Analysis of C. elegans Germline Small RNA Pathways.

Sequence-specific gene regulation by small RNA (sRNA) pathways is essential for the development and function of organisms in all domains of life. These regulatory complexes, containing an Argonaute protein (AGO) guided by a bound sRNA, have the potential to regulate thousands of individual target transcripts at both the co- and post-transcriptional level. Determining the repertoire of transcripts that an AGO is capable of regulating in a particular context is essential to understanding the function of these regulatory modules. Immunoprecipitation (IP) of AGOs and subsequent RNA sequencing of their bound sRNAs allows for the inference of their target transcripts by mapping the sequences of the co-precipitated sRNAs back to their complementary target transcripts. This approach can be complemented by sequencing sRNAs from ago mutants as sRNA transcripts are degraded in the absence of their AGO binding partner. Here, we describe a framework for analyzing AGO/sRNA pathways in the germline, from using CRISPR-Cas9 to tag or mutate AGOs, through protocols for the extraction, sequencing, and analysis of sRNAs from AGO IPs and ago mutants.

PIWI puts spermatogenesis in its place.

In this issue of Developmental Cell, Cornes et al. show that piRNAs initiate transcriptional silencing of spermatogenesis genes in the C. elegans germline via an endogenous nuclear RNAi pathway. This silencing enables a timely transition from spermatogenesis to oogenesis during hermaphrodite development, thus promoting fertility.

Two isoforms of the essential C. elegans Argonaute CSR-1 differentially regulate sperm and oocyte fertility.

The Caenorhabditis elegans genome encodes nineteen functional Argonaute proteins that use 22G-RNAs, 26G-RNAs, miRNAs or piRNAs to regulate target transcripts. Only one Argonaute is essential under normal laboratory conditions: CSR-1. While CSR-1 has been studied widely, nearly all studies have overlooked the fact that the csr-1 locus encodes two isoforms. These isoforms differ by an additional 163 amino acids present in the N-terminus of CSR-1a. Using CRISPR-Cas9 genome editing to introduce GFP::3xFLAG into the long (CSR-1a) and short (CSR-1b) isoforms, we found that CSR-1a is expressed during spermatogenesis and in several somatic tissues, including the intestine. CSR-1b is expressed constitutively in the germline. small RNA sequencing of CSR-1 complexes shows that they interact with partly overlapping sets of 22G-RNAs. Phenotypic analyses reveal that the essential functions of csr-1 described in the literature coincide with CSR-1b, while CSR-1a plays tissue specific functions. During spermatogenesis, CSR-1a integrates into an sRNA regulatory network including ALG-3, ALG-4 and WAGO-10 that is necessary for fertility at 25°C. In the intestine, CSR-1a silences immunity and pathogen-responsive genes, and its loss results in improved survival from the pathogen Pseudomonas aeruginosa. Our findings functionally distinguish the CSR-1 isoforms and highlight the importance of studying each AGO isoform independently.

Tree shape-based approaches for the comparative study of cophylogeny.

Cophylogeny is the congruence of phylogenetic relationships between two different groups of organisms due to their long-term interaction. We investigated the use of tree shape distance measures to quantify the degree of cophylogeny. We implemented a reverse-time simulation model of pathogen phylogenies within a fixed host tree, given cospeciation probability, host switching, and pathogen speciation rates. We used this model to evaluate 18 distance measures between host and pathogen trees including two kernel distances that we developed for labeled and unlabeled trees, which use branch lengths and accommodate different size trees. Finally, we used these measures to revisit published cophylogenetic studies, where authors described the observed associations as representing a high or low degree of cophylogeny. Our simulations demonstrated that some measures are more informative than others with respect to specific coevolution parameters especially when these did not assume extreme values. For real datasets, trees' associations projection revealed clustering of high concordance studies suggesting that investigators are describing it in a consistent way. Our results support the hypothesis that measures can be useful for quantifying cophylogeny. This motivates their usage in the field of coevolution and supports the development of simulation-based methods, i.e., approximate Bayesian computation, to estimate the underlying coevolutionary parameters.