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In silico structural and functional characterization of the RSUME splice variants.

Gerez J, Fuertes M, Tedesco L, Silberstein S, Sevlever G, Paez-Pereda M, Holsboer F, Turjanski AG, Arzt E - PLoS ONE (2013)

Bottom Line: Comparing the structure of the RSUME isoforms we found that, in addition to the previously described RWD domain in the N-terminal, all these RSUME variants also contain an intermediate domain.We found that the C-terminal domain is dispensable for the SUMO-conjugation enhancer properties of RSUME.The results presented here show a degree of redundancy of the RSUME variants on the SUMO pathway.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA)-CONICET, Buenos Aires, Argentina.

ABSTRACT
RSUME (RWD-containing SUMO Enhancer) is a small protein that increases SUMO conjugation to proteins. To date, four splice variants that codify three RSUME isoforms have been described, which differ in their C-terminal end. Comparing the structure of the RSUME isoforms we found that, in addition to the previously described RWD domain in the N-terminal, all these RSUME variants also contain an intermediate domain. Only the longest RSUME isoform presents a C-terminal domain that is absent in the others. Given these differences, we used the shortest and longest RSUME variants for comparative studies. We found that the C-terminal domain is dispensable for the SUMO-conjugation enhancer properties of RSUME. We also demonstrate that these two RSUME variants are equally induced by hypoxia. The NF-κB signaling pathway is inhibited and the HIF-1 pathway is increased more efficiently by the longest RSUME, by means of a greater physical interaction of RSUME267 with the target proteins. In addition, the mRNA and protein levels of these isoforms differ in human glioma samples; while the shortest RSUME isoform is expressed in all the tumors analyzed, the longest variant is expressed in most but not all of them. The results presented here show a degree of redundancy of the RSUME variants on the SUMO pathway. However, the increased inhibition conferred by RSUME267 over the NF-κB signaling pathway, the increased activation over the HIF-1 pathway and the different expression of the RSUME isoforms suggest specific roles for each RSUME isoform which may be relevant in certain types of brain tumors that express RSUME, like human pituitary adenomas and gliomas.

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mRNA and protein expression of RSUME195 and RSUME267 in cells and tumors.A. Semi-quantitative RT-PCR with specific primers to detect mRNA levels of RSUME195 (left panel) and RSUME267 (right panel), was performed in COS-7 cells exposed to normoxic or hypoxic conditions (1% O2, 5% CO2 and 94% N2). B. Semi-quantitative RT-PCR with specific primers to detect mRNA levels of RSUME195 and RSUME267, was performed on an unstimulated human non-functioning pituitary tumor explant sample. C. Semi-quantitative RT-PCR with specific primers to detect mRNA levels of RSUME195 and RSUME267 was performed on human glioma samples. D. Western blot analysis of RSUME protein levels was performed on human glioma samples. β-actin protein levels were used as control.
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pone-0057795-g007: mRNA and protein expression of RSUME195 and RSUME267 in cells and tumors.A. Semi-quantitative RT-PCR with specific primers to detect mRNA levels of RSUME195 (left panel) and RSUME267 (right panel), was performed in COS-7 cells exposed to normoxic or hypoxic conditions (1% O2, 5% CO2 and 94% N2). B. Semi-quantitative RT-PCR with specific primers to detect mRNA levels of RSUME195 and RSUME267, was performed on an unstimulated human non-functioning pituitary tumor explant sample. C. Semi-quantitative RT-PCR with specific primers to detect mRNA levels of RSUME195 and RSUME267 was performed on human glioma samples. D. Western blot analysis of RSUME protein levels was performed on human glioma samples. β-actin protein levels were used as control.

Mentions: In COS-7 cells and in human pituitary tumors, where RSUME195 has been previously shown to be expressed [1], RSUME267 is also evident in normoxic conditions (Fig. 7A y 7B). In COS-7 cells we also observed co-expression of both isoforms under hypoxic conditions (Fig. 7A). We further evaluated the expression pattern of these RSUME variants in human gliomas, in which RSUME195 expression has been previously shown at protein level [1]. We found low basal level expression of RSUME195 mRNAs in all gliomas analyzed. In contrast, RSUME267 is expressed in most but not in all these tumors (Fig. 7C). Furthermore, RSUME195 mRNA levels are similar in all the tumors tested, but the expression levels of RSUME267 mRNA differ. Similarly, in a second serie of tumor samples, all of the gliomas analyzed showed RSUME195 protein expression, while only a few gliomas showed expression of RSUME267 at protein level (Fig. 7D). These results may suggest nonredundant functions between these two RSUME variants and a constitutive role of RSUME195 in this type of human brain tumors.


In silico structural and functional characterization of the RSUME splice variants.

Gerez J, Fuertes M, Tedesco L, Silberstein S, Sevlever G, Paez-Pereda M, Holsboer F, Turjanski AG, Arzt E - PLoS ONE (2013)

mRNA and protein expression of RSUME195 and RSUME267 in cells and tumors.A. Semi-quantitative RT-PCR with specific primers to detect mRNA levels of RSUME195 (left panel) and RSUME267 (right panel), was performed in COS-7 cells exposed to normoxic or hypoxic conditions (1% O2, 5% CO2 and 94% N2). B. Semi-quantitative RT-PCR with specific primers to detect mRNA levels of RSUME195 and RSUME267, was performed on an unstimulated human non-functioning pituitary tumor explant sample. C. Semi-quantitative RT-PCR with specific primers to detect mRNA levels of RSUME195 and RSUME267 was performed on human glioma samples. D. Western blot analysis of RSUME protein levels was performed on human glioma samples. β-actin protein levels were used as control.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585135&req=5

pone-0057795-g007: mRNA and protein expression of RSUME195 and RSUME267 in cells and tumors.A. Semi-quantitative RT-PCR with specific primers to detect mRNA levels of RSUME195 (left panel) and RSUME267 (right panel), was performed in COS-7 cells exposed to normoxic or hypoxic conditions (1% O2, 5% CO2 and 94% N2). B. Semi-quantitative RT-PCR with specific primers to detect mRNA levels of RSUME195 and RSUME267, was performed on an unstimulated human non-functioning pituitary tumor explant sample. C. Semi-quantitative RT-PCR with specific primers to detect mRNA levels of RSUME195 and RSUME267 was performed on human glioma samples. D. Western blot analysis of RSUME protein levels was performed on human glioma samples. β-actin protein levels were used as control.
Mentions: In COS-7 cells and in human pituitary tumors, where RSUME195 has been previously shown to be expressed [1], RSUME267 is also evident in normoxic conditions (Fig. 7A y 7B). In COS-7 cells we also observed co-expression of both isoforms under hypoxic conditions (Fig. 7A). We further evaluated the expression pattern of these RSUME variants in human gliomas, in which RSUME195 expression has been previously shown at protein level [1]. We found low basal level expression of RSUME195 mRNAs in all gliomas analyzed. In contrast, RSUME267 is expressed in most but not in all these tumors (Fig. 7C). Furthermore, RSUME195 mRNA levels are similar in all the tumors tested, but the expression levels of RSUME267 mRNA differ. Similarly, in a second serie of tumor samples, all of the gliomas analyzed showed RSUME195 protein expression, while only a few gliomas showed expression of RSUME267 at protein level (Fig. 7D). These results may suggest nonredundant functions between these two RSUME variants and a constitutive role of RSUME195 in this type of human brain tumors.

Bottom Line: Comparing the structure of the RSUME isoforms we found that, in addition to the previously described RWD domain in the N-terminal, all these RSUME variants also contain an intermediate domain.We found that the C-terminal domain is dispensable for the SUMO-conjugation enhancer properties of RSUME.The results presented here show a degree of redundancy of the RSUME variants on the SUMO pathway.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA)-CONICET, Buenos Aires, Argentina.

ABSTRACT
RSUME (RWD-containing SUMO Enhancer) is a small protein that increases SUMO conjugation to proteins. To date, four splice variants that codify three RSUME isoforms have been described, which differ in their C-terminal end. Comparing the structure of the RSUME isoforms we found that, in addition to the previously described RWD domain in the N-terminal, all these RSUME variants also contain an intermediate domain. Only the longest RSUME isoform presents a C-terminal domain that is absent in the others. Given these differences, we used the shortest and longest RSUME variants for comparative studies. We found that the C-terminal domain is dispensable for the SUMO-conjugation enhancer properties of RSUME. We also demonstrate that these two RSUME variants are equally induced by hypoxia. The NF-κB signaling pathway is inhibited and the HIF-1 pathway is increased more efficiently by the longest RSUME, by means of a greater physical interaction of RSUME267 with the target proteins. In addition, the mRNA and protein levels of these isoforms differ in human glioma samples; while the shortest RSUME isoform is expressed in all the tumors analyzed, the longest variant is expressed in most but not all of them. The results presented here show a degree of redundancy of the RSUME variants on the SUMO pathway. However, the increased inhibition conferred by RSUME267 over the NF-κB signaling pathway, the increased activation over the HIF-1 pathway and the different expression of the RSUME isoforms suggest specific roles for each RSUME isoform which may be relevant in certain types of brain tumors that express RSUME, like human pituitary adenomas and gliomas.

Show MeSH
Related in: MedlinePlus