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Uridylation of Mature Mirnas and Sirnas by the MUT68 Nucleotidyltransferase Promotes Their Degradation in Chlamydomonas

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Uridylation of Mature Mirnas and Sirnas by the MUT68 Nucleotidyltransferase Promotes Their Degradation in Chlamydomonas Uridylation of mature miRNAs and siRNAs by the MUT68 nucleotidyltransferase promotes their degradation in Chlamydomonas Fadia Ibrahima,1, Linda A. Rymarquisb,1, Eun-Jeong Kima, James Beckera, Eniko Balassaa, Pamela J. Greenb, and Heriberto Ceruttia,2 aSchool of Biological Sciences and Center for Plant Science Innovation, University of Nebraska, Lincoln, NE 68588; and bDelaware Biotechnology Institute, University of Delaware, Newark, DE 19711 Edited by James C. Carrington, Oregon State University, Corvallis, OR, and approved December 8, 2009 (received for review November 2, 2009) Regulation of gene expression by small RNAs (∼20–30 nucleotides in 9). Activated RISC uses the remaining single-stranded siRNA as a length) plays an essential role in developmental pathways and guide to identify homologous RNAs, ultimately triggering tran- defense responses against genomic parasites in eukaryotes. Micro- script degradation and/or translation repression (1–3). RNAs (miRNAs) and small interfering RNAs (siRNAs) commonly The biogenesis and the mode of action of sRNAs have direct the inactivation of cognate sequences through a variety of attracted great attention (1–3, 10), but much less is known about mechanisms, including RNA degradation, translation inhibition, and mechanisms of miRNA/siRNA turnover and their role in small transcriptional repression. Recent studies have provided consider- RNA function. Degradation of mature miRNAs in Caeno- able insight into the biogenesis and the mode of action of miRNAs rhabditis elegans, mediated by the 5′-to-3′ exoribonuclease and siRNAs. However, relatively little is known about mechanisms XRN2, has recently been shown to modulate miRNA accumu- of quality control and small RNA decay in RNA interference (RNAi) lation in vivo (11). A conserved nuclease from C. elegans and pathways. Here we show that deletion of MUT68, encoding a ter- Schizosaccharomyces pombe, Eri-1, degrades siRNA duplexes minal nucleotidyltransferase in the alga Chlamydomonas reinhard- with 2-nucleotide 3′ overhangs in vitro and reduces the efficiency tii, results in elevated miRNA and siRNA levels. We found that of RNAi in vivo (12, 13). Another family of 3′-to-5′ exoribonu- PLANT BIOLOGY MUT68 plays a role in the untemplated uridylation of the 3′ ends cleases, encoded by the SMALL RNA DEGRADING NUCLE- of small RNAs in vivo and stimulates their degradation by the RRP6 ASE (SDN) genes, has been implicated in mature miRNA exosome subunit in vitro. Moreover, RRP6 depletion also leads to turnover in A. thaliana (14). In C. elegans and mammalian cells, accumulation of small RNAs in vivo. We propose that MUT68 and Lin-28 (a stem-cell-specific regulator) binds the precursor of the RRP6 cooperate in the degradation of mature miRNAs and siRNAs, as let-7 miRNA in the cytoplasm and promotes its 3′ end uridyla- a quality control mechanism to eliminate dysfunctional or damaged tion by a poly(U) polymerase (15–17). This leads to precursor small RNA molecules. RNA degradation and downregulation of the let-7 miRNA. In contrast, terminal uridylation of mature miR-26 in mammalian exosome | RNA interference | miRNA quality control cells appears to impart functional differences that attenuate miRNA-targeted repression without obvious changes in miRNA NA-mediated silencing is an evolutionarily conserved mech- steady-state levels (18). In A. thaliana,3′ end uridylation has Ranism(s) by which small RNAs (sRNAs) induce the inacti- been postulated, although not demonstrated yet, to stimulate the vation of cognate sequences, although sRNAs may also decay of small RNAs (19, 20). Indeed, the enzymes involved in participate in the activation of gene expression (1–3). At least two untemplated nucleotide additions to the 3′ ends of mature major classes of small RNAs have been identified in many sRNAs and the functional significance of these modifications eukaryotes: microRNAs and small interfering RNAs (1, 2). remain largely uncharacterized in most eukaryotes. Moreover, miRNAs originate from endogenous noncoding RNA transcripts plant miRNAs and siRNAs and animal siRNAs and PIWI- or introns that fold into imperfect stem loop structures and often interacting RNAs (piRNAs) have a 2′-O-methyl group on their modulate the expression of genes with roles in development, 3′ termini, introduced by the RNA methyltransferase HEN1, physiological processes, or stress responses (1–3). siRNAs are which seems to protect them against nucleotide additions and/or produced from long, near-perfect complementarity double- exonucleolytic shortening (19–22). However, the putative stranded RNAs (dsRNAs) of diverse origins, including the tran- enzyme(s) involved in the proposed 3′-to-5′ degradation of scripts of long inverted repeats, the products of convergent unmethylated and uridylated sRNAs is not known because transcription or RNA-dependent RNA polymerase activity, viral Arabidopsis SDN1 is inhibited by 3′ terminal uridylation (14). RNAs, or dsRNAs experimentally introduced into cells (1–5). We report here on the characterization of a C. reinhardtii These siRNAs play various roles in posttranscriptional regulation mutant (Mut-68) that is defective in RNAi and provides further of gene expression, suppression of viruses and transposable ele- insight on the mechanisms of mature miRNA/siRNA degrada- ments, and/or heterochromatin formation (1–5). Hairpin and long dsRNAs are processed into sRNAs by an RNaseIII-like endo- nuclease named Dicer (1, 2). These small RNAs are then incor- Author contributions: F.I., L.A.R., P.J.G., and H.C. designed research; F.I., L.A.R., E.-J.K., J.B., porated into multisubunit effector complexes, such as the RNA- and E.B. performed research; F.I., L.A.R., P.J.G., and H.C. analyzed data; and F.I., L.A.R., induced silencing complex (RISC) (1, 2). Argonaute proteins, and H.C. wrote the paper. which include two main subfamilies of polypeptides named after The authors declare no conflict of interest. Arabidopsis thaliana ARGONAUTE1 (AGO1) and Drosophila This article is a PNAS Direct Submission. melanogaster PIWI, are core components of the RISC and some Data deposition: The deep sequencing libraries reported in this paper have been depos- function as sRNA-guided endonucleases (1, 2, 6, 7). Recent evi- ited in NCBI GEO (accession no. GSE17815). dence suggests that a siRNA duplex is first loaded into RISC and 1F.I. and L.A.R. contributed equally to this work. AGO cleaves one of the siRNA strands (the passenger strand) (1, 2To whom correspondence should be addressed. E-mail: [email protected]. 2). Ribonucleases, such as Trax or QIP, then promote RISC This article contains supporting information online at www.pnas.org/cgi/content/full/ activation by removing the passenger strand cleavage products (8, 0912632107/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0912632107 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 tion. Mut-68, which lacks the MUT68 nucleotidyltransferase because transcripts for the three AGO genes are present at similar (23), displays elevated levels of miRNAs and siRNAs. The amounts in Mut-68 and the control strains (Fig. S3A). MUT68 enzyme is involved in the untemplated uridylation of the To define the step of small RNA biogenesis at which MUT68 3′ termini of small RNAs in vivo and in stimulating their deg- may act to influence sRNA levels, we determined whether the radation by the RRP6 exosome subunit in vitro. Moreover, amounts of the miRNA* [the strand complementary to a guide RRP6 depletion by RNAi also results in the accumulation of miRNA (1, 2)] were also affected by depletion of this enzyme. For miRNAs and siRNAs in vivo. We hypothesize that MUT68, in two miRNA tested, miR912 and miR1157, the miRNA strand association with RRP6, is part of a quality control mechanism for showed 70–90% higher accumulation in the mutant strain (relative the degradation of functionally defective small RNAs. to the controls) whereas the miRNA* was barely detectable (Fig. 1C). In contrast, if the MUT68 deletion were to stabilize simulta- Results neously and to the same degree both strands of a miRNA/miRNA* Mut-68 Mutant Shows Enhanced Levels of Small RNAs and of the duplex, clearly detectable amounts of the miRNA* would be ARGONAUTE3 (AGO3) Protein. We have previously reported that expected, given the observed enhancement in miRNA levels. An MUT68 is required for the addition of untemplated nucleotides to analysis of sRNAs by high-throughput sequencing is also con- the 5′ RNA fragments produced by RISC cleavage of target sistent with a lack of an effect of MUT68 on miRNA*s (see below). transcripts and for their efficient decay (23). Interestingly, a Indeed, miR912 and miR1157 appear to be predominantly in a mutant strain, deleted for the cognate gene, also shows higher single-stranded conformation in both Mut-68 and its parental levels of several miRNAs (Fig. 1A and Fig. S1), relative to the wild- strain. When sRNAs were separated by nondenaturating poly- type (CC-124) and the parental transgenic strain (Maa7-IR44). acrylamide gel electrophoresis, with or without prior denaturation Likewise, Mut-68 displays enhanced accumulation of siRNAs of the RNA samples, miRNA/miRNA* duplexes were undetect- produced from inverted repeat transgenes (Fig. S2). These able (Fig. 1D). This is in contrast to the accumulation of nicked changes are paralleled by increased levels of AGO3 (Fig. 1B), one duplex sRNAs caused by depletion of the QIP nuclease (9), which of the three C. reinhardtii Argonaute proteins (4), and this effect is removes AGO-cleaved passenger strands in Neurospora crassa. likely the result of translational or posttranslational regulation Moreover, because no single-stranded guide sRNA seems to be Fig. 1. Mut-68 and strains depleted for the RRP6 exosome subunit show increased levels of miRNAs and of the AGO3 protein. (A) Northern blot analyses of sRNAs isolated from the indicated strains and detected with probes specificforChlamydomonas miRNAs. Cad112, candidate miRNA 112 (31). The numbers below the blots indicate the relative abundance of the miRNAs.
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