Human
cyclin D1 is expressed as two
isoforms derived by alternate RNA splicing, termed D1a and D1b, which differ for the inclusion of intron 4 in the D1b
mRNA. Both
isoforms are frequently upregulated in human
cancers, but
cyclin D1b displays relatively higher oncogenic potential. The
splicing factors that regulate alternative splicing of
cyclin D1b remain unknown despite the likelihood that they contribute to
cyclin D1 oncogenicity. In this study, we report that Sam68, an
RNA-binding protein frequently overexpressed in
prostate cancer cells, enhances splicing of
cyclin D1b and supports its expression in
prostate cancer cells.
Chromatin immunoprecipitation and
RNA coimmunoprecipitation experiments showed that Sam68 is recruited to the human CCND1 gene encoding
cyclin D1 and that it binds to
cyclin D1 mRNA. Transient overexpression and RNAi knockdown experiments indicated that Sam68 acts to enhance endogenous expression of
cyclin D1b. Minigene reporter assays showed that Sam68 directly affected alternative splicing of CCND1 message, with a preference for the A870 allele that is known to favor
cyclin D1b splicing. Sam68 interacted with the proximal region of intron 4, and its binding correlated inversely with recruitment of the spliceosomal component U1-70K. Sam68-mediated splicing was modulated by signal transduction pathways that elicit phosphorylation of Sam68 and regulate its affinity for CCND1 intron 4. Notably, Sam68 expression positively correlates with levels of
cyclin D1b, but not D1a, in human prostate
carcinomas. Our results identify Sam68 as the first
splicing factor to affect CCND1 alternative splicing in
prostate cancer cells, and suggest that increased levels of Sam68 may stimulate
cyclin D1b expression in human
prostate cancers.