Conserved chromosomal functions of RNA interference

Abstract

RNA interference (RNAi), a cellular process through which small RNAs target and regulate complementary RNA transcripts, has well-characterized roles in post-transcriptional gene regulation and transposon repression. Recent studies have revealed additional conserved roles for RNAi proteins, such as Argonaute and Dicer, in chromosome function. By guiding chromatin modification, RNAi components promote chromosome segregation during both mitosis and meiosis and regulate chromosomal and genomic dosage response. Small RNAs and the RNAi machinery also participate in the resolution of DNA damage. Interestingly, many of these lesser-studied functions seem to be more strongly conserved across eukaryotes than are well-characterized functions such as the processing of microRNAs. These findings have implications for the evolution of RNAi since the last eukaryotic common ancestor, and they provide a more complete view of the functions of RNAi.

Figure 1 |. Small RNA biogenesis pathways and the evolutionary conservation of their components.

a | Small interfering RNAs (siRNAs) can be produced from either double stranded RNA (dsRNA) generated by RNA-dependent RNA polymerase (Rdrp) or other dsRNA substrates. In S. pombe and other eukaryotes possessing a Rdrp (Rdp1), a transcript is targeted for the synthesis of dsRNA, which is subsequently recognized by Dicer (Dcr1) and cleaved into small RNA duplexes. Additionally, dsRNA can occur without the activity of Rdrp in instances such as bi-directional transcription or inverted repeat hairpin formation. The non-Rdrp precursors are also recognized by Dicer and cleaved into small RNA duplexes. In each case the siRNA duplexes are loaded into Argonaute (Ago1) effectors, the passenger strand is discarded, and the guide strand is then used to recognize RNA targets. b | microRNAs (miRNAs) are produced from hairpin structures formed in the pri-miRNA transcript. In metazoans, such as humans, the hairpin structure is processed initially by the DROSHA/DGCR8 complex into the pre-miRNA, which is further processed by DICER1 into the miRNA duplex. In plants, the processing of pri-miRNA to pre-miRNA to miRNA duplex occurs through two successive cleavages by Dicer-like 1 (Dcl1). In both metazoans and plants, miRNA duplex is bound by an Argonaute effector protein (AGO), the passenger strand is discarded, and the guide strand can then direct the effector to complementary targets. c | PIWI-interacting RNAs (piRNAs) are produced in D. melanogaster and M. musculus from single-stranded RNA (ssRNA) transcripts from piRNA precursor loci and their complementary target loci. The piRNA precursor transcripts are initially processed by PIWI proteins and then enter the ping-pong amplification cycle in which the recognition of the ssRNA target transcript by the PIWI-piRNA effector complex generates secondary piRNAs complementary to the piRNA transcripts. These secondary piRNAs are bound by an Argonaute or PIWI effector protein and can again target the piRNA ssRNA precursor transcripts to produce additional piRNAs derived from the piRNA locus. d | A phylogenetic diagram of humans and selected model organisms displays the conservation of RNAi proteins, microRNAs, and centromere structure.

Figure 2 |. Chromosome segregation phenotypes of RNAi are highly conserved.

a| Deletion mutants of either the RdRP, Dicer (shown), or Argonaute homologues in S. pombe, result in lagging chromosomes in mitosis and meiosis. b| The deletion of an Argonaute homologue in T. brucei results in lagging chromosomes and segregation defects in mitosis. c| The deletion of a Dicer homologue in T. thermophila results in lagging chromosomes and bridging of genetic material in mitosis and meiosis. d| While the displayed images are of disruptions of belle, the knockout or knockdown of Dicer and Argonaute homologues in D. melanogaster leads to lagging chromosomes in the same cell types - wing disc cells and S2 cell culture. e| The deletion of Dicer1 in mouse fibroblasts results in bridging during mitosis and the formation of micronuclei most likely derived from missegregated chromosomes. f| In human RPE-1 cells, the knockdown of Dicer1 or Ago2 by siRNAs generated lagging chromosomes. In human HeLa cells, the knockdown of Dicer1 also generated lagging chromosomes. g| The knockdown of an Ago homologue (shown) in C. elegans or other factors in the CSR-1 22G-RNA pathway results in lagging chromosomes in the early worm embryo.

Figure 3 |. Faithful chromosome segregation is ensured by the recruitment and protection of centromeric cohesin.

a| In S. pombe the RNAi pathway directs the heterochromatin machinery via small RNAs and the establishment of pericentromeric heterochromatin results in the recruitment and retention of centromeric cohesin during mitosis and meiosis and the proper segregation of chromosomes. The formation of pericentromeric heterochromatin begins with Pol II transcription of long non-coding RNAs (lncRNAs) from the dg/dh pericentromeric repeats. The RDRC complex (Rdp1, Hrr1, and Cid12) targets the lncRNAs for the synthesis of dsRNA. These dsRNAs are recognized and cleaved by Dcr1 into siRNAs. These small RNAs are loaded into Ago1 which then directs the RNA-induced transcriptional silencing complex (RITS – Ago1, Chp1, and Tas3) to the pericentromere through the targeting of nascent lncRNAs. Chp1 then recognizes H3K9me and stabilizes the complex at the site. The RITS complex recruits the Clr4-Rik1-Cul4 complex (CLRC – Clr4, Rik1, Cul4, Stc1, Raf2, Raf2), which furthers the spread of H3K9me heterochromatin through the activity of the methyltransferase Clr4. The homologue of heterochromatin protein 1 (HP1) Swi6 binds to H3K9 methylation and reinforces the heterochromatin state. Additionally, it directly recruits the cohesin complex that holds sister chromatids together until anaphase during mitosis. Thus, the combined activity of RNAi and the CLRC complex generate a chromatin state at the pericentromere that results in the proper segregation of chromosomes during cell division. b| In M. musculus and other mammals, such as humans, the RNAi pathway ensures the proper segregation of chromosomes presumably through the formation of heterochromatin at the centromeric region. RNA Pol II transcribes the major satellite pericentromeric repeats into lncRNAs in a bi-directional fashion. The resulting dsRNAs are recognized by DICER1 and possibly cleaved into a small RNA population. There is some evidence to suggest that AGO2 may play a role potentially by loading satellite small RNAs, but the effect this has on the chromatin in the region is unknown. Rather the regulation of the major satellite lncRNAs by DICER1 may ensure the appropriate recruitment of recruiting chromatin machinery to the region. The H3K9 methyltransferases SUV39H1 and SUVH39H2 can be recruited directly by major satellite lncRNAs. The H3K9 methylation and major satellite lncRNAs are recognized by HP1, which reinforces the heterochromatic state of the pericentromere. HP1 may play a role in recruiting and/or maintaining cohesin at the centromere to ensure proper separation of sister chromatids, though this function is primarily performed by SGO1 in mammals.

Figure 4 |. RNAi regulates dosage at the transcript, chromatin, and DNA levels.

a| Meiotic silencing of unpaired DNA (MSUD) is an RNAi-based mechanism that identifies and silences transcription products of unpaired loci at the transcript level. During the pairing of homologous chromosomes in meiosis an unidentified recognition complex identifies regions that are unpaired. This stimulates bi-directional transcription by RNA Pol II of aberrant RNAs. The aberrant RNAs are exported to the out of the nucleus and then used as a template by RdRP homologue SAD-1 to generate dsRNA. A Dicer homolog, DCL-1, then targets the dsRNA and generates siRNAs called MSUD-associated small interfering RNAs or (masiRNAs). The Argonaute homologue SMS-2 loads the masiRNAs and targets any transcript from the unpaired locus for degradation. b| The X chromosome dosage compensation mechanism in Drosophila uses endo-siRNAs targeting the 1.688^X satellite repeat to direct chromatin modifications that contribute to increasing transcription from the male X chromosome to match the dosage of the two X chromosomes in female flies. The mechanism most likely begins with RNA Pol II bi-directionally transcribing the 1.688^X loci. The resulting dsRNA is recognized by Dcr-2, which generates 1.688^X endo-siRNAs. Ago2 loads the endo-siRNAs and directs the H3K9 methyltransferase Su(var)3–9 to the 1.688^X loci on the X chromosome. The presence of this mark may recruit the Male Specific Lethal (MSL) complex to the surrounding region, which in turn increases transcription from the X chromosome. c| The meiotic process of DNA elimination relies on the direction of heterochromatin formation by RNAi to identify sequences to be removed from the new macronucleus. The bi-directional transcription of the parental micronuclear genome, most likely by RNA Pol II, generates dsRNA that are processed by the Dicer homologue Dcl1 into early scan-RNAs (scn-RNAs). The PIWI-like effector Twi1 binds the early scnRNAs and translocates to the parental macronucleus where the effector protein and small RNA search for complementary sequences on nascent transcripts. If a Twi1/scn-RNA complex finds a complementary transcript the scn-RNA is degraded. If a target is not found the Twi1/scn-RNA complex translocates to the developing macronucleus and again searches for complementary nascent transcripts. When a target sequence is identified, Twi1 recruits the H3K9 and H3K27 methyltransferase Ezl1 to the region. The recognition of H3K9me/H3K27me by the HP1 homologue Pdd1 strengthens the chromatin state of the region by inducing the transcription of late-scnRNAs which then feedback on the region to spread heterochromatin. This leads to the excision of this DNA sequence by Tpb2. d| The copy number of the ribosomal DNA tandem array loci is maintained by Dcr1 in S. pombe through the resolution of replication/transcription collisions. In the presence of Dcr1 (top panel) the collision of a replication fork with RNA Pol II can be resolved through the action of Dcr1 in Pol II transcription termination and importantly neither the catalytic activity Dcr1 nor small RNAs are required for this function. Rather it may be an RNA:DNA hybrid that stimulates this function of Dcr1. In the absence of Dcr1 (bottom panel), Rad52 and RNA:DNA hybrids accumulate at sites of unresolved replication stress. This leads to repair by homologous recombination and as a consequence a loss of rDNA copy number.

Figure 5 |. The roles of RNAi in the recognition and resolution of DNA damage.

a| In Neurospora QDE-2-associated siRNAs (qiRNAs) participate in the DNA damage response generally through the HR pathway. The RdRP homologue QDE-1, which has DNA-dependent RNA polymerase activity as well, transcribes aberrant RNA from sites of damage and then converts it to dsRNA. The dsRNA is recognized by DCL-1/2 and qiRNAs are produced. These small RNAs are loaded into Argonaute homologue QDE-2 and then promote the resolution of damage through HR. b| The production of DSB-induced siRNAs (diRNAs) in Arabidopsis is dependent on transcription by RNA Pol IV from the site of the damage. These transcripts serve as the template for RdRP homologues RDR2/6 to generate dsRNA. Dicer-like homologues generate the diRNAs, which are then loaded into Ago2. The Ago2/diRNA complex can then recognize nascent transcripts from RNA Pol V and may direct chromatin effectors or repair machinery to the site of damage. c| The endo-siRNA pathway of Drosophila can generate damage-induced small RNAs (and the production of these small RNAs may be enhanced by the splicing machinery. The production of dsRNA recognized by Dcr-2 is enhanced at sites of active transcription, especially sites where the spliceosome is present indicating that the spliceosome may promote antisense transcription. The diRNAs are loaded into Ago2 and it is proposed that the Ago2/diRNA complex may direct downstream repair machinery to the site of the break. d| In mammals, the RNAi pathway can direct both chromatin modifiers and repair machinery to the site of a DNA double-strand break. Pol II transcribes the regions around the break and it may be recruited there by the early-responding MRN complex. The production of anti-sense transcripts may be promoted by the formation of RNA:DNA hybrids in the region and DROSHA may recognize these hybrids. The production of DNA damage-response RNAS (DDRNAs) is dependent on DROSHA and DICER processing dsRNA from the site of the break. These small RNAs can be loaded into AGO2, which can then target the site of DSB and recruit the chromatin effectors MMSET and TIP60 as well as the DNA damage response factors 53BP1 and RAD51 among others to resolve the damage. e| In human cell lines, DICER1 plays a role in the recognition and resolution of DNA damage caused by UV irradiation. In a complex with ZRF1, which can recognize ubiquitinated H2A, DICER1 localizes to the site of damage in an RNA-dependent manner. DICER1 then recruits chromatin effectors, such as MMSET, and repair factors, such as XPA, 53BP1, and RPA2 to the site to promote the resolution of damage.