Abstract

Protein modification by conjugation of small ubiquitin-related modifier (SUMO) is involved in diverse biological functions, such as transcription regulation, subcellular partitioning, stress response, DNA damage repair, and chromatin remodeling. Here, we show that the serine/arginine-rich protein SF2/ASF, a factor involved in splicing regulation and other RNA metabolism-related processes, is a regulator of the sumoylation pathway. The overexpression of this protein stimulates, but its knockdown inhibits SUMO conjugation. SF2/ASF interacts with Ubc9 and enhances sumoylation of specific substrates, sharing characteristics with already described SUMO E3 ligases. In addition, SF2/ASF interacts with the SUMO E3 ligase PIAS1 (protein inhibitor of activated STAT-1), regulating PIAS1-induced overall protein sumoylation. The RNA recognition motif 2 of SF2/ASF is necessary and sufficient for sumoylation enhancement. Moreover, SF2/ASF has a role in heat shock-induced sumoylation and promotes SUMO conjugation to RNA processing factors. These results add a component to the sumoylation pathway and a previously unexplored role for the multifunctional SR protein SF2/ASF.

Highlights

  • Protein modification by conjugation of small ubiquitin-related modifier (SUMO) is involved in diverse biological functions, such as transcription regulation, subcellular partitioning, stress response, DNA damage repair, and chromatin remodeling

  • We show that the serine/arginine-rich protein SF2/ASF, a factor involved in splicing regulation and other RNA metabolism-related processes, is a regulator of the sumoylation pathway

  • SF2/ASF has a role in hyperthermic stress-induced SUMO conjugation and stimulates the sumoylation of RNA processing factors. These results show that SF2/ASF acts as a cofactor stimulating SUMO conjugation: it displays some characteristics of an E3 ligase and regulates the function of the well-characterized E3 ligase PIAS1

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Summary

Results and Discussion

SF2/ASF Regulates Protein Sumoylation in Cultured Cells. Coexpression of SF2/ASF with mature SUMO1 in HEK 293T cells strongly stimulated global protein sumoylation. Another splicing regulator, heterogeneous nuclear ribonucleoprotein (hnRNP) A1, has little or no effect on protein sumoylation (Fig. 1A). The effect of SF2/ASF on sumoylation is dose-dependent and comparable to the one exerted by the SUMO E3 ligase PIAS1 (Fig. S1A). Diminishing SF2/ASF expression by siRNA reduces (Fig. 1C), but its overexpression stimulates SUMO2/3 conjugation in a dose-dependent manner (Fig. 1D). These results demonstrate that SF2/ASF is a hitherto unexplored regulator of the sumoylation pathway. Its physiological expression is required for maintaining normal overall sumoylation levels, as shown by the RNAi strategy, and reminiscent of the effect of the yeast E3 ligases Siz and Siz gene disruption that abolishes modification of most targets [38]

37 WB: b-actin 1 2 3 4 56 7
D GST-p53
15 WB: T7 b-actin
Materials and Methods
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