Identification of Genetic Suppressors of RNA Granules - SUMMARY/ABSTRACT
Polyadenylated (Poly(A)+) RNA containing cytoplasmic granules affect numerous biological functions
and disease states. In normal cells, poly(A)+ granules take many forms, such as stress granules (SGs),
processing bodies (PBs), neuronal granules, and germ cell granules. These granules affect mRNA localization,
stability, and/or translational control under various physiological contexts. Pathogenic protein aggregates are a
unifying feature of many neurodegenerative diseases (ND), including amyotrophic lateral sclerosis (ALS). The
relationships between normal and pathogenic aggregates is still being deciphered, however clear links
between the two are known. Thus, the co-aggregation of poly(A)+ RNA and protein in cytoplasmic granules
under both normal and disease conditions is of broad biological importance. Yet, the genetic suppressors of
poly(A)+ RNA granule formation have not been identified on a genome-wide scale. The long-term goal of this
research program is to understand both the genetic and the environmental factors that regulate SG formation,
and to understand how SGs relate to other types of aggregates and disease states. The overarching
hypothesis is that genetic regulators of poly(A)+ RNA granule formation can be used to modulate RNA granule
formation in specific biological and disease-related contexts. As a first step to testing this hypothesis, the
primary objective of this proposal is to perform whole-genome screening to identify genes that suppress
poly(A)+ RNA granule formation in both Drosophila and human cells. RNA granule suppressing genes, when
depleted by RNAi, will cause the spontaneous formation of poly(A)+ RNA granules. To obtain the primary
objective, the following two specific aims will be pursued. Aim 1: To identify genes that suppress poly(A)+ RNA
granules in Drosophila S2R+ cells. An image analysis pipeline will be adapted to re-analyze images from a
previously published whole-genome RNAi screen in Drosophila S2R+ cells (Farny et al., 2008). Aim 2: To
examine the evolutionary conservation of poly(A)+ RNA granule suppressors in human cells. Using the primary
screen hits from Aim 1 as a guide, a targeted CRISPR knockout library will be screened to identify human
genes that suppress poly(A)+ RNA granules U2OS cells. This aim will also address the working hypothesis
that genetic silencing of poly(A)+ RNA granule suppressors will result in an increase in endogenous TDP-43
aggregation in U2OS cells. The rationale for this approach is that, given the known links between poly(A)+
RNA granules and disease-causing aggregates, suppressors of poly(A)+ aggregation could be harnessed in
future studies to suppress pathogenic aggregates. Given the known links between normal and pathogenic
aggregate formation, understanding the genetic determinants of poly(A)+ RNA granule formation will provide
new avenues of investigation for the genetic control of pathogenic aggregate formation. Thus the knowledge
gained here will be of broad interest and importance.
