Reproducible features of small RNAs in C. elegans reveal NU RNAs and provide insights into 22G RNAs and 26G RNAs

Abstract

Small RNAs regulate gene expression and most genes in the worm Caenorhabditis elegans are subject to their regulation. Here, we analyze small RNA data sets and use reproducible features of RNAs present in multiple data sets to discover a new class of small RNAs and to reveal insights into two known classes of small RNAs-22G RNAs and 26G RNAs. We found that reproducibly detected 22-nt RNAs, although are predominantly RNAs with a G at the 5 ′ end, also include RNAs with A, C, or U at the 5 ′ end. These RNAs are synthesized downstream from characteristic sequence motifs on mRNA and have U-tailed derivatives. Analysis of 26G RNAs revealed that they are processed from a blunt end of double-stranded RNAs and that production of one 26G RNA generates a hotspot immediately downstream for production of another. To our surprise, analysis of RNAs shorter than 18 nt revealed a new class of RNAs, which we call NU RNAs (pronounced "new RNAs") because they have a NU bias at the 5 ′ end, where N is any nucleotide. NU RNAs are antisense to genes and originate downstream from U bases on mRNA. Although many genes have complementary NU RNAs, their genome-wide distribution is distinct from that of previously known classes of small RNAs. Our results suggest that current approaches underestimate reproducibly detected RNAs that are shorter than 18 nt, and theoretical considerations suggest that such shorter RNAs could be used for sequence-specific gene regulation in organisms like C. elegans that have small genomes.

Neuronal Small RNAs Control Behavior Transgenerationally

“…We next filtered for small RNA reads of 20-23 nucleotide in length ( Blumenfeld and Jose, 2016 ). Then we counted reads in antisense orientation to genes as defined by the corresponding Ensembl .gff file, using a python based script HTSeq count ( Anders et al., 2015 ): HTSeq.scripts.count–stranded = reverse–minaqual = 0–mode = intersection-nonempty input.sam GENES.gff > output.txt …”

“…Endo-siRNAs target both protein-coding and non-protein coding loci ( Gu et al., 2009 , Vasale et al., 2010 ). Endo-siRNAs align in the antisense orientation to exons, can tile the entire length of the mature mRNA transcript, and complement the target perfectly ( Blumenfeld and Jose, 2016 ). For simplicity, units of small RNAs targeting a specific gene, will be referred to here as STGs (see Rechavi et al.…”

C. elegans ADARs antagonize silencing of cellular dsRNAs by the antiviral RNAi pathway

“…While such products arise during viral infection, they have not been observed among endo-siRNAs in wild-type C. elegans (Ruby et al 2006;Ashe et al 2013;Billi et al 2014). Rather, characterized DCR-1-dependent endo-siRNAs are 26 nt, have a 5 ′ guanosine monophosphate (26G siRNAs), and are produced by DCR-1 acting in concert with the RNA-dependent RNA polymerase (RdRP) RRF-3 (Thivierge et al 2012;Blumenfeld and Jose 2016). 26G siRNAs occur in embryos and germline tissues and exist in two classes bound to distinct Argonaute proteins.…”

Each cell needs long double-stranded RNA for silencing by feeding RNAi inC. elegans

“…We found that loss of rrf-3 or of eri-1 but not of ergo-1 eliminated inhibition of feeding RNAi by expression from a repetitive transgene within the hypodermis ( Figure 4I). Because RRF-3 and ERI-1 are not required for the production of dsRNA from repetitive transgenes (Kim et al 2005), these results suggest that silencing by feeding RNAi in the presence of expression from a repetitive transgene is enabled by loss of dsRNA production at endogenous loci (see Figure S9 in Blumenfeld and Jose 2016).…”