Cell cycle abnormalities associated with differential perturbations of the human U5 snRNP associated U5-200kD RNA helicase.

Splicing of pre-messenger RNAs into functional messages requires a concerted assembly of proteins and small RNAs that identify the splice junctions and facilitate cleavage of exon-intron boundaries and ligation of exons. One of the key steps in the splicing reaction is the recruitment of a tri-snRNP harboring the U5/U4/U6 snRNPs. The U5 snRNP is also required for both steps of splicing and exon-exon joining. One of the key components of the tri-snRNP is the U5 200kd helicase. The human U5-200kD gene isolated from Hela cells encodes a 200 kDa protein with putative RNA helicase function. Surprisingly, little is known about the functional role of this protein in humans. Therefore, we have investigated the role of the U5-200kD RNA helicase in mammalian cell culture. We created and expressed a dominant negative domain I mutant of the RNA helicase in HEK293 cells and used RNAi to downregulate expression of the endogenous protein. Transient and stable expression of the domain I mutant U5-200kD protein using an ecdysone-inducible system and transient expression of an anti-U5-200kD short hairpin RNA (shRNA) resulted in differential splicing and growth defects in the 293/EcR cells. Cell cycle analysis of the dominant negative clones revealed delayed exit from the G2/M phase of the cell cycle due to a mild splicing defect. In contrast to the domain I dominant negative mutant expressing cells, transient expression of an anti-U5-200kD shRNA resulted in a pronounced S phase arrest and a minute splicing defect. Collectively, this work demonstrates for the first time establishment of differential human cell culture splicing and cell cycle defect models due to perturbed levels of an essential core splicing factor.

Small interfering RNAs targeting cyclin D1 and cyclin D2 enhance the cytotoxicity of chemotherapeutic agents in mantle cell lymphoma cell lines.

Cyclin D1 (CCND1) is a known cell cycle regulator whose overexpression is a hallmark of mantle cell lymphoma (MCL). Although molecular techniques have unified the diagnostic approach to MCL, no therapeutic advances have been made to target this particular pathway. The significance of CCND1 in the pathogenesis and treatment of MCL has yet to be defined. We have taken advantage of RNA interference (RNAi) to down-regulate CCND1 expression in two MCL cell lines (Granta-519 and Jeko-1) to investigate the cytotoxic effect of combining RNAi with conventional chemotherapeutic agents. We designed four small interfering RNAs (siRNAs) specific to CCND1, one specific to CCND2, and one dual-targeting siRNA that simultaneously down-regulates CCND1 and CCND2. Etoposide and doxorubicin were used as chemotherapeutics in combination with the siRNAs. The transfected siRNAs in MCL cell lines triggered 40-60% reduction in target mRNA and protein levels. Importantly, the siRNA-mediated reduction in cyclins resulted in decreased IC(50) (50% inhibitory concentration) values for both doxorubicin and etoposide. The combination of siRNA-mediated inhibition of the cyclins along with chemotherapeutic agents could potentially be used to lower the effective doses of the chemotherapeutic agents and reduce drug-related toxicities.