Regulation of cyclin D1 RNA stability by SNIP1

Abstract

Cyclin D1 expression represents one of the key mitogen-regulated events during the G(1) phase of the cell cycle, whereas Cyclin D1 overexpression is frequently associated with human malignancy. Here, we describe a novel mechanism regulating Cyclin D1 levels. We find that SNIP1, previously identified as a regulator of Cyclin D1 expression, does not, as previously thought, primarily function as a transcriptional coactivator for this gene. Rather, SNIP1 plays a critical role in cotranscriptional or posttranscriptional Cyclin D1 mRNA stability. Moreover, we show that the majority of nucleoplasmic SNIP1 is present within a previously undescribed complex containing SkIP, THRAP3, BCLAF1, and Pinin, all proteins with reported roles in RNA processing and transcriptional regulation. We find that this complex, which we have termed the SNIP1/SkIP-associated RNA-processing complex, is coordinately recruited to both the 3' end of the Cyclin D1 gene and Cyclin D1 RNA. Significantly, SNIP1 is required for the further recruitment of the RNA processing factor U2AF65 to both the Cyclin D1 gene and RNA. This study shows a novel mechanism regulating Cyclin D1 expression and offers new insight into the role of SNIP1 and associated proteins as regulators of proliferation and cancer.

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.

Figure 2. SNIP1 is required for the co-transcriptional stability of Cyclin D1 mRNA

(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.

Figure 3. Identification of SNIP1 associated proteins

(A) Purification of SNIP1 associated proteins. 10μg of expression plasmids encoding GST-SNIP1 or GST alone were transfected into HEK 293 cells and extracts were prepared prior to purification using glutathione agarose, SDS-PAGE and colloidal coomassie staining. Bands specifically present in the SNIP1-GST lane were excised and proteins identified by MALDI-Tof-Tof (MS/MS). (B) SNIP1 associated proteins are all in a high molecular weight complex of similar size. HeLa nuclear extract was resolved by Superose 6 gel filtration and fractions separated by SDS-PAGE and western blotted using the antibodies indicated. The positions of molecular weight markers used during column calibration are shown. (C) Confirmation of SNIP1 associated proteins by western blot analysis. 293 cells were transfected with THRAP3, SNIP1 or SkIP-HA expression vectors and anti-HA immunoprecipitations performed prior to SDS-PAGE and western blotting for endogenous proteins as indicated. (D) Co-immunoprecipitation of endogenous SNIP1 associated proteins. 200μg of U-2 OS extract was analysed by immunoprecipitation, SDS-PAGE and western blotting using the indicated antibodies. Inputs represent 5% of total protein used in the assay.

Figure 4. SNARP complex proteins are upregulated in G1 phase and are required for Cyclin D1 mRNA production

(A) Cell cycle analysis of SNIP1 associated proteins. U-2 OS cells were fractionated by centrifugal elutriation, whole cell extracts prepared and equivalent protein amounts separated by SDS-PAGE and subjected to western blot analysis using the indicated antibodies. Cells from each fraction were also stained with propidium iodide for analysis by FACS and cell cycle percentages of each fraction are shown graphically. (B) Analysis of RNA transcript levels following depletion of SNARP complex components. U-2 OS cells were treated with siRNAs directed against SNIP1, SkIP and THRAP3/BCLAF1 (knocked down together due to their sequence similarity and possible functional redundancy). The relative levels of pre-spliced and post-spliced RNAs were then assessed by qRT-PCR, normalised to GAPDH levels. The mRNA (post-spliced) : hnRNA (pre-spliced) ratio for the Cyclin D1 transcript is also presented. 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.

Figure 5. SNARP complex components bind to the Cyclin D1 gene and RNA and are required for recruitment of U2AF65

(A) Recruitment of SNARP complex components and splicing factors to the Cyclin D1 gene. ChIP analysis was performed on U-2 OS cells using antibodies directed against SNIP1, the SNARP complex proteins SkIP and THRAP3 together with the RNA processing factor U2AF65. PCR analysis was performed with primers directed at various regions within the Cyclin D1 promoter and along the length of the Cyclin D1 gene as indicated. (B) SNARP complex proteins are co-recruited to the Cyclin D1 gene. Re-ChIP assays were performed as indicated, to detect co-occupancy of SNARP components on specific Cyclin D1 gene regions. Data is presented from a region in Fig. 4A which recruited the SNARP proteins. SNIP1 displays co-occupancy with SkIP, THRAP3 and BCLAF1 at this site. (C) THRAP3 recruitment to the Cyclin D1 gene is SNIP1 dependent. ChIP assays were performed using U-2 OS cells in which SNIP1 had been transiently depleted using siRNA. α-SNIP1 and THRAP3 immunoprecipitations were performed and binding to the indicated regions of the Cyclin D1 gene detected by PCR. (D) SNIP1 dependent binding of U2AF65 to the Cyclin D1 gene and endogenous Cyclin D1 mRNA. Upper panel: ChIP assays were performed using U-2 OS cells in which SNIP1 had been transiently depleted using siRNA. α-SNIP1 and U2AF65 immunoprecipitations were performed and binding to the indicated regions of the Cyclin D1 gene detected by PCR. Lower panel: Protein/RNA cross-linking was performed using U2-OS cells treated with control or SNIP1-targeted siRNAs. Immunoprecipitations were performed using the SNIP1, THRAP3 or U2AF65 antibodies and PCR analysis was used to detect the presence of the Cyclin D1 transcript after RNA extraction and DNAseI treatment.