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Transcriptional co-activator protein p100 interacts with snRNP proteins and facilitates the assembly of the spliceosome.

Yang J, Välineva T, Hong J, Bu T, Yao Z, Jensen ON, Frilander MJ, Silvennoinen O - Nucleic Acids Res. (2007)

Bottom Line: Here we identified interaction between p100 and small nuclear ribonucleoproteins (snRNP) that function in pre-mRNA splicing.The TSN domain of p100 specifically interacts with components of the U5 snRNP, but also with the other spliceosomal snRNPs.Thus our results suggest that p100 protein is a novel dual function regulator of gene expression that participates via distinct domains in both transcription and splicing.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Tianjin Medical University, Heping District Qixiangtai Road No.22, 300070 Tianjin, PR China. yangj@tijmu.edu.cn

ABSTRACT
Transcription and pre-mRNA splicing are the key nuclear processes in eukaryotic gene expression, and identification of factors common to both processes has suggested that they are functionally coordinated. p100 protein has been shown to function as a transcriptional co-activator for several transcription factors. p100 consists of staphylococcal nuclease (SN)-like and Tudor-SN (TSN) domains of which the SN-like domains have been shown to function in transcription, but the function of TSN domain has remained elusive. Here we identified interaction between p100 and small nuclear ribonucleoproteins (snRNP) that function in pre-mRNA splicing. The TSN domain of p100 specifically interacts with components of the U5 snRNP, but also with the other spliceosomal snRNPs. In vitro splicing assays revealed that the purified p100, and specifically the TSN domain of p100, accelerates the kinetics of the spliceosome assembly, particularly the formation of complex A, and the transition from complex A to B. Consistently, the p100 protein, as well as the separated TSN domain, enhanced the kinetics of the first step of splicing in an in vitro splicing assay in dose-dependent manner. Thus our results suggest that p100 protein is a novel dual function regulator of gene expression that participates via distinct domains in both transcription and splicing.

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Interaction between p100 protein and U5 snRNP specific proteins. (A) The schematic structure of p100 protein (B) Pulldown of U5 specific proteins using TSN domain of p100. Aliquots of nuclear lysates from HeLa cells were incubated with either GST alone or with GST-p100-TSN. The bound proteins were subjected to SDS–PAGE and visualized by Coomassie blue or silver staining and identified by mass spectrometry. (C) The loading control of GST and GST-p100-TSN proteins.
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Figure 1: Interaction between p100 protein and U5 snRNP specific proteins. (A) The schematic structure of p100 protein (B) Pulldown of U5 specific proteins using TSN domain of p100. Aliquots of nuclear lysates from HeLa cells were incubated with either GST alone or with GST-p100-TSN. The bound proteins were subjected to SDS–PAGE and visualized by Coomassie blue or silver staining and identified by mass spectrometry. (C) The loading control of GST and GST-p100-TSN proteins.

Mentions: Human p100 protein is composed of four repeats of SN-like domains followed by a C-terminal TSN domain (Figure 1A). The SN consists of two subdomains. The first subdomain belongs to the large oligonucleotide/oligosaccharidebinding (OB)-fold superfamily (26), and the second subdomain consists of two independently folded α-helices. The TSN domain is a hybrid SN domain, in which the OB-domain is divided by a domain found in Drosophila melanogaster Tudor protein. According to the crystal structure of p100 TSN domain (Shaw,N. et al., submitted for publication), the Tudor domain is flanked by two segments of SN, thus we refer it as TSN domain instead, to differentiate it from the bona fide Tudor domains.Figure 1.


Transcriptional co-activator protein p100 interacts with snRNP proteins and facilitates the assembly of the spliceosome.

Yang J, Välineva T, Hong J, Bu T, Yao Z, Jensen ON, Frilander MJ, Silvennoinen O - Nucleic Acids Res. (2007)

Interaction between p100 protein and U5 snRNP specific proteins. (A) The schematic structure of p100 protein (B) Pulldown of U5 specific proteins using TSN domain of p100. Aliquots of nuclear lysates from HeLa cells were incubated with either GST alone or with GST-p100-TSN. The bound proteins were subjected to SDS–PAGE and visualized by Coomassie blue or silver staining and identified by mass spectrometry. (C) The loading control of GST and GST-p100-TSN proteins.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1935017&req=5

Figure 1: Interaction between p100 protein and U5 snRNP specific proteins. (A) The schematic structure of p100 protein (B) Pulldown of U5 specific proteins using TSN domain of p100. Aliquots of nuclear lysates from HeLa cells were incubated with either GST alone or with GST-p100-TSN. The bound proteins were subjected to SDS–PAGE and visualized by Coomassie blue or silver staining and identified by mass spectrometry. (C) The loading control of GST and GST-p100-TSN proteins.
Mentions: Human p100 protein is composed of four repeats of SN-like domains followed by a C-terminal TSN domain (Figure 1A). The SN consists of two subdomains. The first subdomain belongs to the large oligonucleotide/oligosaccharidebinding (OB)-fold superfamily (26), and the second subdomain consists of two independently folded α-helices. The TSN domain is a hybrid SN domain, in which the OB-domain is divided by a domain found in Drosophila melanogaster Tudor protein. According to the crystal structure of p100 TSN domain (Shaw,N. et al., submitted for publication), the Tudor domain is flanked by two segments of SN, thus we refer it as TSN domain instead, to differentiate it from the bona fide Tudor domains.Figure 1.

Bottom Line: Here we identified interaction between p100 and small nuclear ribonucleoproteins (snRNP) that function in pre-mRNA splicing.The TSN domain of p100 specifically interacts with components of the U5 snRNP, but also with the other spliceosomal snRNPs.Thus our results suggest that p100 protein is a novel dual function regulator of gene expression that participates via distinct domains in both transcription and splicing.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Tianjin Medical University, Heping District Qixiangtai Road No.22, 300070 Tianjin, PR China. yangj@tijmu.edu.cn

ABSTRACT
Transcription and pre-mRNA splicing are the key nuclear processes in eukaryotic gene expression, and identification of factors common to both processes has suggested that they are functionally coordinated. p100 protein has been shown to function as a transcriptional co-activator for several transcription factors. p100 consists of staphylococcal nuclease (SN)-like and Tudor-SN (TSN) domains of which the SN-like domains have been shown to function in transcription, but the function of TSN domain has remained elusive. Here we identified interaction between p100 and small nuclear ribonucleoproteins (snRNP) that function in pre-mRNA splicing. The TSN domain of p100 specifically interacts with components of the U5 snRNP, but also with the other spliceosomal snRNPs. In vitro splicing assays revealed that the purified p100, and specifically the TSN domain of p100, accelerates the kinetics of the spliceosome assembly, particularly the formation of complex A, and the transition from complex A to B. Consistently, the p100 protein, as well as the separated TSN domain, enhanced the kinetics of the first step of splicing in an in vitro splicing assay in dose-dependent manner. Thus our results suggest that p100 protein is a novel dual function regulator of gene expression that participates via distinct domains in both transcription and splicing.

Show MeSH