The DEAD-Box RNA Helicase Dbp5 Functions in Translation Termination

Gross, Thomas P.; Siepmann, Anja; Sturm, Dorothée; Windgassen, Merle; Scarcelli, John J.; Seedorf, Matthias; Cole, Charles N.; Krebber, Heike

doi:10.1126/science.1134641

Cited by 128 publications

(179 citation statements)

References 12 publications

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“…NS3 RNA helicase also promotes cellular translation, consistent with previous reports in which the best characterized RNA helicase, the eukaryotic translation initiation factor eIF-4A, is believed to disrupt secondary structures in mRNA upstream of the initiation codon, thereby facilitating attachment of the 40S ribosome (Pause & Sonenberg, 1992;Rozen et al, 1990). Helicase activity is also required for efficient translation termination (Gross et al, 2007). Viral RNA helicase enhancing cellular translation has also been found in previous data (Kato et al, 2002).…”

supporting

confidence: 89%

Effect of NS3 and NS5B proteins on classical swine fever virus internal ribosome entry site-mediated translation and its host cellular translation

Xiao

Bai

et al. 2008

Journal of General Virology

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A full-length NS3 (NS3F) and a truncated NS3 protein (NS3H) with an RNA helicase domain possess RNA helicase activity. Using an in vitro system with a monocistronic reporter RNA or DNA, containing the CSFV 59-UTR, we observed that both NS3F and NS3H enhanced internal ribosome entry site (IRES)-mediated and cellular translation in a dose-dependent manner, but NS3 protease (NS3P) that lacks a helicase domain did not. NS3F was stronger than NS3H in promoting both translations. These results showed that viral RNA helicase could promote viral and cellular translation, and higher RNA helicase activity might be more efficient. The NS5B protein, the viral replicase, did not significantly affect the IRES-directed or cellular translation alone. NS5B significantly enhanced the stimulative effect of NS3F on both IRES-mediated and cellular translation, but did not affect that of NS3H or NS3P. This suggests that NS5B and NS3 interact via the protease domain during the enhancement of translation. Classical swine fever virus (CSFV) is a member of the genusPestivirus of the family Flaviviridae. Bovine viral diarrhea virus 1 (BVDV-1), BVDV-2, border disease virus (BDV) and hepatitis C virus (HCV) also belong to this family (Becher & Thiel, 2002;Cuthbert, 1994;Heinz et al., 2000). CSFV is the leading cause of classical swine fever, a highly contagious and sometimes fatal viral disease of pigs, and it can cause a considerable amount of economic loss. CSFV possesses a single plus-strand RNA genome consisting of 59-and 39-untranslated regions (UTRs), and a single large open reading frame (ORF) that encodes a polyprotein of approximately 3900 aa. The polyprotein (NH2-N pro -C-E rns -E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH) is processed into mature proteins by cellular and viral proteases (Moennig & Plagemann, 1992). The 39-UTR is most likely to be involved in the initiation of pestiviral genome replication (Isken et al., 2003;Pankraz et al., 2005;Xiao et al., 2004;Yu et al., 1999), while the 59-UTR is able to regulate translation of the viral genomes (Fletcher & Jackson, 2002). An internal ribosome entry site (IRES) is present in the 59-UTR, and is located between nt 40 and 350 from the 59-terminal end of the genome. The HCV and the pestiviral IRESs direct the binding of ribosomes to the start codon even in the absence of canonical translation initiation factors (Hellen & Sarnow, 2001).NS3 is a multifunctional protein possessing serine protease, RNA helicase and nucleoside triphosphatase (NTPase) activities, which are located in two functionally distinct domains. The N-terminal one-third of NS3 primarily serves as a protease to process the viral polyprotein. The helicase and NTPase activities are localized to the Cterminal end of the NS3 protein (Warrener & Collett, 1995; Xu et al., 1997). The HCV, BVDV and CSFV NS3 proteins have been demonstrated to have RNA helicase activity (Kim et al., 1995;Sheng et al., 2007;Tai et al., 1996;Warrener & Collett, 1995). Some plus-strand RNA viruses, such as poliovirus and rhinovirus, which lack ...

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supporting

confidence: 89%

Effect of NS3 and NS5B proteins on classical swine fever virus internal ribosome entry site-mediated translation and its host cellular translation

Xiao

Bai

et al. 2008

Journal of General Virology

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“…In addition to targeting the genes involved in pigmentation pathways, we also targeted ddx19, which encodes a DEAD-box protein whose Saccharomyces cerevisiae ortholog, Dbp5, is a wellcharacterized protein essential for mRNA export and protein translation (23,24). A null insertional allele (ddx19 hi1464 ) is available in the zebrafish (25).…”

Section: Biallelic Cas9-induced Disruptions Of Endogenous Loci Resultmentioning

confidence: 99%

Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system

Jao¹,

Wente²,

Chen

2013

Proc. Natl. Acad. Sci. U.S.A.

1,163

1,081

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A simple and robust method for targeted mutagenesis in zebrafish has long been sought. Previous methods generate monoallelic mutations in the germ line of F0 animals, usually delaying homozygosity for the mutation to the F2 generation. Generation of robust biallelic mutations in the F0 would allow for phenotypic analysis directly in injected animals. Recently the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been adapted to serve as a targeted genome mutagenesis tool. Here we report an improved CRISPR/Cas system in zebrafish with custom guide RNAs and a zebrafish codon-optimized Cas9 protein that efficiently targeted a reporter transgene Tg(-5.1mnx1:egfp) and four endogenous loci (tyr, golden, mitfa, and ddx19). Mutagenesis rates reached 75-99%, indicating that most cells contained biallelic mutations. Recessive null-like phenotypes were observed in four of the five targeting cases, supporting high rates of biallelic gene disruption. We also observed efficient germ-line transmission of the Cas9-induced mutations. Finally, five genomic loci can be targeted simultaneously, resulting in multiple loss-of-function phenotypes in the same injected fish. This CRISPR/Cas9 system represents a highly effective and scalable gene knockout method in zebrafish and has the potential for applications in other model organisms.genome engineering | RNA-guided mutagenesis | pigmentation R ecent methodological innovations that exploit zinc finger nucleases (ZFNs) and transcription-activator-like effector nucleases (TALENs) have made it possible to introduce sitespecific genomic modifications in cell culture systems and in a wide range of species (1, 2). These designer nucleases target and cleave specific genomic sequences. Error-prone nonhomologous end joining (NHEJ) DNA repair then generates mutations. The engineering of efficient ZFNs requires extensive technical expertise and empirical testing to find efficient enzymes. The TALEN technology provides an attractive alternative to ZFNs, but like ZFNs, it requires the assembly of two relatively large DNA-binding proteins for each target. Most recently, a new class of genome editing tool based on the type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system has been developed (3).CRISPR systems in eubacteria and archaea use small RNAs and CRISPR-associated proteins (Cas) proteins to target and cleave invading foreign DNAs (4-6). One of the simplest CRISPR systems is the type II CRISPR system from Streptococcus pyogenes: an endonuclease Cas9 and two small RNAs, CRISPR RNA (crRNA) and a transacting RNA (tracrRNA), are sufficient for RNA-guided cleavage of foreign DNAs (7). Recent studies show that a single guide RNA (gRNA) chimera that mimics the crRNA:tracrRNA complex can guide Cas9 to introduce sitespecific DNA double-stranded breaks in vitro (3) and in mammalian cell lines (8-11), bacteria (12, 13), yeast (14), zebrafish (15, 16), and mice (17) with ...

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“…Moreover, the extent of functional connections between mRNA export and translation is unknown. Intriguingly, we and others have shown that Dbp5, Gle1, and IP 6 have roles in translation independent from their roles in mRNA export (32,33). Functionally, each of these factors is required for efficient translation termination.…”

mentioning

confidence: 75%

Control of mRNA Export and Translation Termination by Inositol Hexakisphosphate Requires Specific Interaction with Gle1

Alcázar-Román

Bolger

Wente

2010

Journal of Biological Chemistry

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The unidirectional translocation of messenger RNA (mRNA) through the aqueous channel of the nuclear pore complex (NPC) is mediated by interactions between soluble mRNA export factors and distinct binding sites on the NPC. At the cytoplasmic side of the NPC, the conserved mRNA export factors Gle1 and inositol hexakisphosphate (IP 6 ) play an essential role in mRNA export by activating the ATPase activity of the DEAD-box protein Dbp5, promoting localized messenger ribonucleoprotein complex remodeling, and ensuring the directionality of the export process. In addition, Dbp5, Gle1, and IP 6 are also required for proper translation termination. However, the specificity of the IP 6 -Gle1 interaction in vivo is unknown. Here, we characterize the biochemical interaction between Gle1 and IP 6 and the relationship to Dbp5 binding and stimulation. We identify Gle1 residues required for IP 6 binding and show that these residues are needed for IP 6 -dependent Dbp5 stimulation in vitro. Furthermore, we demonstrate that Gle1 is the primary target of IP 6 for both mRNA export and translation termination in vivo. In Saccharomyces cerevisiae cells, the IP 6 -binding mutants recapitulate all of the mRNA export and translation termination defects found in mutants depleted of IP 6 . We conclude that Gle1 specifically binds IP 6 and that this interaction is required for the full potentiation of Dbp5 ATPase activity during both mRNA export and translation termination.Directional transport of mRNA from the nucleus to the cytoplasm is a highly orchestrated process that bridges nuclear mRNA processing events to protein synthesis in the cytoplasm and thus represents a central step in the regulation of gene expression (1-4). Properly capped, spliced, and polyadenylated mRNAs and their associated RNA-binding proteins constitute mature messenger ribonucleoprotein particles (mRNPs). 4 Of importance for nuclear export, soluble mRNA export factors associate with mRNAs throughout this maturation process, some serving dual roles of mRNA processing and transport regulators (5-8). Export requires targeting to and translocation through NPCs, large hetero-oligomeric structures embedded in nuclear envelope pores. The conserved Saccharomyces cerevisiae Mex67/Mtr2 heterodimer (vertebrate TAP/p15 or NXF1/NXT1) is thought to be the major mRNA export receptor, directly binding the mRNP in the nucleus and facilitating NPC docking and translocation by interaction with NPC proteins (Nups) (1, 9, 10). Mex67-Mtr2 interacts preferentially with phenylalanine-glycine repeats found in Nup domains positioned on the nuclear NPC face and within the central NPC channel (11,12).In addition to critical interactions between Mex67-Mtr2 and phenylalanine-glycine repeat domains in the NPC, directional mRNP export is coincident with altered protein-protein and protein-RNA interactions in the mRNP complex (13). For example, Mex67 and the nuclear poly(A) ϩ -binding protein Nab2 are removed from the mRNP during or after the terminal NPC export step (14, 15). Key factors that med...

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The DEAD-Box RNA Helicase Dbp5 Functions in Translation Termination

Cited by 128 publications

References 12 publications

Effect of NS3 and NS5B proteins on classical swine fever virus internal ribosome entry site-mediated translation and its host cellular translation

Effect of NS3 and NS5B proteins on classical swine fever virus internal ribosome entry site-mediated translation and its host cellular translation

Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system

Control of mRNA Export and Translation Termination by Inositol Hexakisphosphate Requires Specific Interaction with Gle1

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