PSF and p54nrb bind a conserved stem in U5 snRNA

Abstract

PTB-associated splicing factor (PSF) has been implicated in both early and late steps of pre-mRNA splicing, but its exact role in this process remains unclear. Here we show that PSF interacts with p54 nrb , a highly related protein first identified based on cross-reactivity to antibodies against the yeast second-step splicing factor Prp18. We performed RNA-binding experiments to determine the preferred RNA-binding sequences for PSF and p54 nrb , both individually and in combination. In all cases, iterative selection assays identified a purine-rich sequence located on the 39 side of U5 snRNA stem 1b. Filter-binding assays and RNA affinity selection experiments demonstrated that PSF and p54 nrb bind U5 snRNA with both the sequence and structure of stem 1b contributing to binding specificity. Sedimentation analyses show that both proteins associate with spliceosomes and with U4/U6.U5 tri-snPNP.

The Splicing Factor PSF is Part of a Large Complex That Assembles in the Absence of pre-mRNA and Contains All 5 snRNPs

“…1B) is consistent with cosedimentation of PSF with splicing complexes. 23 However, to our surprise, similar if not identical results were obtained when immunoprecipitation reactions were performed under splicing conditions but without the addition of AdML pre-mRNA (Fig. 1A, compare lanes 3 and 4).…”

“…41 Previously, we showed that PSF and p54 nrb interact with U5 snRNA. 23 During further analysis of this interaction by immunoprecipitation (IP) with PSF-specific antibodies, we show here that all five U snRNPs co-IP with PSF when HeLa nuclear extracts are adjusted to splicing conditions, regardless of the addition of pre-mRNA. Sedimentation analyses confirmed the formation of large PSF-containing multi-snRNP complexes in nuclear extracts adjusted to conditions normally used for in vitro splicing.…”

“…Previously, we showed that PSF and the closely related protein p54 nrb bind a conserved stem 1b in U5 snRNA and that both proteins, together with other U5-specific factors, assemble onto biotinylated U5 snRNAs in Hela nuclear extracts under splicing conditions (12 mM Tris-HCl, pH 7.9, 60 mM KCl, 0.12 mM EDTA, 0.5 mM ATP, 20 mM phosphocreatine, 2 mM MgCl 2 and 0.5 mM DTT). 23 When we utilized PSF-specific antibodies to further analyze the interaction with U5, we found that all five snRNPs could be immunoprecipitated (Fig. 1A, lane 4).…”

“…It copurifies with both early (H, A, B) and late (C) spliceosomal complexes, the U4/U6.U5 tri-snRNP, U5 snRNP, and is found in nuclear speckles, colocalizing with many splicing factors. 14,[16][17][18][19][20][21][22][23] It is comprised of an N-terminal glycine-rich domain, a proline/glutamine (P/Q) rich domain, two RNA recognition motifs (RRMs), and a C-terminal region with two nuclear localization signals. A closely related protein with a similar domain structure, p54 nrb (and its mouse homolog nonO) 24,25 has been found to associate with PSF.…”

“…A closely related protein with a similar domain structure, p54 nrb (and its mouse homolog nonO) 24,25 has been found to associate with PSF. 23,[26][27][28] Together, these proteins interact with the C-terminal domain (CTD) of RNA Pol II 29 and contact 5' splice sites within large transcription/splicing complexes, 30 consistent with coupling between transcription and splicing. 31,32 Besides splicing, PSF has also been implicated in many other nuclear events.…”