Alladi Ramakrishnan Hall

Exploring the sequence space and 5’ 1U bias of PIWI-interacting RNAs

Sanga Mitra

NIH, USA

In animals, a discrete class of small RNAs, the PIWI-interacting RNAs (piRNAs), guard germ cell genomes against the deleterious activity of mobile genetic elements (transposons) that have colonized about half of our genomes. Their ability to amplify and reinsert into novel genomic locations threatens genome integrity and must be restrained. Together with PIWI protein partners, piRNAs silence transposons at transcriptional and post-transcriptional levels. Mature piRNAs are generated from single or dual-stranded precursors that arise from discrete genomic loci, termed as piRNA clusters. PiRNA clusters are mainly composed of transposon-derived repetitive sequences giving rise to hundreds and thousands of unique piRNA sequences, which are highly diverse in nature.

How this diverse pool of sequences is controlled to achieve specific restriction of transposons while avoiding off-target effects remains elusive. The sequence diversity of piRNAs in flies and mice has been systematically characterized. With increasing sequencing depth, we observe millions of unique piRNA sequences. This enormous diversity is only partly explained by the variability of piRNA 3’ ends and potentially reflects thousands of piRNA-generating genomic regions, piRNA clusters. However, not every sequence seems to be required for efficient transposon restriction. Overall, our exploration of the piRNA sequence space identified subpopulations of sequences with vastly different importance for piRNA biology.

The enormous sequence space of mature piRNAs is restricted by their preference to start with a Uridine (U) at the 5' most position. The source of this “1U bias”, as well as the potential evolutionary significance, has been a long-standing question in the field. Is it because of processing by Zucchini (Zuc) endoribonuclease or specific selection by specificity loop (SL) and loading in PIWI protein? Or else is this secret hidden in the genomic content of piRNA pool? It is hypothesized that the 1U bias is a result of selection by the PIWI specificity loop, a six amino acid stretch of the PIWI
protein that interacts with the first base of mature piRNAs. Designing PIWI proteins with altered specificity loop sequences answers the question. It is uncovered that the 1U bias of Piwi-piRNAs is established by consecutive discrimination in two steps, whereby in the first step ‘1A’ and ‘1G’ containing RNAs are disfavored during piRNA processing, and in the second step ‘1C’ is repulsed by Piwi’s specificity loop (SL) to yield a final 1U bias.