Abstract RNA-protein (RNP) granules are dynamic membrane-less organelles that form during normal growth and in response to stress in a reversible manner. One type of RNP granules is Processing Bodies (P-bodies, or PBs), which contain mRNA together with RNA processing enzymes. Their roles include sequestration of malfunctioning mRNA destined for degradation and storing and silencing of mRNAs when not needed. PBs components and behavior are highly conserved between yeast and human cells. Our understanding of composition and assembly by liquid-phase separation normal PBs has progressed in the last decade. In contrast, our knowledge about involvement of PBs in human disease is scarce as is evidence about the occurrence of aberrant PBs and clearance of normal or aberrant PBS. In this project, we propose to study variants in a PBs component associated with a neurodevelopmental disorder that causes intellectual disability. Currently, there is no cure for such disorders. We hypothesize that the variants cause accumulation of aberrant PBs that, unlike normal PBs, are not reversible. The human variants carry missense mutations in residues identical in the yeast protein. Therefore, we started by modeling the effects of these mutations on RNA processing and accumulation of PBs in yeast. While normal PBs assemble and disassemble according to cellular needs, our preliminary evidence in yeast cells points to accumulation of persistent PBs as the major phenotype of these mutations. We propose to study the effects of these mutations in yeast and in human tissue culture cells, including neuronal cell lines. The phenotypes we propose to assess are on the accumulation and dynamics of persistent PBs, including adverse effects they might have on cell function. In addition, we will explore possible pathways that can clear normal and persistent PBs, such as macro- and micro-autophagy and ubiquitin-associated degradation. For these studies we will use a combination of molecular genetics and cellular approaches. Most approaches for studying RNP granules, granule clearance pathways in yeast and human cells are established in our lab. Achieving the goals of this proposal would provide novel paradigms on existence and behavior of persistent PBs and the role they play in a neurodevelopmental disorder. Moreover, identifying pathways that can clear normal and aberrant PBs would provide novel therapeutic strategies for a neurodevelopmental disorder associated with intellectual disability, in line with the missions of the National Institute of Neurological Disorders and Stroke.

Project Narrative Early-onset neurodevelopmental disorders affect brain development and intellectual disability of 1-3% of children worldwide. De novo mutations in multiple genes have been implicated in causing intellectual disability, for which no cure is currently available. The proposed research is aimed at elucidating a novel role that accumulating aberrant granules play in causing a neurodevelopmental disorder and identifying pathways that clear these granules.