Argonautes ALG-3 and ALG-4 are required for spermatogenesis-specific 26G-RNAs and thermotolerant sperm in Caenorhabditis elegans

Abstract

Gametogenesis is a thermosensitive process in numerous metazoans, ranging from worms to man. In Caenorhabditis elegans, a variety of RNA-binding proteins that associate with germ-line nuage (P granules), including the Piwi-clade argonaute PRG-1, have been implicated in maintaining fertility at elevated temperature. Here we describe the role of two AGO-class paralogs, alg-3 (T22B3.2) and alg-4 (ZK757.3), in promoting thermotolerant male fertility. A rescuing GFP::alg-3 transgene is localized to P granules beginning at the late pachytene stage of male gametogenesis. alg-3/4 double mutants lack a subgroup of small RNAs, the 26G-RNAs which target and appear to down-regulate numerous spermatogenesis-expressed mRNAs. These findings add to a growing number of AGO pathways required for thermotolerant fertility in C. elegans and support a model in which AGOs and their small RNA cofactors function to promote robustness in gene-expression networks. Argonautes can be classified into three clades (2): (i) the AGO clade, which includes the human AGOs1-4, the Caenorhabditis elegans miRNA effectors ALG1/2, and all of the Arabidopsis thaliana AGOs; (ii) the Piwi clade, which more closely resembles Drosophila Piwi and includes the C. elegans piRNA AGO PRG-1; and (iii) an expanded family of worm-specific AGOs (WAGOs) that, with a few exceptions, lack residues in the PIWI domain thought to be necessary for slicer endonuclease activity.In C. elegans, null mutations are available for the entire family of over 24 AGO genes (3), and at least five different combinations of these mutants result in lethal or sterile phenotypes. alg-1/2 mutants display heterochronic defects and lethality that arise from loss of miRNA-mediated regulation of developmentally important transcripts (4). csr-1 mutants have severe chromosome segregation defects that result in embryonic lethality (3, 5), and prg-1 mutants have severe defects in the development of the germline (6-8). In addition, the simultaneous deletion of 12 WAGOs leads to a temperature-sensitive sterile phenotype, where mutants are viable and fertile at 20°C but sterile at 25°C (9).Here we describe two paralogous AGO-clade family members, alg-3 and alg-4, that are together critical for sperm development and function. Single mutants have only a minor reduction in brood size, but double alg-3/4 mutants exhibit drastically reduced brood sizes at elevated temperatures. We show that a rescuing GFP::ALG-3 protein is expressed in germ cells undergoing spermatogenesis and that, even though both male and hermaphrodite alg-3/4 mutants produce near wild-type numbers of spermatids, these spermatids exhibit severe defects in the activation process called spermiogenesis that converts spermatids into motile ameboid sperm.A class of 26nt small RNAs called the 26G-RNAs were first identified in deep-sequencing datasets in C. elegans as part of the previously described Dicer-ribonuclease-dependent endogenous small RNA pathway, called the ERI pathway, and a subset of these were noted to be enriched for spermatogen...

High-throughput sequencing reveals extraordinary fluidity of miRNA, piRNA, and siRNA pathways in nematodes

“…One class is enriched in oocytes and embryos and associates with ERGO-1 (Han et al 2009;Vasale et al 2010;Fischer et al 2011). The other class associates with ALG-3 and ALG-4 during spermatogenesis (Han et al 2009;Conine et al 2010). Both classes are thought to function by triggering 22G siRNA formation and subsequent silencing of target mRNAs.…”

“…CSR-1 class was defined as siRNAs produced from the 4191 genes that yield siRNAs enriched in CSR-1 immunoprecipitates ). ALG-3/4 (401 genes) class and ERGO-1 (75 genes) class were defined as 26G siRNAs enriched during spermatogenesis and oogenesis/embryogenesis, respectively (Han et al 2009;Conine et al 2010;Vasale et al 2010). miRNAs were predicted using miRDeep2 with default settings (Mackowiak 2011;Friedlander et al 2012).…”

“…The 26G siRNAs fall into two classes: a spermatogenesisenriched class which associates with the AGO clade Argonautes, ALG-3 and ALG-4 (Han et al 2009;Conine et al 2010); and an oocyte-and embryo-enriched class which associates with the divergent PIWI-clade Argonaute, ERGO-1 (Han et al 2009;Vasale et al 2010;Fischer et al 2011). Both classes of 26G siRNAs are produced by the RdRP RRF-3 and are thought to trigger secondary 22G siRNA production.…”

Translational Control in the Caenorhabditis elegans Germ Line

“…The two classes of 26G RNAs are suggested to be sorted into distinct RISC complexes, based on the observation that different Ago-proteins are essential for the presence of the different classes of 26G-RNAs in the worm. Whereas ERGO-1 is important for class II, the abundance of class I depends highly on the two paralogs ALG-3 and ALG-4 (Han et al 2009 ;Conine et al 2010 ) . The functional relevance of class II 26G-RNA remains to be demonstrated, but class I 26G-RNAs are linked to gene regulation during spermatogenesis.…”

“…Another group of noncoding RNAs with germline functions are the endogenously produced small interfering RNAs, called 26G-RNAs (Han et al 2009 ;Conine et al 2010 ) . This group of endo-siRNAs is 26 nt in length, preferentially starts with guanine, is exclusively present in the germ line, and shows perfect complementarity to their target transcripts.…”