title: Increased stability of heterogeneous ribonucleoproteins by a deacetylase inhibitor.
abstract: 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 (K(Ac/Ub)) in the protein modification database PhosphoSitePlus. Mutating each of them but not a control non-K(Ac/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 K(Ac/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.
authors.affiliation: Department of Physiology & Pathophysiology, College of Medicine, Faculty of Health Sciences, University of Manitoba, Winnipeg, MB R3E 0J9, Canada.
authors.forename: Geremy A
authors.initials: GA
authors.lastname: Koumbadinga
authors.affiliation: Biochemistry & Medical Genetics, College of Medicine, Faculty of Health Sciences, University of Manitoba, Winnipeg, MB R3E 0J9, Canada.
authors.forename: Niaz
authors.initials: N
authors.lastname: Mahmood
authors.affiliation: Department of Physiology & Pathophysiology, College of Medicine, Faculty of Health Sciences, University of Manitoba, Winnipeg, MB R3E 0J9, Canada.
authors.forename: Lei
authors.initials: L
authors.lastname: Lei
authors.affiliation: Department of Physiology & Pathophysiology, College of Medicine, Faculty of Health Sciences, University of Manitoba, Winnipeg, MB R3E 0J9, Canada; Nanyang Normal University, Nanyang, Henan, PR China.
authors.forename: Yunchao
authors.initials: Y
authors.lastname: Kan
authors.affiliation: Department of Physiology & Pathophysiology, College of Medicine, Faculty of Health Sciences, University of Manitoba, Winnipeg, MB R3E 0J9, Canada.
authors.forename: Wenguang
authors.initials: W
authors.lastname: Cao
authors.affiliation: Department of Physiology & Pathophysiology, College of Medicine, Faculty of Health Sciences, University of Manitoba, Winnipeg, MB R3E 0J9, Canada.
authors.forename: Vincent G
authors.initials: VG
authors.lastname: Lobo
authors.affiliation: Department of Medical Microbiology, College of Medicine, Faculty of Health Sciences, University of Manitoba, Winnipeg, MB R3E 0J9, Canada.
authors.forename: Xiaojian
authors.initials: X
authors.lastname: Yao
authors.affiliation: Department of Biomedical and Molecular Sciences, Queens University, Kingston, ON, Canada.
authors.forename: Shetuan
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Department of Physiology & Pathophysiology, College of Medicine, Faculty of Health Sciences, University of Manitoba, Winnipeg, MB R3E 0J9, Canada; Biochemistry & Medical Genetics, College of Medicine, Faculty of Health Sciences, University of Manitoba, Winnipeg, MB R3E 0J9, Canada. Electronic address: xiej@umanitoba.ca.
authors.forename: Jiuyong
authors.initials: J
authors.lastname: Xie
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000235:genetics / Q000378:metabolism*; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D009154:Mutation; D016716:PC12 Cells; D055550:Protein Stability / Q000187:drug effects; D012326:RNA Splicing / Q000187:drug effects / Q000235:genetics; D051381:Rats; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: D034441:Heterogeneous-Nuclear Ribonucleoproteins; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D053773:Transforming Growth Factor beta1; C067713:lactacystin; C012589:trichostatin A; D000111:Acetylcysteine
keywords: Bcl-x splicing reporter; Breast cancer cell; Lactacystin; TGFβ1; TSA; hnRNP F/H