Nuclear Noncoding RNAs and Genome Stability

Abstract

In modern molecular biology, RNA has emerged as a versatile macromolecule capable of mediating an astonishing number of biological functions beyond its role as a transient messenger of genetic information. The recent discovery and functional analyses of new classes of noncoding RNAs (ncRNAs) have revealed their widespread use in many pathways, including several in the nucleus. This Review focuses on the mechanisms by which nuclear ncRNAs directly contribute to the maintenance of genome stability. We discuss how ncRNAs inhibit spurious recombination among repetitive DNA elements, repress mobilization of transposable elements (TEs), template or bridge DNA double-strand breaks (DSBs) during repair, and direct developmentally regulated genome rearrangements in some ciliates. These studies reveal an unexpected repertoire of mechanisms by which ncRNAs contribute to genome stability and even potentially fuel evolution by acting as templates for genome modification.

Keywords: DNA double-strand break (DSB); DNA repair; diRNA; genome stability; heterochromatin; lncRNA; noncoding RNA (ncRNA); piRNA; siRNA; transposon silencing.

Figure 1

Mechanisms of ncRNA-mediated genome regulation. (A) Protein (orange and gray ovals)-sRNA (blue or red lines) interaction targets proteins to complementary sequences in trans. (B) Tethered ncRNAs form platforms for the assembly of regulatory protein complexes in cis. (C) ncRNAs facilitate protein-protein interactions by acting as a structural component of these complexes. (D) ncRNAs bridge or template the repair of double-strand breaks (DSBs).

Figure 2

ncRNA-mediated silencing and telomere regulation. (A) siRNA-dependent H3K9 methylation at the fission yeast pericentromeric regions. A network of interactions among RNAi complexes (RITS, RDRC, Dcr1), cen lncRNAs (red wavy line), and CLRC (green pentagon) creates siRNA and H3K9me amplification loops at centromeres. This leads nucleation and spreading of H3K9me and the recruitment of the heterochromatin protein 1 (HP1) proteins, Swi6 (blue oval) and Chp2 (turquoise). Chp2 in complex with SHREC represses RNA Pol II (green circle) transcription (TGS), and Swi6 facilitates RNAi-dependent PTGS via its Ers1-dependent interactions with RDRC. Swi6 also interacts cohesin and is required for its recruitment to cen repeats. In the absence of CLRC, RNAi complexes or Swi6, cohesin recruitment is lost and cells display chromosome segregation defects. (B) piRNA silencing in D. melanogaster maternal germ cells. piRNA clusters and transposons are shown in blue and red, respectively. Cytoplasmic degradation of TE transcripts by the Piwi family proteins (Aubergine (Aub), Piwi and Argonaute-3 (Ago3) is depicted. Iterative cycles of Piwi/Aub and Argonaute-3 (Ago3) cleavage events in the cytoplasm amplify the piRNA signal and degrade TE transcripts. Also, piRNA-Piwi complexes can be imported into the nucleus where they mediate TGS silencing via the recruitment of HP1/Su(var)3–9 proteins (yellow oval). (C) TERRA and telomere regulation. TERRA (blue wavy line) is a structural component of telomeres upon which Sheltrin (gray oval), HP1/Suv39H1 (lime green circle), and other silencing complexes assemble. TERRA also facilitates the RPA (pink rectangle)-to-POT1 (gray pentagon) transition via binding to hnRNAP1 (teal octagon). See text for details.

Figure 3

ncRNA-mediated genome reorganization in ciliates. Genome rearrangement in Oxytricha involves the (A) unscrambling of the developing MAC genome. This requires the maternal inheritance of long noncoding template RNAs (blue wavy lines) from the parental MAC nucleus into the developing progeny MAC. Numbers depict segments of a gene which must be ordered to make a functional open reading frame. (B) DNA retention uses maternally inherited piRNA-Otiwi complexes (orange ovals with blue lines) to mark the regions of the genome which are retained in the developing progeny MAC nucleus. (C) DNA elimination in Tetrahymena requires the bidirectional transcription (wavy black lines) of the parental MIC genome. dsRNAs are degraded into scnRNAs (shown in red, blue and green lines) by Dcl1 and exported to the cytoplasm where they are loaded onto the PIWI protein Twi1 (orange oval). scnRNA-Twi1 complexes are imported into the parental MAC nucleus where they find the complementary genomic regions. All ‘self’ scnRNAs (blue) are eliminated, and the remaining scnRNAs (red and green), complementary to TEs (red rectangle) and IESs (green rectangle), are exported into the developing progeny MAC nucleus. scnRNA-Twi1 complexes basepair with complementary TEs and IESs in the MAC genome and recruit Ezl and Ppd1/3 proteins (lime green circle). These regions are packaged into heterochromatin and later eliminated from the MAC genome by an unknown mechanism.

Figure 4

ncRNAs regulation of DSB repair. (A) diRNA-mediated DSB repair. DSBs induce RNAi-dependent diRNA production, which once loaded onto Ago (orange oval) target Ago recruitment to DSBs (pathways 2 and 3) or other sites around the genome (pathway 1). In 1, diRNAs recruit Ago to sites (other than the DSB) around the genome (for example rDNA in N. crassa), potentially regulating their transcription in a manner that favors DSB repair. In 2, diRNAs recruit Ago and DNA polymerases (blue oval) to the DSB to catalyze a synthesis-dependent repair process. In 3, diRNA-programmed Ago complexes recruit repair proteins to the DSB. (B) lncRNA-mediated DSB repair. RNA molecules (blue line) complementary to the DSB may serve as a bridge (left) or template (right) for repairing DSBs. RNase H1/H,2 inhibit this repair mechanism by removing RNA:DNA hybrids. On the other hand, Rad52 (blue oval), which promotes RNA:DNA hybrid formation in vitro, is proposed to stimulate DSB repair by annealing the RNA to the DSB. RNA:DNA hybrids are thought to promote the precise re-ligation of a DSB (left) or reverse transcriptase-dependent synthesis and re-ligation (right) at the DSB. These depicted mechanisms may operate in non-dividing mammalian cells (Wei et al., 2015).