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

Chen YI, Moore RE, Ge HY, Young MK, Lee TD, Stevens SW - Nucleic Acids Res. (2007)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Graduate program in Microbiology, City of Hope Beckman Research Institute, Duarte, CA 91010, USA.

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.

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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.
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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.

Mentions: We discovered that in gently sonicated nuclei treated with low salt (11), the majority of the snRNA, as judged by visual inspection of ethidium bromide stained gels (Figure 2A), was engaged in very large (>80S) ribonucleoprotein complexes that closely resemble supraspliceosomes in sedimentation values and other properties (9,10,30). These particles may also be related to the polyspliceosomes described in salt-extracted nuclei, which sedimented as complexes slightly smaller than our supra/polyspliceosome, likely reflecting salt-induced factor loss during nuclear extraction (12).Figure 2.


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

Chen YI, Moore RE, Ge HY, Young MK, Lee TD, Stevens SW - Nucleic Acids Res. (2007)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919476&req=5

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.
Mentions: We discovered that in gently sonicated nuclei treated with low salt (11), the majority of the snRNA, as judged by visual inspection of ethidium bromide stained gels (Figure 2A), was engaged in very large (>80S) ribonucleoprotein complexes that closely resemble supraspliceosomes in sedimentation values and other properties (9,10,30). These particles may also be related to the polyspliceosomes described in salt-extracted nuclei, which sedimented as complexes slightly smaller than our supra/polyspliceosome, likely reflecting salt-induced factor loss during nuclear extraction (12).Figure 2.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Graduate program in Microbiology, City of Hope Beckman Research Institute, Duarte, CA 91010, USA.

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.

Show MeSH
Related in: MedlinePlus