Molecular Evidence for the Existence of Two Distinct Subgroups in Cucumber Mosaic Cucumovirus

Infectious full-length cDNA clones from the genomic RNAs of a subgroup II cucumber mosaic cucumovirus strain (Trk7) were obtained. Sequence analysis of the whole genome revealed strong homology (99%) to the genome of Q-CMV, the only subgroup II strain whose entire genomic nucleotide sequence had been available in the database, and an overall 75% homology to those of subgroup I strains. We provide sequence comparisons of different parts of 1a, 2a and 2b proteins of Cucumovirus species, and propose phylogenetic trees based on these protein sequences.     

Involvement of RNA helicase p68 in skin wound healing process in rats

Purpose: RNA helicase p68 plays an important role in organ development and maturation through tuning cell proliferation. However, the character and role of p68 in the whole wound healing process need more study. Methods: First, we characterize expression of p68 in normal rat skin development postnatal. Then, we assayed dynamic change of p68 in rat skin from different stage after injury, and explored the role of p68 in proliferation and migration of three types of wound healing related cells. Results: p68 was down-regulated during skin developmental and maturation process, up-regulated after wound, peaked on day 14 and then significantly decreased. Wound fluid enhanced wound healing related cell proliferation and up-regulated expression of p68. Conversely, reducing p68 expression by RNA interference resulted in significantly slower proliferation and migration. Conclusion: Our results define an important role of RNA helicase p68 in skin wound healing process.     

Mouse MOV10L1 associates with Piwi proteins and is an essential component of the Piwi-interacting RNA (piRNA) pathway

Piwi-interacting RNAs (piRNAs) are essential for silencing of transposable elements in the germline, but their biogenesis is poorly understood. Here we demonstrate that MOV10L1, a germ cell–specific putative RNA helicase, is associated with Piwi proteins. Genetic disruption of the MOV10L1 RNA helicase domain in mice renders both MILI and MIWI2 devoid of piRNAs. Absence of a functional piRNA pathway in Mov10l1 mutant testes causes loss of DNA methylation and subsequent derepression of retrotransposons in germ cells. The Mov10l1 mutant males are sterile owing to complete meiotic arrest. This mouse mutant expresses Piwi proteins but lacks piRNAs, suggesting that MOV10L1 is required for piRNA biogenesis and/or loading to Piwi proteins.     

Advances in hepatitis C: What is coming in the next 5 years?

Hepatitis C virus (HCV) is a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Numerous advances have been made in the understanding of HCV replication, including detailed molecular characterization of its viral proteins and genomic RNA. The inability to grow HCV in cell culture had impeded the development of antiviral agents against this virus. To overcome this obstacle, a number of unique tools have been prepared, such as molecular clones that are infectious in the chimpanzee animal model of infection, and the development of a subgenomic replicon system in Huh7 cells. In addition, the major non-structural proteins have been crystallized, thus enabling rational drug design directed to these targets. Current developments in antiviral agents are reviewed in the context of these potential new viral targets for the future treatment of HCV in chronically infected individuals.     

RecQ helicase acts before RuvABC,RecG and XerC proteins during recombination in recBCD sbcBC mutants of Escherichia coli

The RecQ helicase is required by the RecF recombination pathway that is operative in recBC(D) sbcB sbcC(D) mutants of Escherichia coli. Genetic data suggest that RecQ participates in resection of DNA ends during initiation of recombination. In vitro, RecQ can unwind a variety of DNA substrates, including recombination intermediates such as D-loops and Holliday junctions. However, its potential role in processing of recombination intermediates during the late stage of the RecF pathway has not been genetically tested. Here we studied the effect of a recQ mutation on transductional recombination and DNA repair after γ-irradiation in ΔrecBCD ΔsbcB sbcC strains deficient for RuvABC, RecG and XerC proteins. RuvABC and RecG proteins process recombination intermediates in the late stage of recombination, whereas XerC is required to resolve chromosome dimers formed upon recombination. Our results do not reveal any substantial synergistic effect between the recQ mutation, on one hand, and ruvABC, recG and xerC mutations on the other. In addition, the recQ mutation suppresses chromosome segregation defects in γ-irradiated ruvABC recG and xerC mutants. These results suggest that RecQ acts upstream of RuvABC, RecG and XerC proteins, a finding that is compatible with its primary role in initiation of the RecF recombination pathway.     

Human Rhinovirus-induced Proinflammatory Cytokine and Interferon-β Responses in Nasal Epithelial Cells From Chronic Rhinosinusitis Patients

Purpose

Asthma exacerbation from human rhinovirus (HRV) infection is associated with deficient antiviral interferon (IFN) secretion. Although chronic rhinosinusitis (CRS), an inflammatory upper airway disease, is closely linked to asthma, IFN-β responses to HRV infections in human nasal epithelial cells (HNECs) from CRS patients remain to be studied. We evaluated inflammatory and antiviral responses to HRV infection in HNECs from CRS patients.

Methods

HNECs, isolated from turbinate tissue of 13 patients with CRS and 14 non-CRS controls, were infected with HRV16 for 4 hours. The HRV titer, LDH activity, production of proinflammatory cytokines and IFN-β proteins, and expression levels of RIG-I and MDA5 mRNA were assessed at 8, 24, and 48 hours after HRV16 infection.

Results

The reduction in viral titer was slightly delayed in the CRS group compared to the non-CRS control group. IL-6 and IL-8 were significantly increased to a similar extent in both groups after HRV infection. In the control group, IFN-β production and MDA5 mRNA expression were significantly increased at 8 and 24 hours after HRV16 infection, respectively. By contrast, in the CRS group, IFN-β was not induced by HRV infection; however, HRV-induced MDA5 mRNA expression was increased, but the increase was slightly delayed compared to the non-CRS control group. The RIG-I mRNA level was not significantly increased by HRV16 infection in either group.

Conclusions

HRV-induced secretion of proinflammatory cytokines in CRS patients was not different from that in the non-CRS controls. However, reductions in viral titer, IFN-β secretion, and MDA5 mRNA expression in response to HRV infection in CRS patients were slightly impaired compared to those in the controls, suggesting that HRV clearance in CRS patients might be slightly deficient.     

沉默解旋酶BLM基因对结直肠癌细胞伊立替康化疗敏感性的影响及其机制

探讨沉默结直肠癌（CRC）细胞中布卢姆综合征解旋酶（BLM）基因表达对伊立替康（即CPT-11）化疗敏感性的影响,并阐明其相关作用机制。     

Domain within the helicase subunit Mcm4 integrates multiple kinase signals to control DNA replication initiation and fork progression

Eukaryotic DNA synthesis initiates from multiple replication origins and progresses through bidirectional replication forks to ensure efficient duplication of the genome. Temporal control of initiation from origins and regulation of replication fork functions are important aspects for maintaining genome stability. Multiple kinase-signaling pathways are involved in these processes. The Dbf4-dependent Cdc7 kinase (DDK), cyclin-dependent kinase (CDK), and Mec1, the yeast Ataxia telangiectasia mutated/Ataxia telangiectasia mutated Rad3-related checkpoint regulator, all target the structurally disordered N-terminal serine/threonine-rich domain (NSD) of mini-chromosome maintenance subunit 4 (Mcm4), a subunit of the mini-chromosome maintenance (MCM) replicative helicase complex. Using whole-genome replication profile analysis and single-molecule DNA fiber analysis, we show that under replication stress the temporal pattern of origin activation and DNA replication fork progression are altered in cells with mutations within two separate segments of the Mcm4 NSD. The proximal segment of the NSD residing next to the DDK-docking domain mediates repression of late-origin firing by checkpoint signals because in its absence late origins become active despite an elevated DNA damage-checkpoint response. In contrast, the distal segment of the NSD at the N terminus plays no role in the temporal pattern of origin firing but has a strong influence on replication fork progression and on checkpoint signaling. Both fork progression and checkpoint response are regulated by the phosphorylation of the canonical CDK sites at the distal NSD. Together, our data suggest that the eukaryotic MCM helicase contains an intrinsic regulatory domain that integrates multiple signals to coordinate origin activation and replication fork progression under stress conditions.Eukaryotic DNA replication initiates from multiple replication origins within each chromosome to duplicate the large genome efficiently. To ensure DNA synthesis occurs once and only once across the genome, cells adopt a two-step process to activate replication origins during two separate stages of the cell-division cycle. The first step is licensing of replication origins, which occurs only when cyclin-dependent kinase (CDK) activity is low. In Saccharomyces cerevisiae, origins of DNA replication are licensed in G1 by the formation of a prereplicative complex (pre-RC). The process begins with the origin recognition complex binding to replication origins and recruiting the licensing factor Cdc6, which facilitates loading of the Cdt1-bound minichromosome maintenance (MCM) complex composed of Mcm2–Mcm7 (Mcm2–7). The hexameric Mcm2–7 is the core of the replicative helicase that unwinds DNA during replication. Within the pre-RC Mcm2–7 is loaded as an inactive double hexamer. The next step, activation of licensed origins (origin firing), occurs throughout the S phase and requires the continuous presence of two kinases, the S phase CDKs and the Dbf4-dependent Cdc7 kinase (DDK). CDK phosphorylates Sld2 and Sld3 to allow their binding to Dpb11 (1, 2), facilitating recruitment of Cdc45 and GINS (composed of protein subunits Sld5, Psf1, Psf2 and Psf3; Go, Ichi, Nii, and San stand for five, one, two, and three in Japanese, respectively) to Mcm2–7 to create an active helicase. DDK phosphorylates Mcm2–7 and blocks an intrinsic initiation inhibitory activity residing in the N terminus of the Mcm4 subunit (3). The concerted action of these S-phase kinases transforms the inactive Mcm2–7 double hexamer into the active helicase complex composed of Cdc45, Mcm2-7, and GINS (the CMG complex) (4–6). Upon initiation, DNA polymerases and other components of the replication machinery are recruited to form replisomes and establish replication forks, where DNA synthesis ensues.Kinase-signaling pathways target various components of the replication machinery. Both CDK and DDK target replication proteins in addition to their essential targets described above. Furthermore, Ataxia telangiectasia mutated/Ataxia telangiectasia mutated Rad3-related (ATM/ATR) signaling targets components of the CMG helicase complex under replication stress (7–10). In the yeast S. cerevisiae, DNA damage activates the checkpoint kinase Rad53, which phosphorylates both Sld3 and Dbf4 to inhibit late origin firing (11, 12). The yeast ATM/ATR homolog Mec1 also targets Mcm4 (13). The stress-activated protein kinase Hog1 targets an auxiliary replisome component Mrc1 to regulate both origin firing and fork progression (14). Although we now have a better understanding of the essential functions of protein kinases in controlling the initiation of replication, we do not completely understand how the separate kinase signaling pathways are coordinated to regulate both initiation and replication fork progression.The structurally disordered N-terminal serine/threonine-rich domain (NSD) of Mcm4 is a target of multiple kinases, including DDK, CDK, and Mec1 (3, 13, 15, 16). Within this region we have identified two functionally distinct domains that exert different functions and are regulated by different kinase systems even though they overlap extensively in primary amino acid sequences. The segment of the Mcm4 NSD proximal to the DDK-docking domain (DDD) (15), and hence termed “proximal NSD,” blocks initiation until it is phosphorylated by DDK. In contrast, the distal segment of the NSD at the N terminus, away from the DDD, is targeted by additional kinases and contributes positively to promote S-phase progression. In this study we present a comprehensive analysis of the pattern of origin activation, replication fork progression, and the checkpoint response in cells under replication stress caused by the inhibition of ribonucleotide reductase (RNR). We show that the distal and proximal NSD segments contribute differently to origin activation and DNA replication fork progression. Furthermore, they exert opposing effects on checkpoint signaling under replication stress. All these effects are regulated by phosphorylation. We suggest that the Mcm4 NSD, a regulatory domain intrinsic to the replicative helicase, mediates the control of multiple aspects of DNA replication. Our data reveal a sophisticated mechanism to fine-tune S-phase progression in response to changing environments.     

DEAD-box protein CYT-19 is activated by exposed helices in a group I intron RNA

DEAD-box proteins are nonprocessive RNA helicases and can function as RNA chaperones, but the mechanisms of their chaperone activity remain incompletely understood. The Neurospora crassa DEAD-box protein CYT-19 is a mitochondrial RNA chaperone that promotes group I intron splicing and has been shown to resolve misfolded group I intron structures, allowing them to refold. Building on previous results, here we use a series of tertiary contact mutants of the Tetrahymena group I intron ribozyme to demonstrate that the efficiency of CYT-19–mediated unfolding of the ribozyme is tightly linked to global RNA tertiary stability. Efficient unfolding of destabilized ribozyme variants is accompanied by increased ATPase activity of CYT-19, suggesting that destabilized ribozymes provide more productive interaction opportunities. The strongest ATPase stimulation occurs with a ribozyme that lacks all five tertiary contacts and does not form a compact structure, and small-angle X-ray scattering indicates that ATPase activity tracks with ribozyme compactness. Further, deletion of three helices that are prominently exposed in the folded structure decreases the ATPase stimulation by the folded ribozyme. Together, these results lead to a model in which CYT-19, and likely related DEAD-box proteins, rearranges complex RNA structures by preferentially interacting with and unwinding exposed RNA secondary structure. Importantly, this mechanism could bias DEAD-box proteins to act on misfolded RNAs and ribonucleoproteins, which are likely to be less compact and more dynamic than their native counterparts.DEAD-box proteins constitute the largest family of RNA helicases and function in all stages of RNA metabolism (1, 2). In vivo, many DEAD-box proteins have been implicated in assembly and conformational rearrangements of large structured RNAs and ribonucleoproteins (RNPs), including the ribosome, spliceosome, and self-splicing introns (3). Thus, it is important to establish how these proteins use their basic mechanisms of RNA binding and helix unwinding to interact with and remodel higher-order RNA structures.Structural and mechanistic studies have elucidated the basic steps of the ATPase cycle of DEAD-box proteins and have provided an understanding of the coupling between ATPase and duplex unwinding activities (4–11). The conserved helicase core consists of two flexibly linked RecA-like domains that contain at least 12 conserved motifs, including the D-E-A-D sequence in the ATP-binding motif II (3, 12). Binding of ATP and double-stranded RNA to domains 1 and 2, respectively, induces domain closure, which completes the formation of an ATPase active site at the domain interface and introduces steric clashes in the RNA binding site, leading to the displacement of one of the RNA strands (6, 7). ATP hydrolysis and inorganic phosphate release are then thought to regenerate the open enzyme conformation (4, 8, 13). Unlike conventional helicases, DEAD-box proteins have not been found to translocate, limiting the unwinding activity to short helices that can be disrupted in a single cycle of ATP binding and hydrolysis (4, 8, 9, 14–16). This mechanism is compatible with the physiological roles of DEAD-box proteins, because cellular RNAs rarely contain continuous base-paired regions that are longer than one or two helical turns.The interactions of DEAD-box proteins with structured RNAs have been extensively studied using two homologous proteins that function as general RNA chaperones: CYT-19 from Neurospora crassa and Mss116 from Saccharomyces cerevisiae. In vivo, CYT-19 is required for efficient splicing of several mitochondrial group I introns and can promote splicing of group I and group II introns in yeast mutants that lack functional Mss116 (17, 18). Both proteins have been shown to act as general RNA chaperones during group I and group II intron folding in vitro and are thought to act primarily by reversing misfolding of the intron RNAs, although additional mechanisms may be used for some substrates (17–23). Importantly, the chaperone activities of these and other DEAD-box proteins correlate with their ATP-dependent helix unwinding activities, suggesting that DEAD-box proteins function by lowering the energy barriers for transitions between alternative structures that involve disruption of base pairs (24, 25).In vitro studies using the group I intron ribozyme from Tetrahymena thermophila have been instrumental in probing the chaperone mechanism of CYT-19 (17, 26–28). This ∼400-nt RNA folds into a compact, globular structure composed of a conserved core and a series of peripheral elements that encircle the core by forming long-range tertiary contacts (Fig. 1) (29–31). Upon addition of Mg²⁺ ions, the majority of the ribozyme population becomes trapped in a long-lived misfolded conformation, which then slowly refolds to the native state (32). The misfolded intermediate is remarkably similar to the native ribozyme, forming a complete native network of secondary and tertiary interactions and a globally compact fold (33, 34). Despite these similarities, refolding to the native state requires extensive unfolding, including disruption of all five peripheral tertiary contacts and the core helix P3 (33, 35). To explain these results, a topological error has been proposed, wherein two single-stranded joining elements are crossed incorrectly in the core of the misfolded ribozyme, and transient disruption of the surrounding native structure is required for refolding (33, 35).Open in a separate windowFig. 1.The Tetrahymena group I intron ribozyme. (A) Secondary structure and mutations. Peripheral elements are colored and thick arrows mark the long-range peripheral tertiary contacts. Paired regions (P) and loops that were mutated in this study (L) are labeled based on group I intron nomenclature in ref. 31. The mutated regions are enclosed in dashed boxes and labeled in bold, with sequence substitutions indicated nearby. Sequences that were deleted to construct the helix truncation mutants (Fig. 6) are enclosed in gray dashed boxes and the replacement nucleotides are shown in gray italic font. (B) Tertiary structure model of the ribozyme (31). Peripheral elements (colored surface) and the locations of the long-range peripheral tertiary contacts (circles) are highlighted using the same color scheme as in A. The ribozyme core is shown in silver. The block arrows indicate the approximate positions of tertiary contacts not visible in each respective view of the ribozyme. The figures were prepared using PyMOL.Given the structural similarity between the native and misfolded ribozyme, it is interesting that CYT-19 can accelerate refolding of the misfolded intermediate by at least an order of magnitude without detectably unfolding the native ribozyme (26). Insights into this apparent preference for the misfolded ribozyme came from studies of two ribozyme mutants in which the tertiary structure was destabilized, making the stability of the native ribozyme comparable to that of the misfolded intermediate (28). CYT-19 unfolded the native and misfolded conformers of these mutants with comparable efficiencies, suggesting that the efficiency of chaperone-mediated unfolding depended on the stability of ribozyme tertiary structure. However, the mutations studied were concentrated in one region of the ribozyme, leaving open the possibility that CYT-19 recognizes local disruptions rather than global stability.Here we investigate the roles of RNA stability in CYT-19-mediated unfolding of the Tetrahymena ribozyme by using a series of ribozyme mutants with disruptions of each of the five peripheral tertiary contacts. We observe a strong correlation between CYT-19 activity and global stability of ribozyme tertiary structure. Further, we find that the RNA-dependent ATPase activity of CYT-19 depends on the accessibility of secondary structure in the ribozyme. Our results lead to a general model for recognition and remodeling of unstable or incorrectly folded RNAs by a DEAD-box protein.