Biogenesis and function of cyclic phosphate-containing RNAs

Project Summary and Abstract Short non-coding RNAs (sncRNAs) possess a wide range of functions and are indispensable to many biological processes. While the best characterized sncRNAs are those associated with Argonaute family proteins, such as microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs), recent research has uncovered other functional sncRNA classes with distinct properties, biogenesis, and functions. During the biogenesis of sncRNAs, enzymatic cleavages of precursor molecules leave various terminal formations, such as hydroxyl (OH), phosphate (P), or 2′,3′-cyclic phosphate (cP). However, the majority of RNA-seq data for sncRNAs to date has been generated using standard RNA-seq methods, which predominantly capture sncRNAs with 5′-P and 3′-OH ends, like miRNAs. SncRNAs with other terminal structures cannot be ligated to the 5′- and/or 3′-adaptors, and thus remain unrecognized by standard RNA-seq methods. This limitation results in the exclusion of non-miRNA- sncRNAs lacking the 5′-P/3′-OH ends from expression analyses, keeping them uncharacterized. This study specifically focuses on short cP-containing RNAs (cP-RNAs) which contain a cP at their 3′-end and thus are absent in standard RNA-seq data. Despite being largely undetected in standard RNA-seq analyses, numerous cP-RNAs have been identified as functional sncRNAs. A representative example is the 5′-tRNA halves produced through anticodon cleavage of tRNAs by the angiogenin endoribonuclease. These cP-RNAs play significant roles in various biological processes, including stress response, cell proliferation, and immune response. By developing cP-RNA-seq, which can specifically sequence cP-RNAs, we have performed a genome-wide expression analysis of short cP-RNAs across various cells and tissues, revealing that: 1) cP-RNAs are generated not from random degradation but through regulated biogenesis pathways; 2) cP-RNAs constitute a significant portion of sncRNAs (15–60 nucleotides) in cells, tissues, and in extracellular vesicles; and 3) expression levels and profiles of cP-RNAs are regulated by various factors such as hormone signaling pathways, aging, oxidative stress, and infection. We propose cP-RNAs as promising research subjects due to their diverse physiological functions in various biological processes. Moving forward, our research will focus not only on identifying which cP-RNAs are expressed, but more importantly on understanding the regulation of their biogenesis and elucidating their molecular functions. Recently, we have discovered RNase Kappa as a novel enzyme generating cP-RNAs, along with two new proteins that interact with and stabilize cP-RNAs. Our proposed study will involve characterizing the roles of these factors in the regulation of cP-RNA expression and investigating the functional significance of their generated/stabilized cP-RNAs in piRNA biogenesis, regulation of mRNA stability, and immune receptor activations during immune response, which will help achieve our goal of understanding cP- RNA biology and realizing its broader implications for human health and disease.