Increased stability of heterogeneous ribonucleoproteins by a deacetylase inhibitor

• Published in: Biochimica et biophysica acta 2015-08, Vol.1849 (8), p.1095-1103
• Main Authors: Koumbadinga, Geremy A., Mahmood, Niaz, Lei, Lei, Kan, Yunchao, Cao, Wenguang, Lobo, Vincent G., Yao, Xiaojian, Zhang, Shetuan, Xie, Jiuyong
• Format: Article
• Language: English
• Subjects: Acetylcysteine - analogs & derivatives; Acetylcysteine - pharmacology; Amino Acid Sequence; Animals; Bcl-x splicing reporter; Breast cancer cell; Heterogeneous-Nuclear Ribonucleoproteins - genetics; Heterogeneous-Nuclear Ribonucleoproteins - metabolism; Histone Deacetylase Inhibitors - pharmacology; hnRNP F/H; Humans; Hydroxamic Acids - pharmacology; Lactacystin; Mutation; PC12 Cells; Protein Stability - drug effects; Rats; RNA Splicing - drug effects; RNA Splicing - genetics; TGFβ1; Transforming Growth Factor beta1 - pharmacology; TSA; Tumor Cells, Cultured
• ISSN: 1874-9399; 0006-3002; 1876-4320
• DOI: 10.1016/j.bbagrm.2015.05.001

Splicing factors are often influenced by various signaling pathways, contributing to the dynamic changes of protein isoforms in cells. Heterogeneous ribonucleoproteins (hnRNPs) regulate many steps of RNA metabolism including pre-mRNA splicing but their control by cell signaling particularly through acetylation and ubiquitination pathways remains largely unknown. Here we show that TSA, a deacetylase inhibitor, reduced the ratio of Bcl-x splice variants Bcl-xL/xS in MDA-MB-231 breast cancer cells. This TSA effect was independent of TGFβ1; however, only in the presence of TGFβ1 was TSA able to change the splicing regulators hnRNP F/H by slightly reducing their mRNA transcripts but strongly preventing protein degradation. The latter was also efficiently prevented by lactacystin, a proteasome inhibitor, suggesting their protein stability control by both acetylation and ubiquitination pathways. Three lysines K87, K98 and K224 of hnRNP F are potential targets of the mutually exclusive acetylation or ubiquitination (KAc/Ub) in the protein modification database PhosphoSitePlus. Mutating each of them but not a control non-KAc/Ub (K68) specifically abolished the TSA enhancement of protein stability. Moreover, mutating K98 (K98R) and K224 (K224R) also abolished the TSA regulation of alternative splicing of a Bcl-x mini-gene. Furthermore, about 86% (30 of 35) of the multi-functional hnRNP proteins in the database contain lysines that are potential sites for acetylation/ubiquitination. We demonstrate that the degradation of three of them (A1, I and L) are also prevented by TSA. Thus, the deacetylase inhibitor TSA enhances hnRNP F stability through the KAc/Ub lysines, with some of them essential for its regulation of alternative splicing. Such a regulation of protein stability is perhaps common for a group of hnRNPs and RNA metabolism. •TSA/lactacystin inhibited hnRNP F/H degradation in TGFβ1-treated breast cancer cells.•Mutating acetyl/ubiquityl (Ac/Ub) K87/K98/K224 of hnRNP F abolished the TSA effect.•K98R and K224R also abolished TSA-regulated Bcl-x splicing, an hnRNP F target.•KAc/Ub found in the majority of hnRNPs; three (A1, I and L) also stabilized by TSA