Regulation of gene expression by non-coding RNAs
The Pillai group seeks to understand molecular mechanisms involved in piRNA biogenesis and its function in protecting the genome from instability.
Localisation of a tagged insect Piwi protein to perinuclear cytoplasmic granules in insect cell cultures. These are putative piRNA biogenesis sites, similar to the nuage in germ cells.
Previous and current research
Past invasion events from mobile genetic elements have left eukaryotic genomes littered with repeats and other transposon sequences. Much of these are inactive fossils, but some still retain the potential to get activated and cause genome instability. Protection from transposons is achieved by silencing them in the germline, which is then maintained throughout the life of the individual. Animal germ cells express a specialised class of ~30 nt small non-coding RNAs called piwi-interacting RNAs (piRNAs), which are implicated in guiding this silencing. Indeed, one universal feature of piRNAs in all animals is their origin from transposon-rich genomic regions. In mammals, they are believed to recruit DNA methyltransferases to transposon sequences. In Drosophila, maternally produced piRNAs are deposited in the egg and they contribute to protection from new transposons brought in by the paternal genome. Thus, piRNAs constitute an epigenetic component of the genome defence mechanism in animals.
Our lab is interested in understanding the molecular mechanisms involved in piRNA biogenesis and function. A striking feature of piRNAs is their clustered genomic origins. It is believed that a long single-stranded transcript arising from a cluster is processed into thousands of piRNAs. The mechanism of this processing and the identity of factors involved are unknown. We have taken a biochemical approach to identify these factors by isolating mouse Piwi-associated proteins. This led to the identification of Tudor domain-containing protein 1 (Tdrd1), which interacts by recognising symmetrical dimethyl arginine modification marks on Piwi proteins. Another factor is the putative helicase Mov10l, which is an essential piRNA biogenesis factor, as piRNAs fail to accumulate in mutant mice. In all these studies, we have used a variety of techniques ranging from protein biochemistry, cellular imaging, small RNA bioinformatics, and mouse mutants. We are now setting up insect cell culture lines that have an active piRNA pathway, paving the way for potential mechanistic insight into the function of the identified factors. To deepen our understanding, we collaborate with structural biologists to obtain atomic resolution images of the identified pathway components. Recently, this effort resulted in a structure describing the recognition of the 2’-O-methyl mark on piRNAs by the PAZ domain of a Piwi protein.
Future projects and goals
We will continue to analyse additional factors identified in our complex purifications. Another goal is to understand the features that define genomic regions as piRNA clusters, and whether there is a link between transcription from the clusters and piRNA biogenesis. We also hope to use live cell imaging techniques to study assembly of small RNPs in vivo and define the contribution of the individual constituents of the complex to this process. It is our desire to intensify the collaborative work on structural biology of Piwi complexes, adding another dimension to our understanding of germline small RNAs. In addition to small RNAs, our cells express longer non-coding RNAs (ncRNAs), which are implicated in a variety of gene regulatory functions, usually in epigenetic roles. We wish to apply biochemical methods to identify protein components of long ncRNPs to understand their contribution to the molecular function of the RNA.