Methylation of Sm proteins - Roles in snRNP Biogenesis and germline specification

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DESCRIPTION (provided by applicant): Sm class small nuclear ribonucleoproteins (snRNPs) are core components of the spliceosome and are required for splicing in vivo. In addition, Sm proteins appear to have a more ancestral role in specification of the germline. The overall goal of this project is to understand the mechanism by which Sm proteins are assembled into snRNPs for the purpose of splicing or into specialized ribonucleoproteins (RNPs) required to establish germ cell fate. At the nexus of these two seemingly disparate pathways is the PRMT5 complex. The biogenesis of snRNPs is highly orchestrated, requiring several essential co-factors. In particular, cytoplasmic snRNP assembly relies on two heteromeric complexes, the PRMT5 complex and the SMN complex. Importantly, mutations that reduce the level of SMN protein are correlated with a neurodegenerative disease known as Spinal Muscular Atrophy (SMA). Patients with the disease often die very early in childhood. The molecular etiology of SMA is unknown, however, perturbation of snRNP biogenesis is thought to be a major contributing factor. A detailed understanding of the mechanism of snRNP assembly will shed light on the disease process. The PRMT5 complex is responsible for symmetrically dimethylating Sm proteins, and by virtue of this activity, is thought to cooperate with the SMN complex in mediating efficient snRNP assembly. However, an in vivo examination of this process is currently lacking. Furthermore, PRMT5 associates with BLIMP1 and is required for germ cell development in the mouse. Disruption of PRMT5 activity could thus impinge on two distinct but separable pathways - snRNP biogenesis and germ cell development. To gain mechanistic insight into these processes we have developed in vivo protocols making use of Drosophila as a model system as well as mammalian cell culture techniques. Aim 1 explores the requirement for Sm protein methylation in snRNP biogenesis. Since SMN binds with a higher affinity to methylated Sm proteins, this modification may act as a modifier of the SMA phenotype. Specifically, lack of Sm protein methylation in mammals may phenocopy SMA. Alternatively, increased methylation may ameliorate the disease severity. Aim 2 examines the requirement for DartS, the fly ortholog of PRMT5, in germ cell specification and addresses the specific role played by Sm proteins in this process. Lay summary: Spinal Muscular Atrophy, a common genetic disease, results in very early childhood mortality. Whereas the gene responsible for the disease has been identified, the mechanism that ultimately results in the disease pathology is unknown. In order to develop effective therapies, a molecular understanding of the disease gene product is essential.