CDE-1 suppresses the production of risiRNA by coupling polyuridylation and degradation of 26S rRNA

Antisense ribosomal siRNAs (risiRNAs) downregulate pre-rRNAs through the nuclear RNAi pathway in Caenorhabditis elegans. However, the biogenesis and regulation of risiRNAs remain obscure. Previously, we showed that 26S rRNAs are uridylated at the 3’-ends by an unknown terminal polyuridylation polymerase before the rRNAs are degraded by a 3’ to 5’ exoribonuclease SUSI-1(ceDIS3L2). There are three polyuridylation polymerases, CDE-1, PUP-2, and PUP-3, in C. elegans. Here, we found that CDE-1 is specifically involved in suppressing risiRNA production. CDE-1 localizes to perinuclear granules in the germline and uridylates both Argonaute-associated 22G-RNAs and 26S rRNAs at the 3’-ends. Immunoprecipitation followed by mass spectrometry (IP-MS) revealed that CDE-1 interacts with SUSI-1(ceDIS3L2). Consistent with those results, both CDE-1 and SUSI-1(ceDIS3L2) are required for the inheritance of RNAi. Therefore, this work identified a rRNA surveillance machinery of rRNAs that couples terminal polyuridylation and degradation.

single copy transgene CDE-1::mCherry by MosSCI technology. This transgene was 104 able to rescue the cde-1(tm936) defects and redistribute NRDE-3 from the nucleus to 105 the cytoplasm ( Figure S1A). To exclude the possibility that PUP-2 and PUP-3 act 106 redundantly to suppress siRNA generation, we generated pup-2;pup-3 double mutants. 107 In the double mutants, NRDE-3 still accumulated in the cytoplasm ( Figure S1B). These 108 data suggest that NRDE-3 was bound to newly generated siRNAs in cde-1 mutants.

CDE-1 is involved in uridylation of 26S rRNA
Previously we showed that SUSi-1 degrades oligouridylated rRNAs and suppresses 196 the production of risiRNA (Zhou et al., 2017b). To test whether CDE-1 uridylates 197 rRNAs, we used a 3' tail-seq assay to examine whether rRNA that was oligouridylated 198 at 3'-tail was depleted in cde-1(tm936) animals ( Figures 4A and 4B). Total RNA was 199 isolated from embryos and L3-staged control and cde-1 animals, ligated to a barcoded 200 DNA linker and reverse transcribed with a primer complementary to the linker.

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Libraries were then prepared by PCR with a 26S rRNA primer and a primer targeting 202 the linker. Illumina adaptor sequences were subsequently added, which was followed 203 by a number of PCR cycles and high-throughput sequencing.

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The 3'-end of 26S rRNA was extensively modified by all four nucleotides 206 compared to the annotated rRNA sequence (Zhou et al., 2017). Only a small fraction of 207 the 3'-end exactly matched to the annotated 26S rRNA from the Wormbase WS250 208 assembly. Although we did not detect a dramatic change in the nontemplated addition 209 of a single nucleotide, we observed a modest depletion of oligouridylation at the 3'-tail 210 of 26S rRNA, comparing cde-1(tm936) to control animals ( Figure 4B). The 211 introduction of the CDE-1::mCherry transgene can rescue this oligouridylation defect. 212 We conclude that CDE-1 is involved in uridylating rRNAs.

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Since CDE-1 interacts with SUSI-1(ceDIS3L2), we tested whether susi-1 was also 218 required for the inheritance of RNAi. We used a germline-expressed mex-219 5p::GFP::H2B (abbreviated as GFP::H2B) transgene as a reporter, which can inherit 220 RNAi-induced gene silencing for multiple generations. Both hrde-1 and cde-1 were not 221 required for exogenous gfp dsRNA to silence the GFP::H2B transgene in the parental 222 generation, but they were essential for silencing in the F1 generation ( Figures 5A and  5B). Similarly, susi-1(ceDis3L2) was not required for exogenous gfp dsRNA to silence 224 the GFP::H2B transgene in the P0 generation, but was necessary for silencing in F1 225 progeny. We conclude that susi-1(ceDis3L2) is required for the inheritance of RNAi.   However, the typical consequence of uridylation is accelerating the RNA degradation 249 (Pirouz, Du et al., 2016, Ustianenko et al., 2016. In this work, we showed that CDE-1 250 can uridylate 26S rRNAs and recruit the 3'-5' exoribonuclease SUSI-1(ceDIS3L2), 251 which may further promote the degradation of oligouridylated rRNAs. In the absence 252 of either CDE-1 or SUSI-1(ceDIS3L2), erroneous rRNAs will accumulate in cells, 253 which thereafter recruit the RNA-dependent RNA polymerases, including RRF-1 and 254 RRF-2, to initiate risiRNA production ( Figure 6). risiRNAs then bind to both nuclear 255 and cytoplasmic Argonaute proteins and silence rRNAs through both nuclear and 256 cytoplasmic RNAi machinery. Therefore, risiRNA and the RNAi machinery, together 257 with exoribonucleases, act to avoid the accumulation of potentially harmful or 258 unnecessary erroneous rRNA transcripts (Henras et al., 2015, Houseley et al., 2006, 259 Karbstein, 2013, Lafontaine, 2010, Pena, Hurt et al., 2017, Schmidt & Butler, 2013 Vanacova & Stefl, 2007). underlying mechanism remains unclear. Here, we found that CDE-1 interacted with SUSI-1, another protein required for the inheritance of RNAi. Further elucidating the 284 function of SUSI-1 and CDE-1 will shed light on the mechanism of RNAi inheritance. Bistol strain N2 was used as the standard wild-type strain. All strains were grown 289 at 20°C unless otherwise specified. The strains used in this study were listed in 290 supplementary Table S1.  Table S2. eft-3 mRNA was used as an internal control for sample normalization.

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Data analysis was performed using a comparative threshold cycle (ΔΔCT) approach.    The Illumina-generated raw reads were first filtered to remove adaptors, low-378 quality tags and contaminants to obtain clean reads by Novogene. Clean reads ranging 379 from 18 to 30 nt were mapped to the transcriptome assembly WS243 using Bowtie2 Cocktail, 10 mM NaF, and 2 mM Na3VO4), and lysed in a FastPrep-24 5G homogenizer.

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The lysate supernatant was incubated with anti-GFP antibody, which was linked to 393 beads, for one hour at 4 ℃. The beads were then washed three times with cold lysis