Uridylation of mature miRNAs and siRNAs by the MUT68 nucleotidyltransferase promotes their degradation in Chlamydomonas

Abstract

Regulation of gene expression by small RNAs ( approximately 20-30 nucleotides in length) plays an essential role in developmental pathways and defense responses against genomic parasites in eukaryotes. MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) commonly direct the inactivation of cognate sequences through a variety of mechanisms, including RNA degradation, translation inhibition, and transcriptional repression. Recent studies have provided considerable insight into the biogenesis and the mode of action of miRNAs and siRNAs. However, relatively little is known about mechanisms of quality control and small RNA decay in RNA interference (RNAi) pathways. Here we show that deletion of MUT68, encoding a terminal nucleotidyltransferase in the alga Chlamydomonas reinhardtii, results in elevated miRNA and siRNA levels. We found that MUT68 plays a role in the untemplated uridylation of the 3' ends of small RNAs in vivo and stimulates their degradation by the RRP6 exosome subunit in vitro. Moreover, RRP6 depletion also leads to accumulation of small RNAs in vivo. We propose that MUT68 and RRP6 cooperate in the degradation of mature miRNAs and siRNAs, as a quality control mechanism to eliminate dysfunctional or damaged small RNA molecules.

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 specific for Chlamydomonas miRNAs. Cad112, candidate miRNA 112 (31). The numbers below the blots indicate the relative abundance of the miRNAs. CC-124, wild-type strain; Maa7-IR44, CC-124 transformed with an IR transgene designed to induce RNAi of MAA7 (encoding tryptophan synthase β subunit); Mut-68, MUT68 deletion mutant; Rrp6-IR5 and Rrp6-IR11, Maa7-IR44 transformed with IR transgenes inducing RNAi of two distinct genes encoding the RRP6 exosome subunit; Csl4-IR2, strain transformed with an IR transgene triggering RNAi of CSL4 (encoding a core exosome subunit). (B) Immunoblot analysis of AGO3 protein levels. The specificity of the indicated AGO3 band was verified by peptide blocking assays. The asterisk shows a cross-reacting antigen. Coomassie-blue staining of an equivalent gel is shown as a control for similar loading of the lanes (Right). (C) Northern blot analysis of sRNAs isolated from the indicated strains and detected using probes specific for the guide strand (miRNA) or the passenger strand (miRNA*) of Chlamydomonas miR912 or miR1157. Control hybridizations to synthetic oligonucleotides are shown on the Right of each panel. (D) Northern blot analysis of sRNAs from the indicated strains separated by nondenaturing polyacrylamide gel electrophoresis. Isolated RNA was resuspended in a nondenaturing buffer (undenatured) or in formamide and denatured by heating (denatured) before loading the gel. The asterisks indicate uncharacterized RNA forms that are present in both Maa7-IR44 and Mut-68 although, due to reduced sample loading, they are less noticeable in the Mut-68 lanes. Because the intensity of these bands was not affected by denaturation, they do not appear to correspond to dsRNA. RNAi null, uncharacterized Chlamydomonas mutant lacking miRNAs. Synthetic oligoRNAs corresponding to miR912 and its miRNA* were used to demonstrate that an annealed duplex is stable under our extraction and electrophoretic conditions (Right). Annealed dsRNAs were resuspended in formamide and denatured by heating (single-stranded lane) or resuspended in nondenaturing buffer (duplex lane) before loading on the same gel as for the miR912 samples.

Fig. 2.

MUT68 acts as a terminal nucleotidyltransferase and promotes the degradation of oligoribonucleotides by the RRP6 exosome subunit in vitro. (A) Recombinant MUT68 was incubated with a ³²P-labeled oligoRNA and the indicated nucleotide triphosphates for 5 or 20 min. Products were separated on a denaturing polyacrylamide/urea gel and analyzed by autoradiography. Controls included the omission of RNA, protein, or nucleotides as well as the substitution of MUT68 for the catalytically inactive MUT68(DADQ). (B) MUT68 activity, in the presence of UTP, on synthetic oligoRNAs corresponding in sequence to miR912, either unmodified or 2′-O-methylated on the 3′ terminal ribose. (C) 5′-end-labeled C₁₅A₁₀ oligoRNA was incubated with affinity-purified RRP6 alone (buffer) or with the addition of MUT68 or the catalytically inactive MUT68(DADQ). Reactions were stopped at the indicated times, separated by denaturing PAGE, and analyzed by autoradiography. (D) Enzymatic activity of RRP6, RRP6/MUT68, or RRP6/MUT68(DADQ) on an unmodified miR912 oligoRNA. (E) Enzymatic activity of RRP6, RRP6/MUT68, or RRP6/MUT68(DADQ) on miR912 with a 3′ terminal 2′-O-methyl group.

Fig. 3.

Analysis of endogenous small RNA sizes and their 3′ untemplated nucleotide additions in Mut-68 and the parental strain Maa7-IR44. (A) Size distribution of redundant small RNAs, after subtracting putative degradation products and normalization. Two independent libraries were examined for each strain. Redundant sRNA sequences from Maa7-IR44 are represented by black (Maa7-IR44-1) or gray (Maa7-IR44-2) bars. Redundant sRNA sequences from Mut-68 are represented by red (Mut-68-1) or yellow (Mut-68-2) bars. (B) Frequency of the predominant untemplated nucleotides added to the 3′ ends of small RNAs (relative to all sRNAs with 3′ nucleotide additions). (C) Size distribution of redundant small RNAs with an untemplated 3′ terminal U or UU (after removing the tails). Two independent libraries were examined for each strain and the bars are colored as indicated above (A).

Fig. 4.

Proposed model for the role of MUT68 and RRP6 in the degradation of mature miRNAs and siRNAs (see text for details). (A) MUT68/RRP6 may eliminate dysfunctional or subfunctional guide small RNAs in kinetic competition with the methyltransferase HEN1 (after cleavage and dissociation of the passenger strand during RISC loading of siRNAs or perfectly complementary miRNA duplexes). (B) MUT68/RRP6 may also degrade damaged guide sRNAs during the multiple cycles of RISC activity.