Deciphering the molecular role of Spenito, a novel component of the m6A methyltransferase complex

• Applicant: Professor Dr. Jean-Yves Roignant
• Subject Area: Developmental Biology ; General Genetics and Functional Genome Biology ; Biochemistry
• Term: from 2017 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 376428912

Epitranscriptomics is an expanding research area of more than 100 known RNA modifications. The most prevalent modification on messenger RNA (mRNA) is N6-methlyadenosine (m6A). While the functions of m6A in the posttranscriptional regulation of gene expression are beginning to be unveiled, the precise roles of m6A during development of complex organisms remain unclear. We have carried out a comprehensive molecular and physiological characterization of the individual components of the methyltransferase complex as well as of the YTH nuclear reader protein in Drosophila melanogaster. We demonstrated important roles of this complex in neuronal functions and sex determination, and implicate the nuclear reader YT521-B as a main m6A effector in these processes. Importantly, we identified the member of the split ends protein family, Spenito (Nito), as a novel bona fide subunit of the methyltransferase complex. Nito interacts with other components of the complex and its loss leads to strong decrease of m6A levels. RMB15/RBM15B is the vertebrate ortholog of Nito and its role within the methyltransferase complex was also recently demonstrated. Its absence was shown to prevent the recruitment of the complex to XIST mRNA, resulting in a failure to deposit m6A and to promote X-chromosome silencing. Hence, the current model proposes that RBM15/RBM15B interacts directly with mRNA via its RNA binding domains and in this way provides the specificity to the methyltransferase complex by promoting its recruitment to m6A-targeted sites. However, how RBM15/RBM15B interacts with other components of the methyltransferase complex and whether its activity alone is sufficient to guide the complex to RNA remains to be determined. In this proposal, we want to unravel the precise molecular function of Nito within the methyltransferase complex. Combining biochemistry and state-of-the-art ribonomics approaches, we will systematically address the following questions: (1) What are the direct molecular interactions between Nito and the other members of the methyltransferase complex? (2) Which protein domains of Nito are required for m6A functions and its physiological activities? (3) What are the direct Nito targets and is it required to guide the methyltransferase complex to its targeted sites? (4) Is Nito sufficient to recruit the methyltransferase complex and promote m6A de novo. Our study will enable us to obtain detailed insights into Nito assembly within the methlytransferase complex and its precise contribution to m6A biogenesis.

• DFG Programme: Research Grants