A Genome‐wide RNAi screen identifies novel factors involved in the processing of snRNA

Proper 3′ end formation of snRNA depends on a conserved element termed the 3′ box and is thought to be tightly coupled to transcription. Moreover, there is a poorly understood complex of proteins called the “Integrator Complex” that is thought to mediate the processing of snRNAs. This complex contains at least 12 unique polypeptides that are likely loaded onto RNA polymerase II at the promoter and are involved in 3′ end formation. We are using the Drosophila U7 snRNA gene as a model substrate to study pre‐snRNA processing. We designed a cell‐based reporter by cloning the U7 snRNA gene upstream of the GFP ORF. In the event that an essential processing factor is depleted, the polymerase will transcribe past the processing site resulting in expression of GFP. Using this reporter, we performed a genome‐wide RNAi screen in S2 cells to identify factors required for the 3′ end formation of snRNA. We found that nearly all of the Integrator proteins identified biochemically are functionally required for the 3′ end processing of snRNA. Two of these Integrator proteins are indeed required for processing of endogenous U7snRNA in mutant flies and we also found that U4 snRNA biosynthesis is only sensitive to a subset of the Integrator proteins leading to the potential that there are distinct complexes associated at different snRNA genes. We also identified several novel genes that had not been previously implicated in snRNA biogenesis.