Structure-function analysis of a novel, maternally provided, multi-functional RNP complex in C. elegans

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This project presents experiments aimed at characterizing, both in vivo as well as in vitro, a novel protein complex with roles in multiple RNA-related processes. This complex, named PETISCO and identified in C. elegans, is composed of both highly conserved, and more C. elegans specific proteins. Nematode-specific proteins are PID-3 and TOFU-6. PID-3 has a MID- and an RRM domain, and TOFU-6 has a Tudor- and an RRM domain. Strongly conserved proteins are IFE-3, a homolog of eIF4E and ERH-2, one of the nematode homologs of 'enhancer of rudimentary'. The latter is a small protein, with known roles, even though mechanistically unresolved, in mRNA homeostasis in S. pombe. PETISCO is important for the biogenesis of a specific class of small RNAs (piRNAs), but also affects the splice-leader RNA, SL1, and histone mRNA. These RNA species have not mechanistically been linked before, and as such the PETISCO complex represents a novel and unique interface between these RNA species. Interestingly, we identified two proteins, PID-1 and TOST-1, that bind PETISCO mutually exclusively, and that appear to help define the function of the PETISCO complex. This provides us with a unique handle to study different aspects of this multi-protein complex. Furthermore, we already elucidated the interactions between the different PETISCO subunits, providing a ready-to-go framework for the proposed studies. The aim of the project is to understand PETISCO biochemistry in vitro and its function in vivo. Emphasis will be given to understanding PETISCO function in relation to trans-splicing of mRNAs, and to histone mRNA homeostasis. Techniques that will be used will range from recombinant protein technology and structural biology to genome editing and phenotypic analyses in C. elegans.