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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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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.
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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.

Mentions: Using our recently developed CLEP tagging procedure (22), we tagged the SmD3 polypeptide in chicken DT40 cells by introducing a TAP tag (24) at the native genomic locus. For each experiment, 6 l of SmD3-TAP-DT40 cells were harvested and processed as described for purification of supraspliceosomes from HeLa cells. Affinity chromatography was performed according to the TAP procedure (24) and the TEV eluate was sedimented through a glycerol gradient. The material corresponding to the supraspliceosomes was isolated; proteins and nucleic acids are shown in Figures 3A and B, respectively. In Figure S1, we show the proteins (panel C) and RNA (panel D) resulting from an identical affinity purification procedure performed using extracts from untagged DT40 cells. The absence of proteins, beyond the contaminating TEV protease, and the absence of snRNAs indicates that the purification is specific and the proteins identified by mass spectrometry are likely to be bona fide supraspliceosome components. Additional confidence is provided in that there is size-selection as well as one or two steps of affinity chromatography.Figure 3.


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)

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

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

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.
Mentions: Using our recently developed CLEP tagging procedure (22), we tagged the SmD3 polypeptide in chicken DT40 cells by introducing a TAP tag (24) at the native genomic locus. For each experiment, 6 l of SmD3-TAP-DT40 cells were harvested and processed as described for purification of supraspliceosomes from HeLa cells. Affinity chromatography was performed according to the TAP procedure (24) and the TEV eluate was sedimented through a glycerol gradient. The material corresponding to the supraspliceosomes was isolated; proteins and nucleic acids are shown in Figures 3A and B, respectively. In Figure S1, we show the proteins (panel C) and RNA (panel D) resulting from an identical affinity purification procedure performed using extracts from untagged DT40 cells. The absence of proteins, beyond the contaminating TEV protease, and the absence of snRNAs indicates that the purification is specific and the proteins identified by mass spectrometry are likely to be bona fide supraspliceosome components. Additional confidence is provided in that there is size-selection as well as one or two steps of affinity chromatography.Figure 3.

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