RNA function in germ and stem cell biology The integrity of the genome transmitted to the next generation intrinsically relies on cells of the germline. Processes that ensure germ cell development, genomic stability and reproductive lifespan are essential for the long-term health and success of a species. We tackle fundamental questions regarding the mammalian germ line and heredity from an RNA perspective. Specifically, our research explores the contribution of PIWI-interacting RNAs (piRNAs) and RNA modification pathways to germ cell development. We also have a keen interest in characterizing spermatogonial stem cell (SSC) populations that underpin male fertility throughout adult life. Professor Donal O'Carroll Group Leader Contact details Website: Personal Profile Work: +44 (0)0131 651 9631 Email: donal.ocarroll@ed.ac.uk Aims and areas of interestWhy we study the germlineThe germline is the immortal lineage, the cells that transcend generations and initiate the cycle of life. The lineage where evolution occurs. The mammalian germline is subject to major and essential epigenetic reprogramming. Processes that ensure germ cell development, epigenetic integrity, genomic stability and reproductive lifespan are essential for the long-term health and success of a species, or society in a human context. Our research helps understand the basis of infertility and diseases that arise by germline mutation or epimutation. RNA modification Image Figure 1: TUT4/7-mediated 3'terminal mRNA uridylation is required to build a functional maternal transcriptome during oocyte growth whereas m6A-YTHDF2 is required for the metabolism of maternal transcripts during oocyte maturation. Both modifications are essential for oocyte competence and fertility. The germline and regulatory RNA mechanisms have an intimate relationship asseveral stages of both male and female germ cell development are transcriptionally inert and thus rely on post-transcriptional regulation of gene expression. Thus, the study of RNA modification in the germline will give profound insights into both processes. Indeed, the maternal transcriptome is the blueprint for early life and the understanding of the mechanisms that enable its ordered construction, usage and destruction can also serve as a paradigm for understanding basic principles of regulated gene expression. We focus on the function of N6-methyladenosine (m6A) and 3' terminal uridylation mRNA modifications, both of which can promote RNA degradation. We showed essential and specific functions for poly(A) tail length and TUT4/7-mediated 3' terminal uridylation in sculpting a functional maternal transcriptome during oocyte growth (Nature, 2017) (Figure 1). We recently demonstrated that a programmed wave of uridylation-primed mRNA degradation is essential for meiotic progression and mammalian spermatogenesis (Cell Res, 2019). Finally, we demonstrated that the m6A-reader YTHDF2 regulates transcript dosage during oocyte maturation and is an intrinsic determinant of mammalian oocyte competence as well as early zygotic development (Mol Cell, 2017) (Figure 1). We currently try to understand additional functions for these as well as other modifications in the germline and beyond. We have a major collaboration with Prof. Kamil Kranc (Barts Cancer Institute, London) on the role of mRNA m6A and 3'uridylation in leukaemia.The PIWI-piRNA pathway Image Figure 2: Genomic DNA methylation is erased (reprogramming) and reset (de novo methylation) during germ cell development. The mechanism by which the PIWI protein MIWI2 direct de novo methylation of young transposable elements remains unknown. In mammals, the acquisition of the germline from the soma provides the germline with an essential challenge, the necessity to erase and reset genomic methylation. This is one of the most drastic epigenetic events in mammalian life. De novo genome methylation re-encodes the epigenome, imprinting and transposable element (TE) silencing. In the male germline piRNA-directed DNA methylation silences young active TEs. This poorly understood but essential process is central to the immortality of the germline. Upon completion of germline reprogramming with the full erasure of genomic methylation TEs become derepressed. PIWI proteins, MILI & MIWI2, and their associated piRNAs neutralize this threat. Firstly, through base complementarity piRNAs guide the PIWI endonuclease MILI to destroy cytoplasmic transposon RNAs. Secondly, antisense TE-derived piRNAs generated from intricate biogenesis pathways act to guide the nuclear PIWI protein MIWI2 to instruct TE DNA methylation by an unknown mechanism. We have made an important contribution to the mechanism of piRNA biogenesis as well as elucidating the functions of the piRNA pathway during adult spermatogenesis. Our future goal is to understand the elusive mechanism by which MIWI2 instructs TE methylation and epigenetic silencing (Figure 2).Spermatogonial stem cell populationsSpermatogonial stem cells (SSCs) maintain spermatogenesis throughout adult life as well as underpinthe regenerative capacity of the testis. A small population of undifferentiated spermatogonia have SSC activity. We showed that MIWI2 expression defines a population of transit-amplifying spermatogonia that also retain facultative stem cell function and is essential for the efficient regenerative capacity of the adult testis (J Exp Med, 2017). We also recently demonstrated that defective germline de novogenome methylation rewires spermatogonial transcriptomes (Nat Struct Mol Biol, 2017). We are currently using single-cell techniques to define the impact of regeneration and ageing on SSC populations. In addition, we utilize and develop state of the art cellular barcoding techniques to understand the clonality of SSCs and their clonal contribution to spermatogenesis.Publications Publications Group MembersShaunak Burse, Postdoctoral Research FellowTamoghna Chowdhury, PhD StudentYuka Kabayama, Postdoctoral Research AssociateAleksandra (Ola) Kasprowicz, Postdoctoral Research AssociateDavid MacLeod, PhD StudentMartina Schito, PhD StudentMargot Wyroll, DFG Postdoctoral Research FellowJuTing Yang, PhD StudentFundersCRUKWellcome TrustHorizon 2020CollaboratorsProf Kamil Kranc, Barts Cancer Institute, LondonDr Anton Enright, EMBL European Bioinformatics Institute, Cambridge Dr Vladimir Benes, EMBL, Heidelberg, GermanyDr Tania Auchynnikava, Wellcome Centre for Cell Biology, University of EdinburghProf David Tollervey, Wellcome Centre for Cell Biology, University of EdinburghProf Robin Alshire, Wellcome Centre for Cell Biology, University of EdinburghProf Juri Rappsilber, Wellcome Centre fro Cell Biology, University of Edinburgh This article was published on 2024-02-26
