Abstract
The RNA-binding protein Sam68 is involved in apoptosis, but its cellular mRNA targets and its mechanism of action remain unknown. We demonstrate that Sam68 binds the mRNA for Bcl-x and affects its alternative splicing. Depletion of Sam68 by RNA interference caused accumulation of antiapoptotic Bcl-x(L), whereas its up-regulation increased the levels of proapoptotic Bcl-x(s). Tyrosine phosphorylation of Sam68 by Fyn inverted this effect and favored the Bcl-x(L) splice site selection. A point mutation in the RNA-binding domain of Sam68 influenced its splicing activity and subnuclear localization. Moreover, coexpression of ASF/SF2 with Sam68, or fusion with an RS domain, counteracted Sam68 splicing activity toward Bcl-x. Finally, Sam68 interacted with heterogenous nuclear RNP (hnRNP) A1, and depletion of hnRNP A1 or mutations that impair this interaction attenuated Bcl-x(s) splicing. Our results indicate that Sam68 plays a role in the regulation of Bcl-x alternative splicing and that tyrosine phosphorylation of Sam68 by Src-like kinases can switch its role from proapoptotic to antiapoptotic in live cells.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Adaptor Proteins, Signal Transducing / genetics*
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Adaptor Proteins, Signal Transducing / metabolism*
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Alternative Splicing / genetics*
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Binding Sites / genetics
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Cell Line
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DNA-Binding Proteins / genetics*
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DNA-Binding Proteins / metabolism*
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Down-Regulation / physiology
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Heterogeneous Nuclear Ribonucleoprotein A1
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Heterogeneous-Nuclear Ribonucleoprotein Group A-B / genetics
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Heterogeneous-Nuclear Ribonucleoprotein Group A-B / metabolism
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Humans
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Nuclear Proteins / genetics
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Nuclear Proteins / metabolism
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Phosphorylation
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Point Mutation / genetics
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Protein Isoforms / genetics
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Protein Isoforms / metabolism
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RNA, Messenger / genetics
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RNA, Messenger / metabolism
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RNA, Small Interfering
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RNA-Binding Proteins / genetics*
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RNA-Binding Proteins / metabolism*
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Serine-Arginine Splicing Factors
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Up-Regulation / physiology
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bcl-X Protein / genetics*
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bcl-X Protein / metabolism*
Substances
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Adaptor Proteins, Signal Transducing
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BCL2L1 protein, human
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DNA-Binding Proteins
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FYB1 protein, human
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Heterogeneous Nuclear Ribonucleoprotein A1
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Heterogeneous-Nuclear Ribonucleoprotein Group A-B
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KHDRBS1 protein, human
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Nuclear Proteins
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Protein Isoforms
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RNA, Messenger
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RNA, Small Interfering
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RNA-Binding Proteins
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bcl-X Protein
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Serine-Arginine Splicing Factors