title: Cell cycle abnormalities associated with differential perturbations of the human U5 snRNP associated U5-200kD RNA helicase.
abstract: 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.
authors: Department of Molecular and Cellular Biology, and Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, Duarte, California, United States of America.
authors: Ali
authors: A
authors: Ehsani
authors: Jessica V
authors: JV
authors: Alluin
authors: John J
authors: JJ
authors: Rossi
mesh_terms: D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D015536:Down-Regulation / Q000187:drug effects; D004440:Ecdysone / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D008970:Molecular Weight; D009154:Mutation; D020365:RNA Helicases / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D012326:RNA Splicing / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; D017415:Ribonucleoprotein, U5 Small Nuclear; D004440:Ecdysone; D020365:RNA Helicases
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