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Splicosomal and serine and arginine-rich splicing factors as targets for TGF-β.

Hallgren O, Malmström J, Malmström L, Andersson-Sjöland A, Wildt M, Tufvesson E, Juhasz P, Marko-Varga G, Westergren-Thorsson G - Fibrogenesis Tissue Repair (2012)

Bottom Line: Seventy-six of these proteins were associated with mRNA splicing, including 22 proteins involved in splice site selection.Specifically, TGF-β1 significantly induced expression of SRp20, and reduced the expression of SRp30C, which has been suggested to be a prerequisite for generation of alternatively spliced fibronectin.The results show that TGF-β1 induces the expression of proteins involved in mRNA splicing and RNA processing in human lung fibroblasts.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Experimental Medical Science, Lund University, Lund, Sweden. oskar.hallgren@med.lu.se.

ABSTRACT

Background: Transforming growth factor-β1 (TGF-β1) is a potent regulator of cell growth and differentiation. TGF-β1 has been shown to be a key player in tissue remodeling processes in a number of disease states by inducing expression of extracellular matrix proteins. In this study a quantitative proteomic analysis was undertaken to investigate if TGF-β1 contributes to tissue remodeling by mediating mRNA splicing and production of alternative isoforms of proteins.

Methodology/principal findings: The expression of proteins involved in mRNA splicing from TGF-β1-stimulated lung fibroblasts was compared to non-stimulated cells by employing isotope coded affinity tag (ICATTM) reagent labeling and tandem mass spectrometry. A total of 1733 proteins were identified and quantified with a relative standard deviation of 11% +/- 8 from enriched nuclear fractions. Seventy-six of these proteins were associated with mRNA splicing, including 22 proteins involved in splice site selection. In addition, TGF-β1 was observed to alter the relative expression of splicing proteins that may be important for alternative splicing of fibronectin. Specifically, TGF-β1 significantly induced expression of SRp20, and reduced the expression of SRp30C, which has been suggested to be a prerequisite for generation of alternatively spliced fibronectin. The induction of SRp20 was further confirmed by western blot and immunofluorescence.

Conclusions: The results show that TGF-β1 induces the expression of proteins involved in mRNA splicing and RNA processing in human lung fibroblasts. This may have an impact on the production of alternative isoforms of matrix proteins and can therefore be an important factor in tissue remodeling and disease progression.

No MeSH data available.


Related in: MedlinePlus

Classification according to molecular function of the proteins involved in pre-mRNA splicing. Following TGF-β1 stimulation (10 ng/mL) 76 ICAT labeled proteins involved in pre-RNA splicing and RNA processing were identified. The most abundant groups of proteins identified were proteins involved in RNA processing, splicing factors, and other proteins involved in splice site selection.
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Figure 4: Classification according to molecular function of the proteins involved in pre-mRNA splicing. Following TGF-β1 stimulation (10 ng/mL) 76 ICAT labeled proteins involved in pre-RNA splicing and RNA processing were identified. The most abundant groups of proteins identified were proteins involved in RNA processing, splicing factors, and other proteins involved in splice site selection.

Mentions: To address the question whether TGF-β1 stimulation affects nuclear proteins that are involved in the splicing process, we employed isotope coded affinity tag (ICATTM) reagent labeling and tandem mass spectrometry. Our experiments showed that the expression of EDA fibronectin increased from 6 to 48 h of TGF-β1 stimulation (Figure 2A), which indicates that an alternative splice site selection was induced after 24 h. We therefore decided to examine the expression of nuclear proteins at this time point. Figure 3 shows a schematic overview of the workflow used for the identification and relative quantification of proteins in the nuclear fractions [30-32]. Around 2500 proteins were identified in multiple LC-MS/MS experiments. This list was reduced to approximately 2000 unique proteins after consolidating the peptide-protein assignments. However, approximately 1,700 of these could be quantified after the proper normalization of the relative abundances of ‘heavy’ and ‘light’ ICATTM reagent labeled peptides. After classifying the proteins based on gene ontology [33], 76 proteins involved in pre-mRNA splicing and RNA processing were selected and divided into 13 functional groups as shown in Figure 4 and in Additional file 1: Table S1. The most abundant group was classified as heterogeneous ribonucleoproteins and contained 19 proteins (hnRNP). The second most abundant group (12 proteins) was proposed to be involved in splice site selection and the third most abundant group, containing 10 proteins, was classified as splicing factors. Nineteen proteins were subunits of the snRNPs or associated with the U1, U5, or U4/U6 snRNPs. In addition, helicases and peptidyl-prolyl cis-isomerases were detected. The relative standard deviation (RSD) was calculated from all ratios of the total list of identified proteins matched by more than one peptide and the average of this error was found to be 11% +/− 8. The proteome wide error was less than 20%, and in the case of the splicing factors the majority of RSDs was less than 20%.


Splicosomal and serine and arginine-rich splicing factors as targets for TGF-β.

Hallgren O, Malmström J, Malmström L, Andersson-Sjöland A, Wildt M, Tufvesson E, Juhasz P, Marko-Varga G, Westergren-Thorsson G - Fibrogenesis Tissue Repair (2012)

Classification according to molecular function of the proteins involved in pre-mRNA splicing. Following TGF-β1 stimulation (10 ng/mL) 76 ICAT labeled proteins involved in pre-RNA splicing and RNA processing were identified. The most abundant groups of proteins identified were proteins involved in RNA processing, splicing factors, and other proteins involved in splice site selection.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472233&req=5

Figure 4: Classification according to molecular function of the proteins involved in pre-mRNA splicing. Following TGF-β1 stimulation (10 ng/mL) 76 ICAT labeled proteins involved in pre-RNA splicing and RNA processing were identified. The most abundant groups of proteins identified were proteins involved in RNA processing, splicing factors, and other proteins involved in splice site selection.
Mentions: To address the question whether TGF-β1 stimulation affects nuclear proteins that are involved in the splicing process, we employed isotope coded affinity tag (ICATTM) reagent labeling and tandem mass spectrometry. Our experiments showed that the expression of EDA fibronectin increased from 6 to 48 h of TGF-β1 stimulation (Figure 2A), which indicates that an alternative splice site selection was induced after 24 h. We therefore decided to examine the expression of nuclear proteins at this time point. Figure 3 shows a schematic overview of the workflow used for the identification and relative quantification of proteins in the nuclear fractions [30-32]. Around 2500 proteins were identified in multiple LC-MS/MS experiments. This list was reduced to approximately 2000 unique proteins after consolidating the peptide-protein assignments. However, approximately 1,700 of these could be quantified after the proper normalization of the relative abundances of ‘heavy’ and ‘light’ ICATTM reagent labeled peptides. After classifying the proteins based on gene ontology [33], 76 proteins involved in pre-mRNA splicing and RNA processing were selected and divided into 13 functional groups as shown in Figure 4 and in Additional file 1: Table S1. The most abundant group was classified as heterogeneous ribonucleoproteins and contained 19 proteins (hnRNP). The second most abundant group (12 proteins) was proposed to be involved in splice site selection and the third most abundant group, containing 10 proteins, was classified as splicing factors. Nineteen proteins were subunits of the snRNPs or associated with the U1, U5, or U4/U6 snRNPs. In addition, helicases and peptidyl-prolyl cis-isomerases were detected. The relative standard deviation (RSD) was calculated from all ratios of the total list of identified proteins matched by more than one peptide and the average of this error was found to be 11% +/− 8. The proteome wide error was less than 20%, and in the case of the splicing factors the majority of RSDs was less than 20%.

Bottom Line: Seventy-six of these proteins were associated with mRNA splicing, including 22 proteins involved in splice site selection.Specifically, TGF-β1 significantly induced expression of SRp20, and reduced the expression of SRp30C, which has been suggested to be a prerequisite for generation of alternatively spliced fibronectin.The results show that TGF-β1 induces the expression of proteins involved in mRNA splicing and RNA processing in human lung fibroblasts.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Experimental Medical Science, Lund University, Lund, Sweden. oskar.hallgren@med.lu.se.

ABSTRACT

Background: Transforming growth factor-β1 (TGF-β1) is a potent regulator of cell growth and differentiation. TGF-β1 has been shown to be a key player in tissue remodeling processes in a number of disease states by inducing expression of extracellular matrix proteins. In this study a quantitative proteomic analysis was undertaken to investigate if TGF-β1 contributes to tissue remodeling by mediating mRNA splicing and production of alternative isoforms of proteins.

Methodology/principal findings: The expression of proteins involved in mRNA splicing from TGF-β1-stimulated lung fibroblasts was compared to non-stimulated cells by employing isotope coded affinity tag (ICATTM) reagent labeling and tandem mass spectrometry. A total of 1733 proteins were identified and quantified with a relative standard deviation of 11% +/- 8 from enriched nuclear fractions. Seventy-six of these proteins were associated with mRNA splicing, including 22 proteins involved in splice site selection. In addition, TGF-β1 was observed to alter the relative expression of splicing proteins that may be important for alternative splicing of fibronectin. Specifically, TGF-β1 significantly induced expression of SRp20, and reduced the expression of SRp30C, which has been suggested to be a prerequisite for generation of alternatively spliced fibronectin. The induction of SRp20 was further confirmed by western blot and immunofluorescence.

Conclusions: The results show that TGF-β1 induces the expression of proteins involved in mRNA splicing and RNA processing in human lung fibroblasts. This may have an impact on the production of alternative isoforms of matrix proteins and can therefore be an important factor in tissue remodeling and disease progression.

No MeSH data available.


Related in: MedlinePlus