The Spliceosomal Protein SF3B5 is a Novel Component of Drosophila SAGA that Functions in Gene Expression Independent of Splicing

Abstract

The interaction between splicing factors and the transcriptional machinery provides an intriguing link between the coupled processes of transcription and splicing. Here, we show that two components of the SF3B complex that forms part of the U2 small nuclear ribonucleoprotein particle (snRNP), SF3B3 and SF3B5, are also subunits of the Spt-Ada-Gcn5 acetyltransferase (SAGA) transcriptional coactivator complex in Drosophila melanogaster. Whereas SF3B3 had previously been identified as a human SAGA subunit, SF3B5 had not been identified as a component of SAGA in any species. We show that SF3B3 and SF3B5 bind to SAGA independent of RNA, and interact with multiple SAGA subunits including Sgf29 and Spt7 in a yeast two-hybrid assay. Through analysis of sf3b5 mutant flies, we show that SF3B5 is necessary for proper development and cell viability, but not for histone acetylation. Although SF3B5 does not appear to function in SAGA’s histone modifying activities, SF3B5 is still required for expression of a subset of SAGA-regulated genes independent of splicing. Thus, our data support an independent function of SF3B5 in SAGA’s transcription coactivator activity that is separate from its role in splicing.

Molecular basis of differential 3′ splice site sensitivity to anti-tumor drugs targeting U2 snRNP

“…Indeed, dynamic acetylation of an H3K4me3 nucleosome positioned on exon 2 of MCL1 is linked to alternative skipping of this SF3B1-regulated exon 60 , 61 . SF3B complex components have been also found associated with the epigenetic remodeling complexes Polycomb and SAGA 62 – 64 , adding yet another layer of regulatory functions that can be affected by SF3B1 inhibitors.…”

The Drosophila Dbf4 ortholog Chiffon forms a complex with Gcn5 that is necessary for histone acetylation and viability

“…Tandem FLAG-HA affinity purification and MudPIT analysis was conducted from stable Drosophila S2 cell lines as described previously (Stegeman et al, 2016). TAP purifications from S. cerevisiae was performed as described previously (Lee et al, 2004).…”

“…Briefly, shared spectral counts (sSpC) were distributed based on spectral counts unique to each protein (uSpC). Histone acetyltransferase assays were performed as previously described (Stegeman et al, 2016) using Flag-purified complexes and HeLa core histones or human histone H3 peptide (K5-K23) as substrate.…”

“…Using this approach, the Ada2b isoforms could be distinguished by peptide spectra that mapped to their unique C-terminal regions. Ada2b-PB co-purified all other 19 SAGA subunits (Stegeman et al, 2016), but did not copurify any peptide spectra specific to the short Ada2b-PA isoform (Fig. 1B).…”

The SF3b complex: splicing and beyond

“…Subsequently, SF3b5 was additionally identified as a SAGA component together with the above SF3b3, based on an interaction with the conserved Sgf29 subunit of the human SAGA complex [98]. Moreover, a recent study showed that SF3b3 and SF3b5 were also found in drosophila SAGA [99]. This suggests that these two proteins may be intrinsic components of SAGA in higher eukaryotes.…”