Inhibition of post-transcriptional RNA processing by CDK inhibitors and its implication in anti-viral therapy

Abstract

Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle and RNA polymerase II mediated transcription. Several pharmacological CDK inhibitors are currently in clinical trials as potential cancer therapeutics and some of them also exhibit antiviral effects. Olomoucine II and roscovitine, purine-based inhibitors of CDKs, were described as effective antiviral agents that inhibit replication of a broad range of wild type human viruses. Olomoucine II and roscovitine show high selectivity for CDK7 and CDK9, with important functions in the regulation of RNA polymerase II transcription. RNA polymerase II is necessary for viral transcription and following replication in cells. We analyzed the effect of inhibition of CDKs by olomoucine II on gene expression from viral promoters and compared its effect to widely-used roscovitine. We found that both roscovitine and olomoucine II blocked the phosphorylation of RNA polymerase II C-terminal domain. However the repression of genes regulated by viral promoters was strongly dependent on gene localization. Both roscovitine and olomoucine II inhibited expression only when the viral promoter was not integrated into chromosomal DNA. In contrast, treatment of cells with genome-integrated viral promoters increased their expression even though there was decreased phosphorylation of the C-terminal domain of RNA polymerase II. To define the mechanism responsible for decreased gene expression after pharmacological CDK inhibitor treatment, the level of mRNA transcription from extrachromosomal DNA was determined. Interestingly, our results showed that inhibition of RNA polymerase II C-terminal domain phosphorylation increased the number of transcribed mRNAs. However, some of these mRNAs were truncated and lacked polyadenylation, which resulted in decreased translation. These results suggest that phosphorylation of RNA polymerase II C-terminal domain is critical for linking transcription and posttrancriptional processing of mRNA expressed from extrachromosomal DNA.

Figure 1. Olomoucine II differs from roscovitine only by an additional hydroxyl group on 6-benzyl group.

Figure 2. OCII and Rosc suppress T-antigen expression.

(A) OCII and Rosc suppress T-antigen expression and restore p53–mediated transactivation of p21^WAF−1 during SV40 infection. MCF-7 (wild type p53) cell line infected with SV40 was treated either with 8 µM OCII or 20 µM Rosc, either immediately or 16 h post-infection. (OCII 16 h, Rosc 16 h). Infected cells were harvested at 45 h and then analyzed for the expression of large T-antigen, p53, p21^WAF−1 and actin by immunoblotting. (B) Inhibition of expression from viral promoter integrated into cellular genome. Cell lines CV-1 and COS-1 were treated either with 8 µM OCII or 20 µM Rosc and analyzed for the expression of large T-antigen, p53, p21^WAF−1 and actin by immunoblotting. CV-1 cell line was used as SV40-negative control cell line (no large T-antigen expression). The numbers represent the fold changes of samples to untreated controls.

Figure 3. OCII and Rosc suppress expression from extrachromosomal genetic elements.

(A) MCF-7 cells transfected with pSV-lacZ were treated either with OCII (8 µM) or Rosc (20 µM). Relative levels of β-galactosidase activity were determined in a chromogenic (ONPG) enzyme assay. The Y axis represents relative β-galactosidase activity and the error bars illustrate the standard deviation of three independent biological replicates. (B) OCII and Rosc induce wild type p53-mediated transactivation of p21^WAF−1. MCF-7 (wild type p53) cell line was treated either with OCII (8 µM) or Rosc (20 µM) for 24 h and analyzed for the expression of p53, p21^WAF−1 and actin by immunoblotting. The numbers represent the fold changes of samples to untreated controls.

Figure 4. Inhibition of expression from extrachromosomal HIV promoter by PCI.

A β-galactosidase reporter assay was used to measure expression from the promoter within the pHIV-lacZ plasmid following transfection into a Tat-expressing cell line (H1299-Tat). Results were compared with expression from Tat-expressing cell line with pHIV-lacZ stably integrated into the cellular genome (H1299-HIV). The requirement for Tat to promote expression from HIV promoter was illustrated in H1299 (Tat-negative) control cells transiently transfected with pHIV-lacZ. The Y axis represents relative β-galactosidase activity and the error bars illustrate the standard deviation of three independent biological replicates.

Figure 5. The effect of OCII and Rosc on phosphorylation of RNA polymerase II CTD.

(A) Phosphorylation of RNA polymerase II CTD in wild type p53 cells either with stably integrated SV40 (COS-1) or SV40-negative cells (CV-1) was determined. The phosphorylation of both Ser 2 and Ser 5 was inhibited after 12 h treatment either with 8 µM OCII or 20 µM Rosc in all tested cell lines. (B) Comparison of RNA polymerase II CTD phosphorylation in cells expressing Tat with pHIV-lacZ stably integrated into cellular genome (H1299-HIV) or with pHIV-lacZ as a part of extrachromosomal DNA (H1299-Tat). The phosphorylation of Ser 2 and Ser 5 in both cell lines was inhibited after PCI treatment for 12 h. The numbers represent the fold changes of samples to untreated controls.

Figure 6. The experimental setup:

1. Total RNA was extracted. 2. Reverse transcription was performed in two different setups i) using random hexamers to collect all possible types of RNA transcripts and ii) using oligo dT primers to gain polyadenylated types of RNA transcripts. 3) Real-time PCR with primers designed to specifically recognize N- and C-terminus of β-galactosidase cDNA was used to amplify sequences at both 5′-end (PCR-1) and 3′-end (PCR-2) of β-galactosidase RNA transcripts.

Figure 7. The effect of PCIs on the integrity of synthesized RNA.

We compared amounts of total and full length polyadenylated transcripts of β-galactosidase gene after treatments with OLII and Rosc. We quantified total and full length transcripts of β-galactosidase gene in cell lines H1299-HIV (B) and H1299-Tat transiently transfected by pHIV-LacZ (A) by using qRT-PCR. PCIs stimulated the RNA synthesis from viral promoters ((B) H1299-HIV PCR-1, Random hexamers (A) H1299-Tat, PCR-1, Random hexamers) however viral promoters incorporated in the cellular genome generated higher amounts of full length RNA transcripts ((B) H1299-HIV, PCR-2, OLIGO dT). The amount of full length polyadenylated transcripts produced from extrachomosomal viral promoters after treatments with PCIs were significantly lower ((A) H1299-Tat, PCR-2, OLIGO dT). Data were determined by relative quantification with control PCR-1 and control PCR-2 as the calibrators. The Y axes represent the fold change of β-galactosidase RNA transcripts after PCIs treatment to the amount of β-galactosidase RNA transcripts in controls. The error bars illustrate the standard deviation of three independent biological replicates.