A comprehensive road map of small RNA expression during mouse germ cell and zygote development
Adriano Biasini | Zamore Research Group | Swiss National Science Foundation Postdoctoral Mobility Fellowship
"Small noncoding RNAs <70 nucleotides long (sncRNAs) regulate diverse biological processes, including development and epigenetic inheritance. For example, in the germ cells of male placental mammals PIWI-interacting RNAs (piRNAs) and microRNAs (miRNAs) guide Argonaute proteins to silence transposable elements and mRNAs, thereby ensuring the development of functional sperm. As sperm mature, piRNAs and miRNAs are replaced by tRNA fragments, which are believed to mediate epigenetic inheritance. Methods to clone and identify sncRNAs have played a key role in understanding their biogenesis and function. Notably, the strategies typically used to sequence Argonaute protein-associated small RNAs only capture RNAs with 5′-monophosphate (P) and 3′ hydroxyl (OH) termini. Consequently, they fail to detect tRNA fragments or other sncRNAs with different terminal groups. Methods that convert all termini to 5′-P and 3′-OH can capture diverse sncRNA types but fail to retain knowledge of the original terminal groups, information critical to deducing sncRNA biogenesis and function.
To fill this gap, we have developed Terminus-Specific Ligation and sequencing of sncRNAs (TSL-Seq), a high-throughput sequencing framework that captures diverse sncRNAs while identifying the terminal groups of each RNA. Here, I propose to:
1. Maximize the efficiency of the TSL-Seq cloning procedure;
2. Use TSL-Seq to capture the diversity of mouse sncRNAs in developing and mature male germ cells;
3. Use TSL-Seq to capture the diversity of mouse sncRNAs in female germ cells and zygotes.
Optimizing TSL-seq will decrease the amount of input RNA required, allowing characterization of sncRNAs from rare cell types or mutant tissues. This project will produce the first comprehensive catalog of sncRNAs in developing mouse sperm and oocytes, uncover paternally and maternally deposited sncRNAs, and reveal the fates of sncRNAs after fertilization before the activation of zygotic transcription"
