SIAH1 targets the alternative splicing factor T-STAR for degradation by the proteasome.
T-STAR is one of three members of the SAM68 family of RNA-binding proteins that have been shown to be involved in various gene expression pathways including the control of pre-mRNA splicing. We employed a two-hybrid screen to identify proteins that interact with human T-STAR. The predominant interacting proteins were the E3 ubiquitin ligases SIAH1 and SIAH2. We found that SIAH1 bound to an octapeptide sequence in T-STAR targeting it for proteasome-dependent degradation. Rodent T-STAR orthologues (also known as etoile or SLM2) were not targeted for degradation by SIAH1. However a double amino acid substitution of mouse T-STAR that mimics the human SIAH1-binding site brought mouse T-STAR under in vivo control of SIAH1. Using a minigene transfection assay for alternative splicing activity we showed that human T-STAR, like its rodent orthologues can influence splice site choice and that human, but not mouse, T-STAR-dependent alternative splicing is modulated by SIAH1. Western blots of protein from purified germ cells indicated that SIAH1 protein expression peaks in meiosis. In mouse, T-STAR is co-expressed with SIAH1 during meiosis but, in humans, T-STAR is only strongly expressed after meiosis. Comparative sequence analysis showed SIAH-mediated proteasomal degradation of T-STAR has evolved in the primate lineage. Collectively these data suggest that SIAH-mediated down regulation of alternative splicing may be an important developmental difference between otherwise highly conserved T-STAR proteins.
Venables
Julian P
JP
Institute of Human Genetics, International Centre for Life, Newcastle upon Tyne, UK.
Dalgliesh
Caroline
C
Paronetto
Maria Paolo
MP
Skitt
Lindi
L
Thornton
Jared K
JK
Saunders
Philippa T
PT
Sette
Claudio
C
Jones
Keith T
KT
Elliott
David J
DJ
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D019143:Evolution, Molecular; D006801:Humans; D008540:Meiosis; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*; D011489:Protein Denaturation; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D000098563:Seven in Absentia Proteins
C118777:KHDRBS3 protein, human; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D044767:Ubiquitin-Protein Ligases; D000098563:Seven in Absentia Proteins; D046988:Proteasome Endopeptidase Complex