Upregulation of the cell-cycle regulator RGC-32 in Epstein-Barr virus-immortalized cells

Epstein-Barr virus (EBV) is implicated in the pathogenesis of multiple human tumours of lymphoid and epithelial origin. The virus infects and immortalizes B cells establishing a persistent latent infection characterized by varying patterns of EBV latent gene expression (latency 0, I, II and III). The CDK1 activator, Response Gene to Complement-32 (RGC-32, C13ORF15), is overexpressed in colon, breast and ovarian cancer tissues and we have detected selective high-level RGC-32 protein expression in EBV-immortalized latency III cells. Significantly, we show that overexpression of RGC-32 in B cells is sufficient to disrupt G2 cell-cycle arrest consistent with activation of CDK1, implicating RGC-32 in the EBV transformation process. Surprisingly, RGC-32 mRNA is expressed at high levels in latency I Burkitt's lymphoma (BL) cells and in some EBV-negative BL cell-lines, although RGC-32 protein expression is not detectable. We show that RGC-32 mRNA expression is elevated in latency I cells due to transcriptional activation by high levels of the differentially expressed RUNX1c transcription factor. We found that proteosomal degradation or blocked cytoplasmic export of the RGC-32 message were not responsible for the lack of RGC-32 protein expression in latency I cells. Significantly, analysis of the ribosomal association of the RGC-32 mRNA in latency I and latency III cells revealed that RGC-32 transcripts were associated with multiple ribosomes in both cell-types implicating post-initiation translational repression mechanisms in the block to RGC-32 protein production in latency I cells. In summary, our results are the first to demonstrate RGC-32 protein upregulation in cells transformed by a human tumour virus and to identify post-initiation translational mechanisms as an expression control point for this key cell-cycle regulator

Sam68 regulates translation of target mRNAs in male germ cells, necessary for mouse spermatogenesis

Sam68 is a KH-type RNA-binding protein involved in several steps of RNA metabolism with potential implications in cell differentiation and cancer. However, its physiological roles are still poorly understood. Herein, we show that Sam68−/− male mice are infertile and display several defects in spermatogenesis, demonstrating an essential role for Sam68 in male fertility. Sam68−/− mice produce few spermatozoa, which display dramatic motility defects and are unable to fertilize eggs. Expression of a subset of messenger mRNAs (mRNAs) is affected in the testis of knockout mice. Interestingly, Sam68 is associated with polyadenylated mRNAs in the cytoplasm during the meiotic divisions and in round spermatids, when it interacts with the translational machinery. We show that Sam68 is required for polysomal recruitment of specific mRNAs and for accumulation of the corresponding proteins in germ cells and in a heterologous system. These observations demonstrate a novel role for Sam68 in mRNA translation and highlight its essential requirement for the development of a functional male gamete

Spy1 regulation of the cell cycle, checkpoint activation and apoptosis

While the prevailing dogma states that cell cycle progression is associated with the activation of the cyclin-dependent kinases (CDKs), and cell cycle arrest and apoptosis with the inhibition of CDKs, there is growing evidence that regulation of CDK activity is not that straight forward and the balance between proliferation and checkpoint arrest is intricately weaved. This suggests that specific mechanisms of regulation will exist to accomplish this apparent paradoxical regulation. The Speedy/RINGO family of CDK regulators appears to play a role in this specialized regulation. Spy1 is the originally identified member of the Speedy/RINGO family, and has been shown to atypically activate CDKs, even in the face of inhibition. Here I report a role for Spy1- regulation of CDK2 in apoptosis and checkpoint activation in response to Ultraviolet (UV) irradiation. Using an inducible system allowing for regulated expression of Spy1, I show that Spy1 expression suppresses apoptosis in a p53 and p21 dependent fashion. Spy1 expression also allows for UV irradiation resistant DNA synthesis (UVDS) and inhibits the S- and G2/M- checkpoints through the inhibition of checkpoint response proteins. This leads to DNA damage tolerance and prevention of repair of UV-induced cyclobutane pyrimidine dimers through suppression of nucleotide excision repair. Furthermore, knockdown of Spy1 activates intrinsic damage responses indicating that Spy1 is required to promote tolerance of damage that may occur endogenously or exogenously. p27Kip1 binds to cyclin E/ CDK2 complexes inhibiting its kinase activity, yet certain critical events trigger CDK2 to phosphorylate its own inhibitor leading to p27 degradation and cell cycle progression. Utilizing recombinant proteins, we demonstrate that Spy1 activates CDK2 to phosphorylate p27 at T187 in vitro. Spy1 expression in vivo leads to enhanced T187 phosphorylation and degradation of endogenous p27 in late G1 and throughout S-phase. The mechanisms of action conferred by the Speedy/RINGO family represent novel modes by which CDKs are regulated. This type of regulation may be important at cell cycle transitions, in the tolerance of normal intrinsic damage, or in response to exogenous DNA damag

Spy1 enhances phosphorylation and degradation of the cell cycle inhibitor p27

The cyclin dependent kinase inhibitor (CKI) p27(Kip1) binds to cyclin E/CDK2 complexes and prevents premature S-phase entry. During late G(1) and throughout S-phase, p27 phosphorylation at T187 leads to its subsequent degradation, which relieves CDK2 inhibition to promote cell cycle progression. However, critical events that trigger CDK2 complexes to phosphorylate p27 remain unclear. Utilizing recombinant proteins, we demonstrate that human Speedy ( Spy1) activates CDK2 to phosphorylate p27 at T187 in vitro. Addition of Spy1 or Spy1/CDK2 to a preformed, inhibited cyclin E/CDK2/p27 complex also promoted this phosphorylation. Furthermore, Spy1 protected cyclin E/CDK2 from p27 inhibition toward histone H1, in vitro. Inducible Spy1 expression in U2OS cells reduced levels of endogenous p27 and exogenous p27(WT), but not a p27(T187A) mutant. Additionally, Spy1 expression in synchronized HeLa cells enhanced T187 phosphorylation and degradation of endogenous p27 in late G(1) and throughout S-phase. Our studies provide evidence that Spy1 expression enhances CDK2-dependent p27 degradation during late G(1) and throughout S-phase