attract RNA silencing

To grasp the biological relevance of RNA-directed chromatin modifications, it is important to investigate the nature of the DNA sequences that generate the endogenous siRNAs that influence chromatin structure. To date, all natural targets for RNAi-mediated heterochromatin formation appear to involve TEs or repetitive DNA. This suggests that RNA silencing recognizes an intrinsic property common to these sequences in the context of centromeric function or transposon/viral control. But what could

RNA-induced chromatin silencing in metazoans

SiRNAs act to target histone and/or DNA modifications to homologous sequences in plants, ciliates and fission yeast. But do noncoding RNAs play a pivotal role in gene silencing and chromatin modifications in metazoans? Clearly X-inactivation in female mammals requires expression of Xist RNA in cis to effect chromatin modifications that result in gene silencing [64]. In addition, imprinting of paternally derived Igf2r requires expression of the associated Air noncoding RNA [65]. Likewise,

Transposable elements and repeats can influence gene regulation

The action of RNA silencing on centromeric repeat transcripts is important in defining structures and functions associated with these chromosomal regions. However, a large proportion of repetitive sequences are not concentrated in pericentromeric regions but are scattered throughout the genome. TE insertions are known to have dramatic effects on expression levels of surrounding genes by disturbing the transcriptional activity of the affected regions. Moreover, it now seems likely that observed

Concluding remarks

Noncoding RNA is the central player of an ancient and conserved form of silencing. Although the different forms of RNA silencing were initially unearthed as seemingly distinct phenomena, basic machinery is held in common between PTGS, TGS, quelling and RNAi. In addition, these same components are conserved in a large variety of organisms and thus must have arisen early in eukaryotic evolution. Since its discovery several years ago, the biological relevance of RNA-directed silencing mechanisms

Acknowledgements

We apologize to all the colleagues whose work, owing to space limitations, was not mentioned in this review. We wish to thank David Finnegan Alison Pidoux and Vera Schramke for useful discussion and critical review of the manuscript, and all the members of the Allshire laboratory for their support. We are grateful to the EC FP6 ‘The Epigenome’ Network (LSHG-CT-2004–503433) for input. Ricardo Almeida is a student of the 3rd Gulbenkian PhD Programme in Biomedicine and is sponsored by the

Glossary

5-Me-C
5-Methylcytosine
DNMT
DNA de novo methyltransferase
dsRNA
Double-stranded RNA
HDAC
Histone deacetylase
HMT
Histone methyltransferase
H3K9ac
Histone H3 acetylated on lysine 9
H3K9me2/3
Histone H3 di/tri-methylated on lysine 9
LTR
Long terminal repeat
PEV
Position effect variegation
PPD
PAZ/PIWI domain
PTGS
Posttranscriptional gene silencing
RdRP
RNA-dependent RNA polymerase
RISC
RNA-induced silencing complex
RITS
RNA-induced transcriptional silencing complex
RNAi
RNA interference
siRNA
Small interfering RNA
TE

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