Direct and indirect regulation of mecp2 by rna

Methyl CpG Binding Protein 2 (MeCP2) is a vertebrate DNA Reader that, through protein-protein interactions, participates in several molecular processes, such as regulation of transcription, chromatin organization, mRNA splicing, and miRNA biogenesis. However, the mechanism(s) that regulate these causative interactions in differing cellular contexts is/are unclear. Recently, a novel RNA Binding Domain (RBD) was identified in MeCP2, which lacks homology to well known “canonical” RBDs. Interestingly, the MeCP2 RBD represents a hotspot of missense mutations which cause the neurodevelopmental disorder known as Rett Syndrome, suggesting functional significance to this domain. Characterizing the molecular interactions with this novel, non-canonical RNA binding domain will help close the knowledge gap of how MeCP2 participates in myriad processes, and will contribute to the development of tailored therapies of MeCP2-related disorders, through understanding the downstream effects of MeCP2-RNA interaction. Here, Wild Type and RBD mutant MeCP2 proteins were expressed in human cells and in vitro. Biochemical methodologies including enzymatic chromatin fractionation as well as phase separation, in addition to immunoprecipitation and molecular docking were used to begin defining the type of RNA interaction occurring at the RBD of MeCP2, and to explore the biological implications of this interaction. I have shown that MeCP2-RNA and not MeCP2-chromatin interaction is mediated by its RNA Binding Domain, and I validated binding to a candidate RNA target, the lncRNA NEAT1L, in human cells. I then began to map where MeCP2 binds on the NEAT1L transcript, to postulate the structure- or sequence-specificity of MeCP2 for RNA. Finally, Co-immunoprecpitation data indicate that MeCP2-RNA interaction occurring at the RBD, directly and indirectly impacts MeCP2-protein interaction with two of its important transcriptional regulation complex partners, respectively. The elusive functional behaviour of MeCP2 has burdened researchers and families of children with MeCP2-related disorders world-wide for decades. I suggest that RNA interaction of the non-canonical RBD of MeCP2 may be one of the missing links to understanding and overcoming its related pathologies. The data from this proposal also represents a step towards understanding these emerging non-canonical RNA binding domains as well as a framework for determining RNA-based MeCP2 targets.