Small interfering RNAs regulate gene expression and map to many genes in C. elegans. The most abundant of these small RNAs are generated by RNA-dependent RNA polymerases (RdRPs) and have a 5'-triphosphate. Many labs have used a 5'-monophosphate-independent ligation of 18-nt to 28-nt RNAs followed by RNA-Seq to identify these RNAs, which are typically called "22G RNAs" because most of them are 22-nt long and begin with a 5' G. However, it remains unclear if such RNAs form a single class and if the sets of RNAs identified by different labs differ in their composition. Here, we analyze RNA-Seq data from multiple labs to discover reliable features of abundant small RNAs in C. elegans. We find that these datasets have a characteristic and reproducible distribution of 15-nt to 26-nt RNA. These reproducibly detected RNAs are derived from at least two distinct classes: 20-nt to 22-nt RNAs (class A) and 15-nt and longer RNAs that are found as 1-nt-staggered clusters (class B). The 5' end of class A RNAs are associated with a YGW motif where G is the 5'-nt of the RNA, Y = C or U, and W = A or U. Furthermore, RNAs with a 5' A or U have a strong preference for an upstream C and RNAs with a 5' C have a strong preference for an upstream U. Consistent with earlier proposals, these biases associated with class A RNAs likely reflect the requirements for initiation of synthesis by RdRPs. Class B RNAs are independent of all known RdRPs and their 5' ends have no detectable nucleotide bias. Preliminary analyses suggest that class A and class B RNAs originate from distinct sets of genes. The reproducible detection of RNAs that are as short as 15-nt suggests that current methods that typically select for RNAs longer than 18-nt underestimate stable and potentially functional shorter RNAs. Given the size of the C. elegans genome, sequences as small as 13-nt can potentially be used for sequence-specific regulation and even shorter RNAs may be used for gene regulation if a meaningful fraction of the genome (e.g. the exome) is selected for targeting through other mechanisms.

Affiliation:
- Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742