Small RNA in situ hybridization in Caenorhabditis elegans, combined with RNA-seq, identifies germline-enriched microRNAs

Abstract

Over four hundred different microRNAs (miRNAs) have been identified in the genome of the model organism the nematode Caenorhabditis elegans. As the germline is dedicated to the preservation of each species, and almost half of all the cells in an adult nematode are germline, it is likely that regulatory miRNAs are important for germline development and maintenance. In C. elegans the miR35 family has strong maternal effects, contributing to normal embryogenesis and to adult fecundity. To determine whether any particular miRNAs are greatly enriched in the C. elegans germline we used RNA-seq to compare the miRNA populations in several germline-defective strains of adult C. elegans worms, including glp-4(germline proliferation-4), glh-1(germline helicase-1) and dcr-1(dicer-1). Statistical analyses of RNA-seq comparisons identified 13 miRNAs that are germline-enriched, including seven members of the well-studied miR35 family that were reduced as much as 1000-fold in TaqMan qRT PCR miRNA assays. Along with the miR35s, six others: miR-56 (a member of the miR51 family),−70, −244, −260 , −788 and −4813, none of which previously considered as such, were also identified by RNA-seq as germline-enriched candidates. We went on to develop a successful miRNA in situ hybridization protocol for C. elegans, revealing miR35s specifically concentrate during oogenesis in the pachytene region of the gonad, and persist throughout early embryogenesis, while in adult animals neither let-7 nor miR-228 has a germline-bias.

Tissue- and sex-specific small RNAomes reveal sex differences in response to the environment

“…In addition, miRNAs expressed in the C. elegans somatic gonad maintain germline proliferation and differentiation [41]. We identified female germline-biased expression of the miR35-41 cluster in isolated gonads, which is in line with studies conducted on whole animals [27,29,70].…”

“…Many miRNAs are expressed in a tissue-and sex-specific specific manner reflecting functional sex-differences [23–26]. For instance, miR-35 family activity in the female germline is important for progeny viability and fecundity [27–29]. Furthermore, distinct Argonaute proteins associate with additional types of endogenous siRNAs in the male and female germline, suggesting functional sex-differences [19,30–34].…”

Functional analysis of microRNA pathway genes in the somatic gonad and germ cells during ovulation in C. elegans

“…miRNAs are found in germ cells across the animal kingdom including worms, flies, mice, and cows (Gilchrist et al, 2016; Gu et al, 2009; Lee et al, 2014; Ma et al, 2010; McEwen et al, 2016). In worms, the mir-35 family is one of several miRNA families found to be highly enriched in germ cells, specifically in oocytes (Gu et al, 2009; McEwen et al, 2016).…”

“…In worms, the mir-35 family is one of several miRNA families found to be highly enriched in germ cells, specifically in oocytes (Gu et al, 2009; McEwen et al, 2016). Like other miRNAs that are expressed in oocytes, the mir-35 family is required for embryonic development but has no known function in oocyte formation or maturation (McJunkin and Ambros, 2014).…”

“…In worms, Dicer is phosphorylated and localized to the nucleus during most of oogenesis in worms, thereby preventing its normal cytoplasmic processing role (Drake et al, 2014). However, miRNA biogenesis likely functions at an early stage of germ cell development because mature, processed miRNAs are present in oocytes (Gu et al, 2009; McEwen et al, 2016). It is clear that some maternal miRNAs that are present in oocytes, including the miR-35 family, are essential for early development (Alvarez-Saavedra and Horvitz, 2010).…”