In silico structural and functional characterization of the RSUME splice variants

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.

Figure 1. The human RSUME splice variants.

Schematic illustration of the RSUME pre-mRNA (above) and the four human RSUME mRNA transcripts splice variants documented to the date. The variant 1 (NM_015485) encodes the longest isoform of RSUME containing 267 aminoacids (RSUME267); the variant 2 (NM_ 001128142) encodes the shortest isoform of RSUME containing 195 aminoacids (RSUME195); the variant 3 (NM-001199682) encodes a 200 aminoacids RSUME (RSUME200); and the variant 4 (NR_037643) is a non-coding RNA because it presents a premature stop codon in the exon 2 that goes to degradation via NMD. Boxes, exons with the exon number inside; filled black lines, introns; gray lines, splicing events (filled, splice event that originates the variant 1; dashed, variant 2; dotted, variant 3; streak and point, variant 4); +1, transcription start site; AUG, translation start site; UAA, UGA and UAG, translation stop sites. This information was obtained from published sequences in the University of California, Santa Cruz, genome browser (http://genome.ucsc.edu/).

Figure 2. RSUME structural features.

A. BLAST multiple sequence alignment for RSUME267 (NP_056300), RSUME195 (NP_001121614) and RSUME200 (NP_001186611) isoforms. The identical aminoacids in the alignment are showed in gray. B. Ribbon Representation showing the secondary structure elements (alpha helixes are purple, beta-sheets are yellow and loops are cyan) of the RWD domain of RSUME obtained from the PDB (PDBid: 2EBK). The figure was made with the program VMD .

Figure 3. Domain characterization of RSUME.

A. Secondary structure prediction with PHYRE Server of the whole RSUME267 amino acid sequence. B. Ribbon representation of the RSUME267 structure as predicted by the PHYRE Server, showing the secondary structure elements: alpha helixes (purple), beta-sheets (yellow), loops (cyan). The Figure was made with the program VMD . C. The same structure that in B but different colors encompassing the different regions of the RSUME267 protein. Blue residues 1 to 123, red residues 124 to 137, green residues 137 to 195, brown residues 196 to 267. D. Schematic comparison of the domains present in the three RSUME isoforms and in the RWD domain containing superfamily. Boxes, structural domains; lines, loops or unstructured regions; numbers, amino acid position.

Figure 4. RSUME267 and RSUME195 similar actions on the SUMO pathway.

A. 1 µg of recombinant RSUME195 or RSUME267 was co-precipitated by GST-Ubc9 in vitro. Pull down experiments were performed and the samples were subjected to Western blot. To compare, the ratio between input and pull-down intensities measured by Image-J Software was calculated. As internal control, to detect unspecific binding, GST alone was used to pull-down any RSUME isoform. Ø, control elution extract purified from E.coli transformed with the empty pQE30 vector. B. 300 ng HA-SUMO-1, expression vector was co-transfected with 500 ng of V5-RSUME195 or V5-RSUME267 and V5-Ubc9 expression vectors. 48 h post-transfection, cell extracts were subjected to western blot with anti-HA to detect sumoylated proteins. RSUME expression was confirmed with anti-RSUME antibodies. C. Sumoylation assay of Topoisomerase I (TOPOI) to test enhancer properties of RSUME isoforms. Experiments were performed in triplicates.

Figure 5. Effect of RSUME195 and RSUME267 over NF-κB signaling pathway.

A. COS-7 cells were co-transfected with RSUME and IκBα, inmunoprecipitated with anti-Flag antibody, and subjected to western blot with anti-V5 or anti-Flag antibodies. *, band corresponding to an IgG used in the inmunoprecipitation. B. and C. COS-7 cells were co-transfected with 500 ng of NF-κB-LUC reporter vector (B), or IL-8-LUC or IL-8(NF-κBmut)-LUC reporter vector (C), 300 ng of Gaussia as control and different concentrations of RSUME195 or 267 vectors (100 or 500 ng). After 24 h cells were stimulated with 10 ng/ml TNF-α for 6 h and luciferase (LUC) activity was measured in the cell extracts. Each value was normalized to Gaussia value. Results are expressed as mean ± SEM from triplicates of one representative experiment of three experiments with similar results. *, p<0.05 compared with cells transfected with the empty vector (Vect) stimulated with TNF-α (ANOVA with Scheffè’s test). ^#, p<0.05 compared each concentration of cells, stimulated with TNF-α, transfected with RSUME195 vs. RSUME267 (ANOVA with Scheffè’s test). D. IL-8 mRNA level was analyzed by quantitative real-time RT-PCR in triplicates in HepG2 cell stimulated with 10 ng/ml TNF-α for 6 h, and the values are given as mean ± SEM after normalization to RPL19. *, p<0.05 compared with cells transfected with the empty vector (Vect) (ANOVA with Scheffè’s test). ^#, p<0.05 compared the condition transfected with RSUME195 vs. RSUME267 (ANOVA with Scheffè’s test).

Figure 6. Effect of RSUME195 and RSUME267 over HIF-1 signaling pathway.

A. COS-7 cells were co-transfected with 500 ng of HRE-LUC report vector, 300 ng of Gaussia report vector and different concentrations of RSUME195 or 267 (100 or 300 ng) to evaluate their effect in HIF-1 transcriptional activity. Twenty-four hours after transfection cells were subjected to hypoxic conditions (1% O_2, 5% CO₂ and 94% N₂) for 16 h. Then LUC activity was measured in the cell extracts. Each value was normalized to Gaussia value. Results are expressed as mean ± SEM from triplicates of one representative experiment of three experiments with similar results. *, p<0.05 compared with cells transfected with the empty vector (Vect) (ANOVA with Scheffè’s test). ^#, p<0.05 compared each concentration of cells, under hypoxia, transfected with RSUME195 vs. RSUME267 (ANOVA with Scheffè’s test). NMX, normoxia; HPX, hypoxia. B. COS-7 cells were co-transfected with 300 ng of Flag-HIF-1alpha and/or 500 ng of V5-RSUME195 or V5-RSUME267, or the corresponding empty vector (were both are absent). Twenty-four hours post-transfection, cells were subjected to hypoxic conditions (1% O_2, 5% CO₂ and 94% N₂) for 16 h. Cell extracts were subjected to western blot. NMX, normoxia; HPX, hypoxia; Vect, cells transfected with the corresponding empty vectors. C. COS-7 cells were co-transfected with RSUME and HIF-1alpha. Twenty-four hours post-transfection, cells were subjected to hypoxic conditions (1% O_2, 5% CO₂ and 94% N₂) for 16 h. RIPA cell extracts were inmunoprecipitated with anti-Flag antibody, and subjected to western blot with anti-V5 or anti-Flag antibodies. D. COS-7 cells were co-transfected with 500 ng of VEGF-LUC report vector, 300 ng of CMV-β Gal report vector and RSUME195 or 267. Twenty-four hours after transfection cells were subjected to hypoxic conditions (1% O_2, 5% CO₂ and 94% N₂) for 16 h. Then LUC activity was measured in the cell extracts. Each value was normalized to β-galactosidase value. Results are expressed as mean ± SEM from triplicates of one representative experiment of three experiments with similar results. *, p<0.05 compared with cells transfected with the empty vector (Vect) under hypoxia (ANOVA with Scheffè’s test). ^#, p<0.05 compared the condition transfected with RSUME195 vs. RSUME267, under hypoxia (ANOVA with Scheffè’s test). NMX, normoxia; HPX, hypoxia. E. Semi-quantitative RT-PCR of endogenous VEGF and β-actin mRNA in HepG2 cells transfected with empty vector (Vect), RSUME195 or RSUME267, and subjected to hypoxia (1% O_2, 5% CO₂ and 94% N₂) for 16 h, twenty-four hours after transfection. F. VEGF mRNA level was analyzed by quantitative real-time RT-PCR in triplicates in HeLa cell stimulated with hypoxia for 16 h, and the values are given as mean ± SEM after normalization to RPL19. *, p<0.05 compared with cells transfected with the empty vector (Vect) (ANOVA with Scheffè’s test). ^#, p<0.05 compared the condition transfected with RSUME195 vs. RSUME267 (ANOVA with Scheffè’s test). G. HepG2 cells were transfected with empty vector (Vect), RSUME195 or RSUME267, and subjected to hypoxia (1% O_2, 5% CO₂ and 94% N₂) for 16 h, twenty-four hours after transfection. VEGF protein levels were analysed by western blot.

Figure 7. 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% O_2, 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.