Biogenesis and Stability of Germline Small RNAs in C. elegans.

Abstract

Across the animal kingdom, small, noncoding RNAs preserve and promote fertility by engaging Argonaute effector proteins to silence deleterious genetic elements. Generated in germline and inherited into progeny, endogenous small interfering RNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs) regulate vast suites of gametic and zygotic genes, yet remarkably little is known about how they are regulated. With an expanded repertoire of small RNA classes, Caenorhabditis elegans provides an ideal model for investigating how animals drive epigenetic inheritance of fertility-preserving germline small RNAs.

The conserved methyltransferase HEN1 methylates small RNAs to prevent their degradation. Methylation of germline small RNAs enhances accumulation, promoting robust inheritance into progeny. All plant small RNAs are methylated, but animal HEN1 methylates only some small RNAs. The mechanisms of selective methylation were unknown. I identified the functional C. elegans ortholog of HEN1 and demonstrated that it methylates all piRNAs but only select subclasses of endo-siRNAs. I further found that particular endo-siRNAs are methylated in maternal, but not paternal, germlines. Through genetic and biochemical analyses, I showed that small RNA methylation status is likely dictated by the associated Argonaute. This established selective expression of divergent Argonautes as a novel mechanism for differentially stabilizing germline small RNAs, with significant implications for preferential inheritance of maternal epigenetic information.

piRNAs are essential for animal fertility, but their expression mechanisms are poorly characterized. In collaboration with bioinformatician Mallory Freeberg, I showed that C. elegans male and female germlines express distinct piRNA subsets that evolve independently and differ in inheritance. A common sequence motif lies upstream of nematode piRNA loci. We discovered that this motif varies significantly between male and female piRNAs. Using a novel transgenic approach, I established that C. elegans piRNAs represent thousands of tiny, autonomous transcriptional units, rivaling coding genes in number. I further demonstrated that the upstream motif is required for piRNA expression and that variation at a single nucleotide position within this motif orchestrates selective male versus female germline enrichment and inheritance of piRNAs.

These and additional included studies define novel factors and mechanisms involved in regulation of germline small RNAs and transgenerational transmission of their crucial epigenetic information.