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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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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.
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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.

Mentions: Consistent with this view, other investigators have shown that endogenous pre-mRNA is processed in extremely large ribonucleoprotein particles, called supraspliceosomes (9–11) or polyspliceosomes (12). Biochemical and structural analyses of these complexes have demonstrated the presence of RNA Pol II transcripts (13,14) and the pre-mRNA splicing machinery components (15,16) as well as functional interactions that mirror those in active splicing complexes assembled in vitro (12). The higher order particles formed in vivo partly reflect the presence of multiple introns, an average of eight per pre-mRNA (17) with some transcripts possessing as many as 147 introns [Nebulin (18)], that need to be faithfully removed prior to nuclear export. In Figure 1, we present a schematic model of the pre-mRNA processing pathway in vivo that encompasses the concept of the supra/polyspliceosome. Whether the individual ‘spliceosome’ moieties are formed via stepwise snRNP assembly on individual introns (2) or via pre-formed penta-snRNPs (19) in vertebrates is still a matter of considerable debate, although recent chromatin immunoprecipitation experiments in human cells provide support for the penta-snRNP model (20).Figure 1.


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)

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

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

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.
Mentions: Consistent with this view, other investigators have shown that endogenous pre-mRNA is processed in extremely large ribonucleoprotein particles, called supraspliceosomes (9–11) or polyspliceosomes (12). Biochemical and structural analyses of these complexes have demonstrated the presence of RNA Pol II transcripts (13,14) and the pre-mRNA splicing machinery components (15,16) as well as functional interactions that mirror those in active splicing complexes assembled in vitro (12). The higher order particles formed in vivo partly reflect the presence of multiple introns, an average of eight per pre-mRNA (17) with some transcripts possessing as many as 147 introns [Nebulin (18)], that need to be faithfully removed prior to nuclear export. In Figure 1, we present a schematic model of the pre-mRNA processing pathway in vivo that encompasses the concept of the supra/polyspliceosome. Whether the individual ‘spliceosome’ moieties are formed via stepwise snRNP assembly on individual introns (2) or via pre-formed penta-snRNPs (19) in vertebrates is still a matter of considerable debate, although recent chromatin immunoprecipitation experiments in human cells provide support for the penta-snRNP model (20).Figure 1.

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