ALG-5 is a miRNA-associated Argonaute required for proper developmental timing in the Caenorhabditis elegans germline

Abstract

Caenorhabditis elegans contains 25 Argonautes, of which, ALG-1 and ALG-2 are known to primarily interact with miRNAs. ALG-5 belongs to the AGO subfamily of Argonautes that includes ALG-1 and ALG-2, but its role in small RNA pathways is unknown. We analyzed by high-throughput sequencing the small RNAs associated with ALG-5, ALG-1 and ALG-2, as well as changes in mRNA expression in alg-5, alg-1 and alg-2 mutants. We show that ALG-5 defines a distinct branch of the miRNA pathway affecting the expression of genes involved in immunity, defense, and development. In contrast to ALG-1 and ALG-2, which associate with most miRNAs and have general roles throughout development, ALG-5 interacts with only a small subset of miRNAs and is specifically expressed in the germline where it localizes alongside the piRNA and siRNA machinery at P granules. alg-5 is required for optimal fertility and mutations in alg-5 lead to a precocious transition from spermatogenesis to oogenesis. Our results provide a near-comprehensive analysis of miRNA-Argonaute interactions in C. elegans and reveal a new role for miRNAs in the germline.

Unravelling the developmental and functional significance of an ancient Argonaute duplication

“…For example, vertebrate AGO2, but not the others, contains an active catalytic domain that enables to promote slicing of fully complement targets 36 and to enable non-canonical maturation of some miRNAs [37][38][39] . In C. elegans, Alg5 was shown to carry some unique miRNAs that are not carried by the other two miRNA-specific AGOs of this species 40 . In this case, the specificity is enabled via tissue specific co-expression of these particular miRNAs with Alg5 in the germline.…”

miRNA activity contributes to accurate RNA splicing inC. elegansintestine and body muscle tissues

“…Student t-test *p<0.05 **p<0.01. (B) A comparison of the splice junction usage in unc-60 as observed in transcriptome data for alg-1 and alg-2 knockout strains (Brown et al 2017). The numbers above each slice junction indicates the number of reads mapped to that splice junction.…”

“…We downloaded from the GEO database the following transcriptome datasets published by Brown et al, 2017 (Brown et al 2017): Project number GSE98935, Wild type Rep 1-3 (GSM2628055, GSM2628056, GSM2628057); alg-1 ( gk214 ) Rep 1-3 (GSM2628061, GSM2628062, GSM2628063); alg-2 ( ok304 ) Rep 1-3 (GSM2628064, GSM2628065, GSM2628066). We used in-house Perl scripts to prepare the reads for mapping, and then these reads as input to the TopHat algorithm (Trapnell et al 2012) to map splice junctions in all nine datasets independently.…”

“…Based on these algorithms it was initially predicted that each miRNA controls hundreds of gene products (Chen and Rajewsky 2007). Recent high-throughput wet bench approaches, have validated and expanded on these initial predictions, and provide further evidence that miRNAs can indeed target hundreds of genes, and regulate molecular pathways throughout development and in diseases (Selbach et al 2008; Helwak et al 2013; Wolter et al 2014; Brown et al 2017; Wolter et al 2017).…”

“…In C. elegans there are three known Argonaute proteins that execute the miRNA pathway, and are named alg-1 , alg-2 and alg-5 . A recent transcriptome analysis in strains deficient in each of these members show a remarkable difference in function, where alg-1 and alg-2 are mostly expressed in somatic tissue and are functionally redundant, and alg-5 is expressed exclusively in the gonads, interacts with only a subset of miRNAs and is required for optimal fertility (Brown et al 2017). A more recent study used a novel methylation-dependent sequencing approach (mime-Seq), and identified high quality tissue-specific miRNAs in intestine and body muscle tissues (Alberti et al 2018).…”