Interaction of FLASH with Arsenite Resistance Protein 2 Is Involved in Cell Cycle Progression at S Phase

Abstract

FLASH has been shown to be required for S phase progression and to interact with a nuclear protein, ataxia-telangiectasia locus (NPAT), a component of Cajal bodies in the nucleus and an activator of histone transcription. We investigated the role of human FLASH by using an inducible FLASH knockdown system in the presence or absence of various mutant forms of mouse FLASH. While carboxyl-terminal deletion mutants of FLASH, which do not interact with NPAT, can support S phase progression, its amino-terminal deletion mutants, which are unable to self associate, cannot support S phase progression, replication-dependent histone transcription, or the formation of Cajal bodies. Furthermore, FLASH was shown to be associated with arsenite resistance protein 2 (ARS2) through its central region, which is composed of only 13 amino acids. The expression of ARS2 and the interaction between FLASH and ARS2 are required for S phase progression. Taking these results together, FLASH functions in S phase progression through interaction with ARS2.FADD-like interleukin-1␤-converting enzyme/caspase-8-associated huge protein (FLASH)/CASP8AP2, originally identified as a component of the Fas-caspase-8-mediated apoptosis-inducing pathway (13), was reported to be involved in Fas-mediated apoptotic signaling through translocation from the nucleus to the cytoplasm (20). FLASH also was shown to act in the nucleus as an enhancer and/or repressor of steroid hormone receptor-mediated transcription (14,15,21). On the other hand, FLASH was found to be essential for cell division by the high-throughput screening of a genome-scale library of short interfering RNAs (16). FLASH also was reported to be a potential prognostic marker in cases of acute lymphoblastic leukemia (10). Recently, it was shown that FLASH is an essential component of Cajal bodies in the nucleus, playing an important role in histone transcription and S phase progression (3, 4). In addition, these reports indicated that FLASH interacts with a nuclear protein, ataxia-telangiectasia locus (p220 NPAT [NPAT]), a component of Cajal bodies. The phosphorylation of NPAT by cyclin E/Cdk2 was reported to regulate the activation of histone gene transcription in S phase and to be necessary to maintain the structure of Cajal bodies during the cell cycle (7,19,27,29,30). Therefore, FLASH has been thought to play an essential role in S phase progression, probably through interaction with NPAT. However, the molecular mechanisms underlying the role of FLASH in cell cycle progression largely have remained unknown.Cajal bodies are small subnuclear organelles, originally described by Santiago Ramon Y Cajal in 1903, that are involved in several nuclear functions, including the biogenesis and trafficking of small nuclear and nucleolar ribonucleoprotein particles (snRNPs and snoRNPs, respectively) and the processing of pre-rRNA and replication-dependent histone mRNA (6, 11). Recently, a number of Cajal bodies were found to contain FLASH and NPAT but not coilin, a conventional marker of such bodies, sugges...

A Conserved Interaction That Is Essential for the Biogenesis of Histone Locus Bodies

“…Previous studies by others demonstrated that the endogenous FLASH and NPAT co-immunoprecipitated but failed to determine whether they interact directly or exist in a common complex containing additional proteins (34,50). We now conclude that FLASH and NPAT directly contact each other via their C-terminal regions.…”

“…It has been previously shown that down-regulation of FLASH by RNAi in human cells results in a gradual redistribution of NPAT to the nucleoplasm (34,50). Thus, NPAT on its own is not able to form HLBs in human cells.…”

“…Its N-terminal region of about 135 amino acids interacts with Lsm11, a component of the U7 snRNP, and together these two proteins recruit a subset of polyadenylation factors, including the endonuclease, to histone pre-mRNA for 3Ј end cleavage (9,53,57). A short motif located in the center of FLASH mediates its interaction with Ars2, a stable component of the Cap binding complex (50). The structural and functional organization of FLASH outside these regions has not been characterized.…”

Protease Activity of Procaspase-8 Is Essential for Cell Survival by Inhibiting Both Apoptotic and Nonapoptotic Cell Death Dependent on Receptor-interacting Protein Kinase 1 (RIP1) and RIP3

“…NM_009812.2). The oligonucleotides of shRNA for silencing lacZ (32), and for silencing ATG7 (23) were previously described. For the specific knockdown of RIP3, shRNA for mouse RIP3 was generated from nucleotides 413 to 431 of RIP3 cDNA (GenBank TM no NM_019955.2).…”

CASP8AP2 (caspase 8 associated protein 2)

“…It participates in TNF-alpha-induced blockade of glucocorticoid receptor transactivation at the nuclear receptor coactivator level, upstream and independently of NF-kappa-B (Kino and Chrousos, 2003). It also contributes to cell cycle progression at S phase (Kiriyama et al, 2009;Barcaroli D et al, 2006).…”