pre-piRNA trimming and 2′-O-methylation protect piRNAs from 3′ tailing and degradation in C. elegans

Abstract

SUMMARY The Piwi-interacting RNA (piRNA) pathway suppresses transposable elements and promotes fertility in diverse organisms. Maturation of piRNAs involves pre-piRNA trimming followed by 2′-O-methylation at their 3′ termini. Here, we report that the 3′ termini of Caenorhabditis elegans piRNAs are subject to nontemplated nucleotide addition, and piRNAs with 3′ addition exhibit extensive base-pairing interaction with their target RNAs. Animals deficient for PARN-1 (pre-piRNA trimmer) and HENN-1 (2′-O-methyltransferase) accumulate piRNAs with 3′ nontemplated nucleotides. In henn-1 mutants, piRNAs are shortened prior to 3′ addition, whereas long isoforms of untrimmed piRNAs are preferentially modified in parn-1 mutant animals. Loss of either PARN-1 or HENN-1 results in modest reduction in steady-state levels of piRNAs. Deletion of both enzymes leads to depletion of piRNAs, desilenced piRNA targets, and impaired fecundity. Together, our findings suggest that pre-piRNA trimming and 2′-O-methylation act collaboratively to protect piRNAs from tailing and degradation.

Dual roles for piRNAs in promoting and preventing gene silencing in C. elegans

“…A subset of genes produced highly elevated levels of 22G-RNAs in prg-1 mutants ( Figure 1B ; Table S1 ). We did not observe elevated levels of 22G-RNAs in animals containing a mutation in henn-1 , which also acts within the piRNA pathway but has only a modest impact on 22G-RNA production in the germline ( Figure 1C ; Table S1 ) ( Billi et al, 2012 ; Kamminga et al, 2012 ; Montgomery et al, 2012 ; Pastore et al, 2021 ; Svendsen et al, 2019 ). Nor did we observe elevated levels of 22G-RNAs in mut-16 mutants, in which WAGO-class 22G-RNAs are nearly completely lost, including those that are piRNA-dependent, but which retain CSR-1-class 22G-RNAs ( Figures 1A and S1A ; Table S1 ).…”

Comparative analysis of piRNA sequences, targets and functions in nematodes

“…To further validate authenticity of piRNA genes, we generated a presumptive null allele of Cbr-prg-1 that bears a 5-nt deletion downstream of the start codon and shifts the open reading frame [15]. Consistent with previous findings that the stability of piRNAs and Piwi protein are co-dependent in C. elegans [18, 21, 23, 48], the overall abundance of 21U-RNAs in the Cbr-prg-1 mutant animals was reduced to ∼1% of wild-type (Fig 1C).…”

Inheritance and maintenance of small RNA‐mediated epigenetic effects

“…[15] The RdRP synthesis of 22G-RNAs serves to amplify the silencing signal initiated by siRNAs derived from dsRNAs. [17,18] In C. elegans, there is an extended clade of Argonaute proteins known as Worm-specific Argonautes (WAGOs). The 22G-RNAs produced by the RdRP are loaded into WAGOs, which are the effector of the silencing responses.…”

“…The 22G-RNAs produced by the RdRP are loaded into WAGOs, which are the effector of the silencing responses. [17,18] The WAGOs loaded with 22G-RNAs repress the expression of the mRNA target at the post-transcriptional and the transcriptional level. The transcriptional silencing is usually associated with the deposition of histone post-translational modifications (PTMs) (reviewed in Ref.…”

“…[10,11] In addition, many cellular factors and nucleases can also contribute to degrading small RNAs. [12][13][14][15][16][17] Therefore, organisms might have evolved mechanisms to protect functional small RNAs to ensure their delivery via gametes to the next generation and avoid their degradation and loss. In the current section, we discuss recent advances in the field to elucidate different modes of small RNA transfer employed by animals.…”