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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

Relative change of proteins involved in splice site selection. The relative change of proteins involved in splice site selection and splicing factors following TGF-β1 stimulation (10 ng/mL) was calculated. The result, when comparing the relative expression between every protein, is visualized as a color grid. Black box represents no statistically significant difference and light grey represents a significant difference of at least P <0.05. 1a denotes: RNA-binding region containing protein 2, 1b: splicing factor, proline-and-glutamine-rich (PTB-associated splicing factor, 1c: splicing factor 3 subunit 1, 1d: splicing factor 3A subunit 3, 1e: splicing factor 3B subunit 1, 1f: U2 small nuclear ribonucleoprotein auxiliary factor 35 Kda subunit related-protein 2, 1 g: splicing factor 3A subunit 2, 1 h: polyadenylate-binding protein 4, 1i: 54 Kda nuclear RNA- and DNA-binding protein (P54(Nrb)), 1j: FUSE binding protein 2, 1 k: splicing factor 3B subunit 3 (spliceosome associated protein 130), and 1 l: splicing factor 3B subunit 793. 2a denotes: splicing factor, arginine/serine-rich 1 (ASF-1), 2b: splicing factor, arginine/serine-rich 7 (splicing factor 9 G8, 2c: splicing factor, arginine/serine-rich 3 (pre-Mrna splicing factor SRP20), 2d: splicing factor U2AF 35 splicing factor U2AF 35 Kda subunit (U2 auxiliary factor 35 Kda subunit), 2e: splicing factor U2AF 65 Kda subunit, 2f: splicing factor, arginine/serine-rich 9 (pre-Mrna splicing factor SRP30c), 2 g: splicing factor, arginine/serine-rich 6 (pre-Mrna splicing factor SRP55), 2 h: splicing factor, arginine/serine-rich 4 (pre-Mrna splicing factor SRP75), 2i: splicing factor arginine/serine-ich 5 (HRS) (pre-Mrna splicing factor SRP40).
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Figure 5: Relative change of proteins involved in splice site selection. The relative change of proteins involved in splice site selection and splicing factors following TGF-β1 stimulation (10 ng/mL) was calculated. The result, when comparing the relative expression between every protein, is visualized as a color grid. Black box represents no statistically significant difference and light grey represents a significant difference of at least P <0.05. 1a denotes: RNA-binding region containing protein 2, 1b: splicing factor, proline-and-glutamine-rich (PTB-associated splicing factor, 1c: splicing factor 3 subunit 1, 1d: splicing factor 3A subunit 3, 1e: splicing factor 3B subunit 1, 1f: U2 small nuclear ribonucleoprotein auxiliary factor 35 Kda subunit related-protein 2, 1 g: splicing factor 3A subunit 2, 1 h: polyadenylate-binding protein 4, 1i: 54 Kda nuclear RNA- and DNA-binding protein (P54(Nrb)), 1j: FUSE binding protein 2, 1 k: splicing factor 3B subunit 3 (spliceosome associated protein 130), and 1 l: splicing factor 3B subunit 793. 2a denotes: splicing factor, arginine/serine-rich 1 (ASF-1), 2b: splicing factor, arginine/serine-rich 7 (splicing factor 9 G8, 2c: splicing factor, arginine/serine-rich 3 (pre-Mrna splicing factor SRP20), 2d: splicing factor U2AF 35 splicing factor U2AF 35 Kda subunit (U2 auxiliary factor 35 Kda subunit), 2e: splicing factor U2AF 65 Kda subunit, 2f: splicing factor, arginine/serine-rich 9 (pre-Mrna splicing factor SRP30c), 2 g: splicing factor, arginine/serine-rich 6 (pre-Mrna splicing factor SRP55), 2 h: splicing factor, arginine/serine-rich 4 (pre-Mrna splicing factor SRP75), 2i: splicing factor arginine/serine-ich 5 (HRS) (pre-Mrna splicing factor SRP40).

Mentions: Interestingly, TGF-β1 altered the expression of splicing factors and additional proteins involved in splice site selection. The expression of splicing factor U2 small nuclear ribonucleoproteins auxiliary factor (65 kDa) (U2AF), and SRp20 were significantly increased by TGF-β1 (1.09-fold and 1.24-fold, respectively) (Additional file 1: Table S1). Furthermore, splicing factor SRp30c was significantly repressed (0.79-fold) following TGF-β1 stimulation. The relative expression of all proteins involved in splice site selection (grouped as splice site selection and splicing factors in Additional file 1: Table S1) was further statistically analyzed as shown in Figure 5. The data show that TGF-β1 significantly repressed the expression of SRp30C labeled ‘2d’ compared to all other splicing factors except SRp 9 G8 (2b). SRp20 (2c) was significantly increased compared to SRp1 (2a) and SRp30c (2d). When comparing the levels of SRp20 with SRp30c, the actual difference between these two splicing factors was 1.6-fold. Several other proteins were significantly changed upon TGF-β1 stimulation, such as RNA-binding region containing proteins, U5 snRNP 100 kDa protein, U5 small nuclear ribonucleoprotein 200 kDa, CGI-59 protein, and Cisplatin resistance-associated over expressed protein. In addition the expression of the helicase Nucleolar RNA helicase II (Nucleolar RNA helicase Gu) was also significantly different.


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)

Relative change of proteins involved in splice site selection. The relative change of proteins involved in splice site selection and splicing factors following TGF-β1 stimulation (10 ng/mL) was calculated. The result, when comparing the relative expression between every protein, is visualized as a color grid. Black box represents no statistically significant difference and light grey represents a significant difference of at least P <0.05. 1a denotes: RNA-binding region containing protein 2, 1b: splicing factor, proline-and-glutamine-rich (PTB-associated splicing factor, 1c: splicing factor 3 subunit 1, 1d: splicing factor 3A subunit 3, 1e: splicing factor 3B subunit 1, 1f: U2 small nuclear ribonucleoprotein auxiliary factor 35 Kda subunit related-protein 2, 1 g: splicing factor 3A subunit 2, 1 h: polyadenylate-binding protein 4, 1i: 54 Kda nuclear RNA- and DNA-binding protein (P54(Nrb)), 1j: FUSE binding protein 2, 1 k: splicing factor 3B subunit 3 (spliceosome associated protein 130), and 1 l: splicing factor 3B subunit 793. 2a denotes: splicing factor, arginine/serine-rich 1 (ASF-1), 2b: splicing factor, arginine/serine-rich 7 (splicing factor 9 G8, 2c: splicing factor, arginine/serine-rich 3 (pre-Mrna splicing factor SRP20), 2d: splicing factor U2AF 35 splicing factor U2AF 35 Kda subunit (U2 auxiliary factor 35 Kda subunit), 2e: splicing factor U2AF 65 Kda subunit, 2f: splicing factor, arginine/serine-rich 9 (pre-Mrna splicing factor SRP30c), 2 g: splicing factor, arginine/serine-rich 6 (pre-Mrna splicing factor SRP55), 2 h: splicing factor, arginine/serine-rich 4 (pre-Mrna splicing factor SRP75), 2i: splicing factor arginine/serine-ich 5 (HRS) (pre-Mrna splicing factor SRP40).
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Figure 5: Relative change of proteins involved in splice site selection. The relative change of proteins involved in splice site selection and splicing factors following TGF-β1 stimulation (10 ng/mL) was calculated. The result, when comparing the relative expression between every protein, is visualized as a color grid. Black box represents no statistically significant difference and light grey represents a significant difference of at least P <0.05. 1a denotes: RNA-binding region containing protein 2, 1b: splicing factor, proline-and-glutamine-rich (PTB-associated splicing factor, 1c: splicing factor 3 subunit 1, 1d: splicing factor 3A subunit 3, 1e: splicing factor 3B subunit 1, 1f: U2 small nuclear ribonucleoprotein auxiliary factor 35 Kda subunit related-protein 2, 1 g: splicing factor 3A subunit 2, 1 h: polyadenylate-binding protein 4, 1i: 54 Kda nuclear RNA- and DNA-binding protein (P54(Nrb)), 1j: FUSE binding protein 2, 1 k: splicing factor 3B subunit 3 (spliceosome associated protein 130), and 1 l: splicing factor 3B subunit 793. 2a denotes: splicing factor, arginine/serine-rich 1 (ASF-1), 2b: splicing factor, arginine/serine-rich 7 (splicing factor 9 G8, 2c: splicing factor, arginine/serine-rich 3 (pre-Mrna splicing factor SRP20), 2d: splicing factor U2AF 35 splicing factor U2AF 35 Kda subunit (U2 auxiliary factor 35 Kda subunit), 2e: splicing factor U2AF 65 Kda subunit, 2f: splicing factor, arginine/serine-rich 9 (pre-Mrna splicing factor SRP30c), 2 g: splicing factor, arginine/serine-rich 6 (pre-Mrna splicing factor SRP55), 2 h: splicing factor, arginine/serine-rich 4 (pre-Mrna splicing factor SRP75), 2i: splicing factor arginine/serine-ich 5 (HRS) (pre-Mrna splicing factor SRP40).
Mentions: Interestingly, TGF-β1 altered the expression of splicing factors and additional proteins involved in splice site selection. The expression of splicing factor U2 small nuclear ribonucleoproteins auxiliary factor (65 kDa) (U2AF), and SRp20 were significantly increased by TGF-β1 (1.09-fold and 1.24-fold, respectively) (Additional file 1: Table S1). Furthermore, splicing factor SRp30c was significantly repressed (0.79-fold) following TGF-β1 stimulation. The relative expression of all proteins involved in splice site selection (grouped as splice site selection and splicing factors in Additional file 1: Table S1) was further statistically analyzed as shown in Figure 5. The data show that TGF-β1 significantly repressed the expression of SRp30C labeled ‘2d’ compared to all other splicing factors except SRp 9 G8 (2b). SRp20 (2c) was significantly increased compared to SRp1 (2a) and SRp30c (2d). When comparing the levels of SRp20 with SRp30c, the actual difference between these two splicing factors was 1.6-fold. Several other proteins were significantly changed upon TGF-β1 stimulation, such as RNA-binding region containing proteins, U5 snRNP 100 kDa protein, U5 small nuclear ribonucleoprotein 200 kDa, CGI-59 protein, and Cisplatin resistance-associated over expressed protein. In addition the expression of the helicase Nucleolar RNA helicase II (Nucleolar RNA helicase Gu) was also significantly different.

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