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

Abstract

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.In eukaryotes, the major transcripts produced by RNA polymerase II (RNAPII) include the polyadenylated [poly (A) ϩ ] mRNAs, the replication-dependent histone mRNAs, and the Sm class of small nuclear RNAs (snRNAs). The 3Ј ends of these three general classes of RNAs are all formed by cotranscriptional cleavage, but each one has a distinct mechanism for 3Ј-end formation (for reviews, see references 29 and 32). In poly(A) ϩ and histone pre-mRNAs there are conserved upstream and downstream sequences that flank the cleavage site; factors bind to these sites and then recruit additional factors that initiate cleavage (53). In the case of poly(A) ϩ pre-mRNA, the upstream element is the canonical AAUAAA polyadenylation signal (PAS) and the downstream sequence is the G/Urich downstream element (DSE). Recognition of the PAS is carried out by the cleavage and polyadenylation specificity complex (CPSF) component CPSF160 via its RNA recognition motifs (RRM) (36), whereas the DSE is bound by the RRM of the cleavage stimulation factor (CstF) component CstF64 (28). Subsequent to this recognition event is recruitment of additional factors that activate the endonucleolytic cleavage between the PAS and the DSE.Histone pre-mRNA contains a distinct set of flanking elements. Upstream of the cleavage site is a conserved stem-loop structure (SL) and downstream a purine-rich element called the ...

3′-End processing of histone pre-mRNAs in Drosophila: U7 snRNP is associated with FLASH and polyadenylation factors

“…We therefore constructed a reporter gene that relies on a promoter derived from the Drosophila U7 snRNA gene. snRNA genes associate with a distinct set of transcriptional factors, and primary transcripts generated from these genes (pre-snRNAs) are cleaved at the 3 ′ end by a distinct machinery termed Integrator that shares no common components with the two types of 3 ′ -end processing machineries operating on premRNAs (Baillat et al 2005;Egloff et al 2008;Ezzeddine et al 2011).…”

The host Integrator complex acts in transcription-independent maturation of herpesvirus microRNA 3′ ends

“…Perhaps the longer 3 ′ box evolved for snRNA processing because it is the only RNA sequence element required for Integrator cleavage. Although the 3 ′ -terminal stem-loop of the Drosophila U7 snRNA is important for 3 ′ end processing with a GFP reporter attached (Ezzeddine et al 2011), others reported that such a stem-loop is not always essential for Integrator-dependent cleavage of snRNAs (Hernandez 1985;Yuo et al 1985). We likewise conclude that the HSUR4 terminal stem-loop is not essential for snRNA 3 ′ end processing, since removing the entire HSUR4 sequence does not affect miR-HSUR4 production, which requires 5 ′ cleavage by Integrator (Cazalla et al 2011).…”

An RNAi screen identifies additional members of the Drosophila Integrator complex and a requirement for cyclin C/Cdk8 in snRNA 3′-end formation

“…Although the 39 box is highly tolerant of mutation (Hernandez 1985;Ach and Weiner 1987;Ezzeddine et al 2011), it is likely to be a binding site in the RNA for the machinery that governs snRNA 39-end formation. Studies have also shown that substituting either human or Drosophila snRNA promoters with mRNA promoters results in total loss of snRNA 39-end formation (de Vegvar et al 1986;Hernandez and Weiner 1986;Ezzeddine et al 2011). In addition, the 39-end formation of viral-encoded snRNAlike transcripts found in Herpesvirus saimiri also exhibits a dependency on an snRNA promoter (Cazalla et al 2011).…”

“…New insight to these snRNA processing events has come from the biochemical purification of the Integrator complex and establishment of its functional requirement in snRNA 39-end formation (Baillat et al 2005;Ezzeddine et al 2011). Integrator subunit 9 (IntS9) and IntS11 (Albrecht and Wagner 2012) are homologous to CPSF100 and CPSF73, which are components of the cleavage factor for mRNA 39-end formation (Mandel et al 2006;Sullivan et al 2009).…”