A Subset of Drosophila Integrator Proteins Is Essential for Efficient U7 snRNA and Spliceosomal snRNA 3′-End Formation

摘要

Proper gene expression relies on a class of ubiquitously expressed, uridine-rich small nuclear RNAs (snRNAs) transcribed by RNA polymerase II (RNAPII). Vertebrate snRNAs are transcribed from a unique promoter, which is required for proper 3′-end formation, and cleavage of the nascent transcript involves the activity of a poorly understood set of proteins called the Integrator complex. To examine 3′-end formation in Drosophila melanogaster, we developed a cell-based reporter that monitors aberrant 3′-end formation of snRNA through the gain in expression of green fluorescent protein (GFP). We used this reporter in Drosophila S2 cells to determine requirements for U7 snRNA 3′-end formation and found that processing was strongly dependent upon nucleotides located within the 3′ stem-loop as well as sequences likely to comprise the Drosophila equivalent of the vertebrate 3′ box. Substitution of the actin promoter for the snRNA promoter abolished proper 3′-end formation, demonstrating the conserved requirement for an snRNA promoter in Drosophila. We tested the requirement for all Drosophila Integrator subunits and found that Integrators 1, 4, 9, and 11 were essential for 3′-end formation and that Integrators 3 and 10 may be dispensable for processing. Depletion of cleavage and polyadenylation factors or of histone pre-mRNA processing factors did not affect U7 snRNA processing efficiency, demonstrating that the Integrator complex does not share components with the mRNA 3′-end processing machinery. Finally, flies harboring mutations in either Integrator 4 or 7 fail to complete development and accumulate significant levels of misprocessed snRNA in the larval stages.