(A) SNIP1 is not required for Cyclin D1 polyadenylation. U2-OS cells were transfected with control or SNIP1 siRNA and RACE-PAT (rapid amplification of cDNA ends-polyA test) PCR was performed to analyse the potential for SNIP1 to regulate Cyclin D1 polyadenylation.
(B) SNIP1 does not regulate Cyclin D1 nucleo-cytoplasmic RNA shuttling. U2-OS cells were transfected with control or SNIP1 siRNAs and separated into nuclear and cytoplasmic fractions prior to RNA extraction, DNAseI treatment and RT-PCR.
(C) SNIP1 is required for the co-transcriptional Cyclin D1 stability. U2-OS cells were transfected with control or SNARP-targeted siRNA oligonucleotides, then treated with 10μg/ml actinomycin D prior to RNA extraction at the time points indicated. Transcript levels were then analysed by qRT-PCR and normalised to rpLP32, a transcript with a half-life in excess of 25h.