The central role of 5′-triphosphate small RNAs in Caenorhabditis elegans.

Small RNA biology in Caenorhabditis elegans occupies a unique position in the RNA world. While early discoveries in this model organism helped define canonical RNA interference, subsequent work revealed a small RNA landscape that differs from that of most multicellular organisms.

In mammals, the majority of small RNAs (including miRNAs, piRNAs, and many siRNAs) carry a 5′-monophosphate, reflecting their generation through Dicer-dependent cleavage or other endonucleolytic processing events. By contrast, C. elegans expresses large and diverse populations of secondary siRNAs, most prominently the so-called 22G-RNAs, that are generated by RNA-dependent RNA polymerases (RdRPs). Because RdRPs synthesize RNA de novo using nucleoside triphosphates, these small RNAs initiate with and retain a 5′-triphosphate. 22G-RNAs are the most abundant small RNAs in this organism, dominating its endogenous small RNA landscape¹.

This blog reviews what has been experimentally established about 5′-triphosphate small RNAs in C. elegans, including their biogenesis, molecular characteristics, and biological roles, and discusses how small RNA sequencing technologies and library preparation strategies have been adapted to accurately capture these non-canonical RNA species.

Biological significance of 5′-triphosphate small RNAs.

The C. elegans small RNA repertoire includes canonical miRNAs, Dicer-derived primary siRNAs, and several endogenous siRNA pathways dominated by RdRP-dependent secondary siRNAs. A widely used and experimentally supported framework describes five major small RNA classes in worms (Table 1).

Table 1. Small RNA classes present in C. elegans.

Deep sequencing analyses have shown that 22G-RNAs constitute the majority of endogenous small RNA reads in wild-type C. elegans, whereas miRNAs and piRNAs represent a minor fraction1,2. These RNAs are synthesized by RdRPs, primarily EGO-1 and RRF-1, which copy RNA templates in an antisense orientation^5,6. Genetic disruption of these RdRPs, as well as associated factors such as DRH-3 and EKL-1, leads to a profound loss of 22G-RNAs across endogenous RNAi pathways, demonstrating that these components are essential for their biogenesis⁷. Because RdRPs initiate RNA synthesis using nucleoside triphosphates, 22G-RNAs are produced with a 5′-triphosphate, a biochemical feature validated through genetic, biochemical, and sequencing-based approaches^6,8.

Functionally, 22G-RNAs act through association with specific Argonaute proteins, defining distinct regulatory pathways. WAGO-family Argonautes bind 22G-RNAs targeting transposons, pseudogenes, and other aberrant sequences, and loss of WAGO pathway components results in derepression of these targets, supporting a role in genome surveillance and transcriptional silencing^1,9. In contrast, a major population of 22G-RNAs associates with the Argonaute CSR-1, which preferentially targets germline-expressed protein-coding genes and is required for proper chromosome segregation and fertility⁵. A key experimentally supported feature of 22G-RNA biology is that they are produced through a signal-amplifying RNAi pathway. Secondary siRNA production is initiated downstream of defined triggering events (such as 26G endo-siRNAs generated by RdRP/Dicer pathways or piRNA-mediated targeting) and results in the synthesis of large numbers of secondary 22G-RNAs from individual target transcripts^7,10.

Best practices to sequence 5′-triphosphate small RNAs.

Most small RNA sequencing protocols rely on sequential ligation of adapters to the 3′ and 5′ ends of RNA molecules using T4 RNA ligases. These enzymes require a 5′-monophosphate and are inefficient or inactive on RNAs bearing a 5′-triphosphate. Therefore, untreated C. elegans RNA samples yield libraries that are heavily biased toward microRNAs and other monophosphorylated species, while largely excluding 22G-RNAs.

To enable efficient sequencing of 5′-triphosphate small RNAs, enzymatic pretreatment steps are now routinely incorporated. RNA 5′ pyrophosphohydrolase (RppH) is the most widely used enzyme for this purpose, as it removes the β and γ phosphates from 5′-triphosphate RNAs, converting them into 5′-monophosphates compatible with adapter ligation. Experimental comparisons showed that RppH treatment selectively enriched for 22-nt RNAs with the characteristic 5′-G bias of 22G-RNAs without distorting the overall small RNA composition^8,11. As a result, RppH treatment has become a standard best practice for comprehensive profiling of RdRP-derived secondary siRNAs. RppH treatment efficacy is assessed by the appearance of a ~22 nt, 5′-G–biased antisense small RNA population, absent in –RppH controls.

The same enzymatic conversion and ligation-based sequencing workflows can be in theory applied to any context in which 5′-triphosphate RNAs are expected, including in vitro–transcribed short RNA molecules, viral RNAs, or synthetic RNAs generated by phage RNA polymerases.