Recognizing the enemy within: licensing RNA-guided genome defense

Abstract

How do cells distinguish normal genes from transposons? Although much has been learned about RNAi-related RNA silencing pathways responsible for genome defense, this fundamental question remains. The literature points to several classes of mechanisms. In some cases, double-stranded RNA (dsRNA) structures produced by transposon inverted repeats or antisense integration trigger endogenous small interfering RNA (siRNA) biogenesis. In other instances, DNA features associated with transposons--such as their unusual copy number, chromosomal arrangement, and/or chromatin environment--license RNA silencing. Finally, recent studies have identified improper transcript processing events, such as stalled pre-mRNA splicing, as signals for siRNA production. Thus, the suboptimal gene expression properties of selfish elements can enable their identification by RNA silencing pathways.

Keywords: PIWI-interacting RNA; RNAi; genome defense; small RNA; small interfering RNA; transposon.

Figure I. Examples of common eukaryotic transposons

Common Class I transposons include LTR retrotransposons, which generally encode a capsid protein (GAG), protease (PRO), reverse transcriptase (RT), RNaseH (RH), and integrase (INT). By contrast, LINEs typically contain two open reading frames, one of unknown function and one that encodes endonuclease and reverse transcriptase activities. SINEs are non-autonomous elements whose sequence features are recognized by transposon-derived proteins acting in trans. Most Class II elements are TIR transposons, which can mobilize either autonomously or non-autonomously. Blue rectangles indicate protein-coding regions.

Figure 1. Transposon features recognized by RNA silencing pathways

A) Transposon-derived dsRNA provides a substrate for Dicer activity and thereby triggers endo-siRNA pathways. Double-stranded RNA can be generated intermolecularly by the action of convergent promoters encoded either by the transposon (blue bar) or by the host. Intramolecular dsRNA is generated from a single transposon when a transcript contains both of its inverted repeat sequences. Inverted duplications of transposons can also cause intramolecular dsRNA formation. B) Unusual chromosomal arrangements of transposons allow their identification by genome defense pathways. In N. crassa, the quelling pathway (left) silences repetitive sequences. The mechanism by which these sequences are detected and used to template siRNA production is unclear, but may involve the formation of unusual DNA repair intermediates, because quelling requires proteins, such as RAD-51, that mediate homologous DNA recombination. Subsequent siRNA production requires QDE-1—a DNA- and RNA-dependent RNA polymerase—and its binding partners Replication protein A (RPA) and QDE-3. The meiotic silencing of unpaired DNA pathway (right) detects loci that lack a partner during homologous chromosome pairing in meiosis I. siRNA production from these loci requires SAD-1, an RdRP that is paralogous to QDE-1.

Figure 2. Stalled spliceosomes license RNA silencing in an endo-siRNA pathway

In the yeast C. neoformans, transcripts targeted by RNA silencing, which primarily include transposons, exhibit sequence features predictive of poor splicing and tend to stall in spliceosomes. The stalled splicing of transposon mRNA precursors is required for siRNA biogenesis mediated by SCANR, a protein complex that contains an RdRP and physically associates with the spliceosome. Lariat debranching enzyme (Dbr1) is also required for siRNA production, suggesting that transposon mRNA precursors in the lariat intermediate stage are linearized to enable dsRNA formation by SCANR. In this hypothetical example, splicing of a transcript’s first intron stalls at the lariat intermediate stage, whereas downstream introns remain partially spliced.

Figure 3. piRNA production occurs at specialized genomic loci

A) In D. melanogaster, the chromatin environment at dual-strand piRNA clusters enables piRNA production from these loci. This chromatin is associated with H3K9 methylation and the specialized heterochromatin protein 1 (HP1) homolog Rhino (Rhi). Primary transcripts produced from piRNA clusters are bound by UAP56, a DEAD box protein required for piRNA processing specificity [87]. The ping pong amplification cycle subsequently promotes the generation of piRNAs complementary to active transposons (see text for details). Mobilization of transposons into piRNA clusters contributes to the enrichment of foreign sequences at these loci. B) In C. elegans, 21U-RNAs are encoded in genomic clusters, but each is expressed by its own promoter. 21U-RNA sequences are very diverse, and not strongly enriched in transposon sequences, suggesting that transposon mobilization into 21U-RNA clusters is not a major mechanism by which 21U-RNA specificity is achieved. 21U-RNAs loaded in the PIWI protein PRG-1 are potentially complementary to both non-self and self transcripts. In the former case, PRG-1 triggers the production of repressive 22G-RNAs to silence the non-self transcript. In the latter case, PRG-1-mediated silencing appears to be less efficient, potentially due to a protective mechanism. This protective mechanism is hypothesized to involve targeting of self transcripts by 22G-RNAs loaded in the Argonaute protein CSR-1.