Figure 1. SNIP1 regulates Cyclin D1 RNA processing.

(A) Cyclin D1 protein expression is SNIP1 dependent. Protein extracts were prepared from U-2 OS cells treated with SNIP1 and control siRNAs. SDS-PAGE and western blot analysis was performed using antibodies directed against Cyclin D1, Cyclin E, SNIP1, IκBα and β-actin.

(B - D) SNIP1 is required for the production of mature Cyclin D1 mRNA. U-2 OS cells were treated with a SNIP1 siRNA. The relative levels of pre-spliced and post-spliced RNAs were then assessed by semi-quantitative PCR. The pre-spliced transcript is detected using primers within intron 1 and the post-spliced transcript detected using primers situated within exons 3 and 5. The same results are also seen with primers directed to different regions of Cyclin D1 RNA (for example, within intron 3 for pre-spliced message, or within exons 1 and 3 or 1 and 5 for post-spliced message). Also shown are the levels of Cyclin D2 and CDK4 mRNAs. In (C) analysis included the alternately spliced Cyclin D1 transcript. In (D) a no reverse-transcriptase control was used to ensure the PCR signal from pre-spliced Cyclin D1 results from RNA and not DNA contamination.