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Tip110 interacts with YB-1 and regulates each other's function.

Timani KA, Liu Y, He JJ - BMC Mol. Biol. (2013)

Bottom Line: The interaction of Tip110 with YB-1 was further dissected and confirmed to be specific and involve the N-terminal of both Tip110 and YB-1 proteins.We showed that YB-1 potentiates the Tip110/Tat-mediated transactivation of the HIV-1 LTR promoter while Tip110 promotes the inclusion of the exon 5 in CD44 minigene alternative splicing.Tip110 and YB-1 interact to form a complex and mutually regulate each other's biological functions.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of North Texas Health Science Center, Fort Worth, TX 76107, USA.

ABSTRACT

Background: Tip110 plays important roles in tumor immunobiology, pre-mRNA splicing, expression regulation of viral and host genes, and possibly protein turnover. It is clear that our understanding of Tip110 biological function remains incomplete.

Results: Herein, we employed an immunoaffinity-based enrichment approach combined with protein mass spectrometry and attempted to identify Tip110-interacting cellular proteins. A total of 13 major proteins were identified to be complexed with Tip110. Among them was Y-box binding protein 1 (YB-1). The interaction of Tip110 with YB-1 was further dissected and confirmed to be specific and involve the N-terminal of both Tip110 and YB-1 proteins. A HIV-1 LTR promoter-driven reporter gene assay and a CD44 minigene in vivo splicing assay were chosen to evaluate the functional relevance of the Tip110/YB-1 interaction. We showed that YB-1 potentiates the Tip110/Tat-mediated transactivation of the HIV-1 LTR promoter while Tip110 promotes the inclusion of the exon 5 in CD44 minigene alternative splicing.

Conclusions: Tip110 and YB-1 interact to form a complex and mutually regulate each other's biological functions.

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Related in: MedlinePlus

Effects of Tip110 expression on YB-1-mediated alternative splicing of the CD44 minigene. A. Schematic of the CD44 minigene containing the variable exon 5 (V5). The locations of the primer set used for RT-PCR were marked by arrows, the dotted lines indicated two alternative splicing possibilities: one with V5 (top, +V5), the other without V5 (bottom, -V5). B. 293T cells were transfected with the CD44 minigene plasmid, pYB-1-Myc, pTip110-HA, or both. Input amounts of plasmids were shown at the top, all were in micrograms (μg). Total RNA was isolated and used for RT-PCR. The ratio of V5 inclusion (+V5/–V5) was determined by densitometric scanning and the ImageJ software and was normalized to that transfected with the CD44 minigene only. Expression of YB-1 and Tip110 was determined by Western blotting. β-actin was the loading control. C. 293T cells were transfected with the CD44 minigene, pYB-1-Myc, pTip110-HA, or each of the Tip110 mutants. The V5 inclusion and quantitation were performed as stated above.
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Figure 5: Effects of Tip110 expression on YB-1-mediated alternative splicing of the CD44 minigene. A. Schematic of the CD44 minigene containing the variable exon 5 (V5). The locations of the primer set used for RT-PCR were marked by arrows, the dotted lines indicated two alternative splicing possibilities: one with V5 (top, +V5), the other without V5 (bottom, -V5). B. 293T cells were transfected with the CD44 minigene plasmid, pYB-1-Myc, pTip110-HA, or both. Input amounts of plasmids were shown at the top, all were in micrograms (μg). Total RNA was isolated and used for RT-PCR. The ratio of V5 inclusion (+V5/–V5) was determined by densitometric scanning and the ImageJ software and was normalized to that transfected with the CD44 minigene only. Expression of YB-1 and Tip110 was determined by Western blotting. β-actin was the loading control. C. 293T cells were transfected with the CD44 minigene, pYB-1-Myc, pTip110-HA, or each of the Tip110 mutants. The V5 inclusion and quantitation were performed as stated above.

Mentions: One of the well-characterized functions of YB-1 is regulation of the alternative splicing of the CD44 gene through interaction with the A/C-rich exon enhancer element[24]. Therefore, we next determined the effects of Tip110 on YB-1-mediated CD44 alternative splicing. We took advantage of a CD44 minigene (Figure 5A) and performed in vivo RT-PCR-based splicing assay. Initial experiments were performed to optimize the input amount of CD44 minigene, YB-1 and Tip110 expression plasmids to ensure the RT-PCR-based detection of the alternative splicing (data not shown). As expected[24], YB-1 expression led to increased inclusion of the variable exon 5 (V5) of the CD44 minigene (Figure 5B, top panels). Tip110 expression alone appeared to have the similar enhancement effects. In the presence of YB-1, Tip110 increased inclusion of the V5 exon of CD44 minigene in a dose-dependent manner. Tip110 and YB-1 expression were determined by Western blotting to ensure that Tip110 expression did not alter YB-1 stability (Figure 5B, bottom panels). We next determined the requirement of Tip110 binding to YB-1 for this function. Similar in vivo splicing assay was performed with Tip110ΔNT and Tip110ΔCT mutants. Compared to the full-length Tip110, Tip110ΔNT and Tip110ΔCT expression alone showed little changes in alternative splicing of the CD44 minigene (Figure 3C). Interestingly, in the presence of YB-1, Tip110ΔNT also showed little effects, while Tip110ΔCT had considerable increase in V5 inclusion of the CD44 minigene. These results suggest that Tip110 binding with YB-1 plays some roles in YB-1 function.


Tip110 interacts with YB-1 and regulates each other's function.

Timani KA, Liu Y, He JJ - BMC Mol. Biol. (2013)

Effects of Tip110 expression on YB-1-mediated alternative splicing of the CD44 minigene. A. Schematic of the CD44 minigene containing the variable exon 5 (V5). The locations of the primer set used for RT-PCR were marked by arrows, the dotted lines indicated two alternative splicing possibilities: one with V5 (top, +V5), the other without V5 (bottom, -V5). B. 293T cells were transfected with the CD44 minigene plasmid, pYB-1-Myc, pTip110-HA, or both. Input amounts of plasmids were shown at the top, all were in micrograms (μg). Total RNA was isolated and used for RT-PCR. The ratio of V5 inclusion (+V5/–V5) was determined by densitometric scanning and the ImageJ software and was normalized to that transfected with the CD44 minigene only. Expression of YB-1 and Tip110 was determined by Western blotting. β-actin was the loading control. C. 293T cells were transfected with the CD44 minigene, pYB-1-Myc, pTip110-HA, or each of the Tip110 mutants. The V5 inclusion and quantitation were performed as stated above.
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Figure 5: Effects of Tip110 expression on YB-1-mediated alternative splicing of the CD44 minigene. A. Schematic of the CD44 minigene containing the variable exon 5 (V5). The locations of the primer set used for RT-PCR were marked by arrows, the dotted lines indicated two alternative splicing possibilities: one with V5 (top, +V5), the other without V5 (bottom, -V5). B. 293T cells were transfected with the CD44 minigene plasmid, pYB-1-Myc, pTip110-HA, or both. Input amounts of plasmids were shown at the top, all were in micrograms (μg). Total RNA was isolated and used for RT-PCR. The ratio of V5 inclusion (+V5/–V5) was determined by densitometric scanning and the ImageJ software and was normalized to that transfected with the CD44 minigene only. Expression of YB-1 and Tip110 was determined by Western blotting. β-actin was the loading control. C. 293T cells were transfected with the CD44 minigene, pYB-1-Myc, pTip110-HA, or each of the Tip110 mutants. The V5 inclusion and quantitation were performed as stated above.
Mentions: One of the well-characterized functions of YB-1 is regulation of the alternative splicing of the CD44 gene through interaction with the A/C-rich exon enhancer element[24]. Therefore, we next determined the effects of Tip110 on YB-1-mediated CD44 alternative splicing. We took advantage of a CD44 minigene (Figure 5A) and performed in vivo RT-PCR-based splicing assay. Initial experiments were performed to optimize the input amount of CD44 minigene, YB-1 and Tip110 expression plasmids to ensure the RT-PCR-based detection of the alternative splicing (data not shown). As expected[24], YB-1 expression led to increased inclusion of the variable exon 5 (V5) of the CD44 minigene (Figure 5B, top panels). Tip110 expression alone appeared to have the similar enhancement effects. In the presence of YB-1, Tip110 increased inclusion of the V5 exon of CD44 minigene in a dose-dependent manner. Tip110 and YB-1 expression were determined by Western blotting to ensure that Tip110 expression did not alter YB-1 stability (Figure 5B, bottom panels). We next determined the requirement of Tip110 binding to YB-1 for this function. Similar in vivo splicing assay was performed with Tip110ΔNT and Tip110ΔCT mutants. Compared to the full-length Tip110, Tip110ΔNT and Tip110ΔCT expression alone showed little changes in alternative splicing of the CD44 minigene (Figure 3C). Interestingly, in the presence of YB-1, Tip110ΔNT also showed little effects, while Tip110ΔCT had considerable increase in V5 inclusion of the CD44 minigene. These results suggest that Tip110 binding with YB-1 plays some roles in YB-1 function.

Bottom Line: The interaction of Tip110 with YB-1 was further dissected and confirmed to be specific and involve the N-terminal of both Tip110 and YB-1 proteins.We showed that YB-1 potentiates the Tip110/Tat-mediated transactivation of the HIV-1 LTR promoter while Tip110 promotes the inclusion of the exon 5 in CD44 minigene alternative splicing.Tip110 and YB-1 interact to form a complex and mutually regulate each other's biological functions.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of North Texas Health Science Center, Fort Worth, TX 76107, USA.

ABSTRACT

Background: Tip110 plays important roles in tumor immunobiology, pre-mRNA splicing, expression regulation of viral and host genes, and possibly protein turnover. It is clear that our understanding of Tip110 biological function remains incomplete.

Results: Herein, we employed an immunoaffinity-based enrichment approach combined with protein mass spectrometry and attempted to identify Tip110-interacting cellular proteins. A total of 13 major proteins were identified to be complexed with Tip110. Among them was Y-box binding protein 1 (YB-1). The interaction of Tip110 with YB-1 was further dissected and confirmed to be specific and involve the N-terminal of both Tip110 and YB-1 proteins. A HIV-1 LTR promoter-driven reporter gene assay and a CD44 minigene in vivo splicing assay were chosen to evaluate the functional relevance of the Tip110/YB-1 interaction. We showed that YB-1 potentiates the Tip110/Tat-mediated transactivation of the HIV-1 LTR promoter while Tip110 promotes the inclusion of the exon 5 in CD44 minigene alternative splicing.

Conclusions: Tip110 and YB-1 interact to form a complex and mutually regulate each other's biological functions.

Show MeSH
Related in: MedlinePlus