Proteomic analysis of in vivo-assembled pre-mRNA splicing complexes expands the catalog of participating factors

Abstract

Previous compositional studies of pre-mRNA processing complexes have been performed in vitro on synthetic pre-mRNAs containing a single intron. To provide a more comprehensive list of polypeptides associated with the pre-mRNA splicing apparatus, we have determined the composition of the bulk pre-mRNA processing machinery in living cells. We purified endogenous nuclear pre-mRNA processing complexes from human and chicken cells comprising the massive (>200S) supraspliceosomes (a.k.a. polyspliceosomes). As expected, RNA components include a heterogeneous mixture of pre-mRNAs and the five spliceosomal snRNAs. In addition to known pre-mRNA splicing factors, 5' end binding factors, 3' end processing factors, mRNA export factors, hnRNPs and other RNA binding proteins, the protein components identified by mass spectrometry include RNA adenosine deaminases and several novel factors. Intriguingly, our purified supraspliceosomes also contain a number of structural proteins, nucleoporins, chromatin remodeling factors and several novel proteins that were absent from splicing complexes assembled in vitro. These in vivo analyses bring the total number of factors associated with pre-mRNA to well over 300, and represent the most comprehensive analysis of the pre-mRNA processing machinery to date.

Figure 1.

Model describing the role of vertebrate supraspliceosomes in gene expression. (A) Co-transcriptional assembly of spliceosomes, 5′ end modification machinery and other pre-mRNA binding factors on RNA polymerase II transcripts. (B) The released transcript is partially spliced and bound by numerous spliceosome moieties as well as the 5′ cap-binding complex and 3′ end processing factors. (C) The mature mRNA is associated in the nucleus with RNA binding proteins, 5′- and 3′-end stabilizing factors (the CBP heterodimer and poly(A)-binding protein), and proteins that promote export to the cytoplasm.

Figure 2.

Human supraspliceosome-associated polypeptides and snRNAs. RNA (A) and protein (B) were extracted from preparative glycerol gradient fractions and electrophoretically resolved through urea-PAGE (A) or SDS-PAGE (B) gels stained with silver (RNA) or coomassie blue (protein). Bar below B represents the fractions of the material pooled for immunopurification with Y12 antibody. (C) Affinity-purified supraspliceosomal proteins run under two SDS-PAGE conditions to resolve either large or small polypeptides. Gels were aligned to show all polypeptides in the affinity-purified fractions and are delineated by the marking between them. The entire gel lanes shown from the two gels in (C) were dissected and each gel slice was subjected to mass spectrometry protein identification. The proteins identified are reported under the Hs PS column in Tables 1–3 and in Supplemental Table S1.

Figure 3.

Chicken supraspliceosome-associated polypeptides and snRNAs. Fractions corresponding to the CLEP-tag purified, glycerol gradient-sedimented chicken supraspliceosomes were separated into protein (A) and RNA (B) fractions and electrophoresed through SDS-PAGE (A) or urea-PAGE (B) gels and stained with coomassie blue (protein) or ethidium bromide (RNA). The identities of the snRNAs are indicated on the right of panel B. The entire gel lane from (A) was dissected and each gel slice was subjected to mass spectrometry for protein identification. The proteins identified are reported under the Gg PS column in Tables 1–3 and in Supplemental Table S1.

Figure 4.

Novel spliceosome-associated polypeptides with predicted RNA binding motifs. Polypeptides from Table 2 with no known function in the pre-mRNA processing pathway are shown with graphical representations of the various RNA interaction or other noted motifs listed at the bottom of the Figure.

Figure 5.

The novel Zn finger protein ZFR is a bona fide spliceosomal component. (A) ZFR sediments with the 200S particle in glycerol gradients. HeLa nuclear extract was subjected to glycerol velocity gradient sedimentation analysis as in Figure 2. Proteins from the indicated fractions were electrophoresed through SDS-PAGE gels and subjected to western blot analysis using anti-ZFR antiserum. The bar below the gel denotes the 200S region. (B) ZFR is specifically associated with spliceosomal snRNAs. Equal amounts of HeLa nuclear extract were incubated with protein-A beads (beads), pre-immune serum and protein-A beads (pre-immune), anti-ZFR antiserum and protein-A beads (anti-ZFR) or anti-SR140 antiserum and protein-A beads (anti-SR140) according to the Materials and Methods. Recovered nucleic acids were subjected to northern blot analysis and probed with antisense probes to human snRNAs (identities noted to the right of the Figure). (C) ZFR is specifically associated with complexes containing spliceosomal proteins. Immunoprecipitation conditions and lanes are as described in (B). Proteins were subjected to western blot analysis using hPrp43 antiserum.