PIWI proteins and PIWI-interacting small RNAs guard the genomic integrity of germ cells

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Small non-coding RNAs play crucial roles in development and disease. Globally referred to as RNA interference (RNAi), conserved small RNA pathways operate from yeast to human. They regulate gene expression, defend against viruses and control repetitive genetic elements. Additionally, their elegant mechanisms are widely harnessed for biotechnology and targeted therapy. Our group studies a particular class of small RNAs that represses transposon activity in the germline. The ability of transposons to mobilize and insert into new genomic locations threatens genomic integrity and must be suppressed. Integrity of genomic information is particularly important in germ cells that determine the genetic make-up of a species. Therefore germ cells employ specialized small RNA pathways -PIWI proteins and PIWI-interacting RNAs (piRNAs)- to silence transposons and thus ensure genomic stability and fertility in animals. Mature piRNAs guide their associated PIWI complexes to silence transposons at transcriptional or post-transcriptional levels, directing chromatin modifications or promoting RNA decay. We aim to elucidate molecular mechanisms of piRNA silencing through an integrated approach combining Drosophila genetics, biochemistry and next-generation sequencing. While recent advances have provided a framework for what resembles a small RNA-based immune system, further studies are required to elucidate the many molecular innovations that enable discrimination of transposons from host genes and efficient selective silencing of genetic mobility. Successfully meeting our goals will bolster our understanding of fundamental mechanisms that survey and guard genomic integrity. How the millions of piRNA sequences faithfully discriminate between self and nonself, and adapt to novel genomic invaders remain key outstanding questions in genome biology. PiRNAs are largely confined to germ cells but the initial epigenetic restriction they impose is maintained in adult somatic cells. However, age and disease weaken epigenetic maintenance and unleashed transposons trigger toxicity and drive mutagenesis. Understanding how transposons are controlled has fundamental implications for age-related diseases, cancer biology and auto-immune disorders, all of which are associated with progressive loss of transposon control. While the sequences of piRNAs vary in different species, key biogenesis factors and processing signatures are conserved from insects to humans. A hallmark of piRNAs is their preference to harbor a uridine in the 5 most position (1U). This 1U-bias is the only known restriction to the enormous piRNA sequence space. To understand how this 1U-bias is established, we combined fly genetics and genomics. We identified a conserved two-step mechanism that establishes the 1U-bias during piRNA processing and formation of functional PIWI-piRNA complexes in flies and mice. Our next project focused on piRNA 3end formation. Variable 3 ends have the potential to modify specificity and efficacy of piRNA-guided silencing. We uncovered that each piRNA has a single major 3end, thus revealing an unanticipated precision in 3 end formation. Our results delineate a conserved hierarchy of sequence and length determinants, and identified a murine-specific road-block that established requirement for exonucleolytic trimming. Finally, our new insights into the precision of piRNA biogenesis provided the foundation to systematically dissect the entire piRNA sequence space for the first time. We uncovered that while piRNAs are highly abundant -with about one million molecules per cell-, a single cell can only accommodate a fraction of these diverse sequences. We observed a skewed distribution of sequence abundance, that results in a few abundant and many low-abundant piRNA sequences. Our genomics and direct molecular experiments revealed that abundant sequences are robust and drive piRNA-guided silencing. In contrast, the diverse group of low-abundant sequences is sporadic and does not contribute to target restriction. We revealed the conserved mechanisms that regulate sequence abundance and established that abundance is key to function in piRNA silencing.