The Dbp5 cycle at the nuclear pore complex during mRNA export II: nucleotide cycling and mRNP remodeling by Dbp5 are controlled by Nup159 and Gle1

Noble, Kristen N.; Tran, Elizabeth; Alcázar-Román, Abel R.; Hodge, Christine A.; Cole, Charles N.; Wente, Susan R.

doi:10.1101/gad.2040611

Cited by 102 publications

(172 citation statements)

References 51 publications

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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.

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1,186

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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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.

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1,186

1,081

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“…Nevertheless, only a third of yeast DEAD-box proteins have been tested for unwinding activity (26,29,31,(48)(49)(50)(51)(52)(53)(54), and several are not efficient unwinding enzymes (21,55). In contrast, some have the ability to promote duplex formation and to dissociate RNA-protein complexes or function as ATP-dependent RNA-binding proteins (56)(57)(58)(59). The ability to unwind duplexes is thought to arise from the exclusion of dsRNAs when the two RecA domains are closed upon each other (33,60,61; reviewed in refs.…”

Section: Resultsmentioning

confidence: 99%

Cofactor-dependent specificity of a DEAD-box protein

Young

Khoshnevis

Karbstein

2013

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

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DEAD-box proteins, a large class of RNA-dependent ATPases, regulate all aspects of gene expression and RNA metabolism. They can facilitate dissociation of RNA duplexes and remodeling of RNA-protein complexes, serve as ATP-dependent RNA-binding proteins, or even anneal duplexes. These proteins have highly conserved sequence elements that are contained within two RecA-like domains; consequently, their structures are nearly identical. Furthermore, crystal structures of DEAD-box proteins with bound RNA reveal interactions exclusively between the protein and the RNA backbone. Together, these findings suggest that DEAD-box proteins interact with their substrates in a nonspecific manner, which is confirmed in biochemical experiments. Nevertheless, this contrasts with the need to target these enzymes to specific substrates in vivo. Using the DEAD-box protein Rok1 and its cofactor Rrp5, which both function during maturation of the small ribosomal subunit, we show here that Rrp5 provides specificity to the otherwise nonspecific biochemical activities of the Rok1 DEAD-domain. This finding could reconcile the need for specific substrate binding of some DEAD-box proteins with their nonspecific binding surface and expands the potential roles of cofactors to specificity factors. Identification of helicase cofactors and their RNA substrates could therefore help define the undescribed roles of the 19 DEAD-box proteins that function in ribosome assembly.RNA helicases | annealing D EAD-box proteins are RNA-binding ATPases that are involved in all aspects of RNA metabolism: translation initiation, pre-mRNA splicing, mRNA export and decay, and ribosome biogenesis. In these processes, their functions include RNA duplex unwinding, RNA-protein complex remodeling, RNA duplex annealing, and ATP-dependent RNA binding (1-5).In vivo, these enzymes have specific functions that often involve the recognition of specific RNAs and the discrimination against a myriad of nonsubstrates. Nevertheless, only DbpA, a DEAD-box protein involved in bacterial ribosome assembly (6-8), has sequence-specific ATPase activity (6, 9), which arises from unique sequences in its C terminus (10). Such sequence specificity has not been demonstrated for any other DEAD-box protein and is consistent with their highly conserved structures and the almost universal conservation of residues that contact the RNA. Furthermore, analysis of the structures of RNA-bound DEAD-box proteins reveals that RNA contacts are made with the sugarphosphate backbone (11-17), largely precluding sequence-specific interactions between DEAD-box proteins and RNA.Many DEAD-box and related DEAH-box proteins function in conjunction with cofactors (ref. 18 and references therein). These cofactors can regulate the activity of DEAD-box or DEAH-box proteins by stimulating or inhibiting their ATPase, helicase, RNAbinding, or nucleotide-binding activities. Interestingly, cofactors are often RNA-binding proteins (19)(20)(21)(22)(23)(24)(25). We and others therefore postulated that these cofactors could als...

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“…Binding of Nup159 blocks the RNA-binding site of Dbp5 (REFS 17,121) (FIG. 5) and this is required for the release of ADP, adding a new level of DEAD box protein regulation by partner proteins 123,124 . So far, such an ADP-release factor has not been described for any other DEAD box protein, but it may not be unique to Dbp5.…”

Section: Nuclear Specklesmentioning

confidence: 99%

From unwinding to clamping — the DEAD box RNA helicase family

Linder

Jankowsky

2011

Nat Rev Mol Cell Biol

956

1,069

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Nearly all aspects of RNA metabolism, from transcription and translation to mRNA decay, involve RNA helicases, which are enzymes that use ATP to bind or remodel RNA and RNA-protein complexes (ribonucleoprotein (RNP) complexes) 1 . RNA helicases are found in all three domains of life, and many viruses also encode one or more of these proteins 2,3 . Together with the structurally related DNA helicases that function in replication, recombination and repair, the RNA helicases are classified into superfamilies and families, based on sequence and structural features 3,4 . DEAD box proteins form the largest helicase family, with 37 members in humans and 26 in Saccharomyces cerevisiae 3 , and are characterized by the presence of an Asp-Glu-Ala-Asp (DEAD) motif.DEAD box helicases have central and, in many cases, essential physiological roles in cellular RNA metabolism 5 (FIG. 1). The proteins generally function as part of larger multicomponent assemblies, such as the spliceosom e or the eukaryotic translation initiation machinery 6 . Mutations and deregulation of several DEAD box proteins have been linked to disease states, including cancer 7 . Although all DEAD box proteins contain a structurally highly conserved core with conserved ATP-binding and RNA-binding sites, different proteins have been associated with diverse and seemingly unrelated functions, including the disassembly of RNPs, chaperoning during RNA folding and even stabil ization of protein complexes on RNA 5,6 . How these highly conserved proteins fulfil such an array of different functions has been a long-standing question.Research has now started to illuminate the molecular basis for this functional diversity. In this Review, we summarize how structural data, together with biochemical and biophysical studies, have revealed unexpected modes by which these proteins function. We also discuss how novel molecular and cell biological approaches have better defined the cellular roles of DEAD box helicases. We outline our current view on common structural and mechan istic themes that have emerged, and on physical models of how these 'unusual' RNA helicases work.Structures of DEAD box RNA helicases A number of structural studies have universally shown that members of the DEAD box family contain a highly conserved helicase core that harbours the binding sites for ATP and RNA 3,4,8 . The core is surrounded by variable auxiliary domains, which are thought to be critical for the diverse functions of these enzymes.Structure of the helicase core. DEAD box proteins belong to helicase superfamily 2 (SF2) 2 . Similarly to all SF2 helicases, DEAD box proteins are built around a highly conserved helicase core of two virtually identical domains that resemble the bacterial recombination protein recombinase A (RecA) 3,4,8 (FIG. 2a,b). Within this helicase core, at least 12 characteristic sequence motifs are located at conserved positions (FIG. 2a,b). Some of these motifs are conserved across the entire SF2 family, whereas others are found only in the DEAD box family 3 ....

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The Dbp5 cycle at the nuclear pore complex during mRNA export II: nucleotide cycling and mRNP remodeling by Dbp5 are controlled by Nup159 and Gle1

Cited by 102 publications

References 51 publications

Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system

Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system

Cofactor-dependent specificity of a DEAD-box protein

From unwinding to clamping — the DEAD box RNA helicase family

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