RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond

Abstract

A growing number of functions are emerging for RNA interference (RNAi) in the nucleus, in addition to well-characterized roles in post-transcriptional gene silencing in the cytoplasm. Epigenetic modifications directed by small RNAs have been shown to cause transcriptional repression in plants, fungi and animals. Additionally, increasing evidence indicates that RNAi regulates transcription through interaction with transcriptional machinery. Nuclear small RNAs include small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs) and are implicated in nuclear processes such as transposon regulation, heterochromatin formation, developmental gene regulation and genome stability.

Figure 1. Generalized pathways depicting the biogenesis of nuclear small RNA

a | siRNA processing takes place in the nucleus in S. pombe and Drosophila and the nucleoulus in Arabidopsis. dsRNA can be produced by convergent transcription, complementary transcripts, structured loci, or by RdRP activity in Arabidopsis and S. pombe. Dicer proteins generate siRNA that is loaded into an Argnoaute protein. In Arabidopsis siRNA are transported to the cytoplasm where Argonaute is loaded and then imported into the nucleus. b | In C. elegans siRNA processing occurs in the cytoplasm in a two-step fashion. Primary trigger dsRNA arises from nuclear transcription or the RdRP activity of RRF-3, which acts on transcripts in the cytoplasm. Primary processing by DCR-1 produces primary 26 nt siRNA which are loaded into ERGO-1. Loaded ERGO-1 can both facilitate PTGS in the cytoplasm and with RRF-1 generate secondary 22G siRNA siRNA. Secondary 22G siRNA is loaded into the nuclear Argonaute NRDE-3 in the cytoplasm that is then transported into the nucleus. c | piRNA biogenesis via the ping-pong cycle in the Drosophila female germline. Primary precursor piRNA antisense to active transposons (blue) is transcribed from heterochromatic piRNA clusters, and sense mRNA from active transposons (pink). In the cytoplasm primary processing generates antisense piRNA from primary precursor that is then loaded into Aub or Piwi and cleaves sense transposon mRNA to produce sense piRNA. Additional antisense piRNA is produced by Ago3 mediated cleavage of antisense primary piRNA transcripts, completing the cycle. Only loaded Piwi is imported into the nucleus.

Figure 2. Co-Transcriptional Gene Silencing in S. pombe

a | RNA Pol II initiates transcription at loci targeted by RNAi. b | During the elongation phase of transcription a Ago1 is guided to the nascent transcript and inhibits RNA Pol II transcription via an unknown mechanism. c | RNAi can lead to a release of RNA Pol II. d | A mechanistic model of RNAi acting during the elongation phase (2b) in S. pombe. The RITSC is localized through siRNA base-pairing with the nascent transcript, and chromatin interaction mediated by the chromodomain of Chp1. The RDRC couples dsRNA production by Rdp1 and siRNA cleavage by Dcr1 and is also associated with the nascent RNA Pol II transcript. The RITSC interacts with the CLRC that catalyzes H3K9 methylation at target loci. This histone modification serves as a binding site for Swi6, the S. pombe ortholog of the highly conserved heterochromatin protein 1 (HP1), which is a defining feature of heterochromatin. The RITSC promotes RNA Pol II release via an unknown mechanism. The dashed grey lines indicate interactions between complexes.

Figure 3. The RNA-directed DNA Methylation pathway in Arabidopsis

RNA Pol IV transcribes ssRNA from repetitive heterochromatic loci. RDR2 physically associates with RNA Pol IV to produce dsRNA. DCL3 cleaves dsRNA to produce siRNA that are transported to the cytoplasm for AGO4 loading, facilitated by HSP90, which is then imported back into the nucleus. In the nucleus AGO4 targets nascent RNA Pol V transcripts through complementarity to siRNA and forms the RdDM complex presumably containing the catalytically active de novo DNA methyltransferase DRM2. The Pol V associated GW/WG protein KTF1 may act as an organizer by interacting with AGO4 and 5meC. Similarly, the AGO4 associated protein RDM1 can bind single stranded methylated DNA and interacts with DRM2. Both could contribute to a positive feedback loop between AGO4 localization and DNA methylation (circular arrows). DRM3, a catalytically inactive paralog of DRM2 is required for RdDM however its role is unknown. Once localized, DRM2 catalyzes methylation of cytosine in all sequence contexts. The dashed grey lines indicate interactions.

Figure 4. RNAi mediated transposon silencing in the germline

a | In the supportive vegetative nucleus of the Arabidopsis male gametophyte ddm1 expression is repressed which leads to the loss of cytosine methylation and reveals transposons. Transposons are processed into 21nt siRNA that are mobile and can direct PTGS in the sperm nuclei. They may also impact transposons transcriptionally by directing or inhibiting epigenetic modification. Red lollipops represent 5meC. b | The supportive central cell of the Arabidopsis female gametophyte reveals transposons for transcription by downregulating the maintenance DNA methyltransferase MET1 and expressing the DNA glycosylase DEMETER causing a loss of cytosine methylation. This activates the RdDM pathway and produces 24nt siRNA that may be transported to the egg cell to enforce transcriptional silencing through AGO9. Red lollipops represent 5meC. c | In the Drosophila ovariole the flamenco cluster is expressed in somatic follicle cells, and generates piRNA independently of the ping-pong cycle. Loaded Piwi silences the gypsy family of retrotransposons which could otherwise form infectious particles. In oocytes and surrounding nurse cells all piRNA clusters are expressed and the primary transcripts enter the ping-pong cycle to produce piRNA. Active transposons are post-transcriptionally silenced, and nuclear Piwi promotes transcriptional silencing via H3K9 methylation, and HP1a localization. The HP1a homolog Rhino binds to heterochromatic piRNA clusters in place of HP1a and promotes transcription.

Figure 5. piRNA (21U) Pathway in the C. elegans germline

a | The “21U” piRNA of C. elegans originate from two broad clusters on chromosome IV, however little is known about their biogenesis. They act with the Piwi family Argonaut PRG-1 to target mRNA in the cytoplasm. Targeting of PRG-1 to mRNA recruits a RdRP to produce abundant 22G siRNA. b | 22G siRNA is loaded into the germline specific nuclear Argonautes WAGO-9/10, which are closely related to NRDE-3, the nuclear Argonaute involved in somatic TGS. Loaded WAGO-9/10 is transported into the nucleus where it targets nascent transcripts of RNA Pol II and directs H3K9me that is dependent on the nuclear RNAi components NRDE-1/2/4. H3K9 methylation is catalyzed by two putative histone methyltransferases SET-25/32. The HP1 ortholog HPL-2 binds H3K9me and is required for multi-generational silencing.