Zuotin, a DnaJ molecular chaperone, stimulates cap-independent translation in yeast

Raychaudhuri, Santanu; Fontanes, Vanessa; Banerjee, Rajeev; Bernavichute, Yana; Dasgupta, Asim

doi:10.1016/j.bbrc.2006.09.124

Cited by 5 publications

(4 citation statements)

References 29 publications

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“…NS5A has previously been reported to alter HCV IRES‐mediated translation,24, 25 and a HSP40 family member has been implicated in IRES‐dependent translation in yeast 26. To determine whether HSP40 and HSP70 were involved with NS5A alteration of IRES activity, we used a cell culture–based bicistronic reporter system (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…HSP70 is implicated in NS5A‐mediated IRES translation, as demonstrated by decreased IRES activity with HSP70 knockdown in the IRES reporter system. A yeast HSP40 homolog, and indirectly HSP70 by virtue of their existence as a complex, have previously been shown to be important for cellular IRES specific translation in yeast 26. The reduction in the IRES activity with knockdown of HSP40 is minimal, and with HSP70 is modest compared with Quercetin treatment and may be related to several factors.…”

Section: Discussionmentioning

confidence: 99%

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The heat shock protein inhibitor Quercetin attenuates hepatitis C virus production

et al. 2009

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The hepatitis C viral (HCV) genome is translated through an internal ribosome entry site (IRES) as a single polyprotein precursor that is subsequently cleaved into individual mature viral proteins. Non-structural protein 5A (NS5A) is one of these proteins that has been implicated in regulation of viral genome replication, translation from the viral IRES and viral packaging. We sought to identify cellular proteins that interact with NS5A and determine whether these interactions may play a role in viral production. Mass spectrometric analysis of coimmunoprecipitated NS5A complexes from cell extracts identified heat shock proteins (HSPs) 40 and 70.Weconfirmed anNS5A/HSPinteraction by confocal microscopy demonstrating colocalization of NS5A with HSP40 and with HSP70. Western analysis of coimmunoprecipitated NS5A complexes further confirmed interaction of HSP40 and HSP70 with NS5A.Atransient transfection, luciferase-based, tissue culture IRES assay demonstrated NS5A augmentation of HCV IRES-mediated translation, and small interfering RNA (siRNA)-mediated knockdown of HSP70 reduced this augmentation. Treatment with an inhibitor of HSP synthesis, Quercetin, markedly reduced baseline IRES activity and its augmentation by NS5A. HSP70 knockdown also modestly reduced viral protein accumulation, whereas HSP40 and HSP70 knockdown both reduced infectious viral particle production in an HCV cell culture system using the J6/JFH virus fused to the Renilla luciferase reporter. Treatment with Quercetin reduced infectious particle production at nontoxic concentrations. The marked inhibition of virus production by Quercetin may partially be related to reduction of HSP40 and HSP70 and their potential involvement in IRES translation, as well as viral morphogenesis or secretion. Conclusion Quercetin may allow for dissection of the viral life cycle and has potential therapeutic use to reduce virus production with low associated toxicity.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

The heat shock protein inhibitor Quercetin attenuates hepatitis C virus production

et al. 2009

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“…Originally, the HAP4, YAP1, and TAF145 elements were identified based on the ability of translationally competent yeast extracts to conduct internal initiation on these mRNAs. Since the identification of these elements, they have all been shown to be active in living cells (13,19,20). Of the aforementioned list of yeast cellular IRES elements, only the TIF4631 IRES has been defined by deletion analysis, but no structural data exists for the minimal IRES element (13).…”

Section: Discussionmentioning

confidence: 99%

A Small Stem Loop Element Directs Internal Initiation of the URE2 Internal Ribosome Entry Site in Saccharomyces cerevisiae

Reineke

Komar

Caprara

et al. 2008

Journal of Biological Chemistry

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Internal initiation of translation is the process of beginning protein synthesis independent of the m 7 G cap structure at the 5-end of an mRNA molecule. We have previously shown that the URE2 mRNA in the yeast Saccharomyces cerevisiae contains an internal ribosome entry site (IRES) whose activity is suppressed by eukaryotic initiation factor 2A (eIF2A; YGR054W). In this study, the minimal sequence required to efficiently direct internal initiation was determined using a system that abrogates cap-dependent scanning of the 40 S ribosomal subunit in both wild-type and eIF2A knock-out cells. Subsequently, secondary structural elements within the minimal sequence were determined by probing with RNases T1 and V1 and the small molecule diethylpyrocarbonate. It was found that the URE2 minimal IRES comprises a 104 nucleotide A-rich stem loop element encompassing the internal AUG codon. Interestingly, the internal AUG seems to be involved in base-pairing interactions that would theoretically hamper its ability to interact with incoming initiator tRNA molecules. Furthermore, none of the truncations used to identify the minimal IRES element were capable of abrogating the suppressive effect of eIF2A. Our data provide the first insight into the RNA structural requirements of the yeast translational machinery for cap-independent initiation of protein synthesis.There are two modes of translation initiation: cap-dependent and cap-independent initiation of protein synthesis. Cap-dependent initiation relies on the presence of a 7-methyl guanosine moiety at the 5Ј terminus of the mRNA. A protein complex, eIF4F, consisting of eukaryotic initiation factors 4E, 4G, and 4A recognizes this structure and recruits the 40 S ribosomal subunit. The 40 S subunit, in complex with associated factors, then scans the mRNA until it recognizes an AUG in the correct context for translation to begin (1). Unlike cap-dependent initiation, cap-independent or internal initiation does not require the presence of the m 7 G structure (1, 2). Instead, complex secondary structure elements, termed internal ribosome entry sites (IRESs), 2 serve to recruit the ribosome for protein synthesis. Often proteins act in trans to either recruit the ribosome or reorganize the structure of the RNA for translation (2). Many cases of internal initiation have been documented in mammalian cells and viruses, but few have been described in the yeast Saccharomyces cerevisiae. Activity of viral IRES elements in yeast has not been obtained without perturbing general translation or using an in vitro system (3, 4). For the hepatitis C virus IRES, some sequence in addition to what has been defined as the minimal IRES seems to be required for activity in yeast (5). Gilbert et al. (6) recently reported the presence of several yeast IRES elements active under glucose starvation conditions. Several of these IRES elements depend on poly(A) stretches upstream of the internal AUG codon for activity. Furthermore, poly(A)-binding protein and one allele of eIF4G were implicated in the mecha...

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“…However, purified Zuo1 unspecifically interacts with a variety of nucleic acids. Initially, Zuo1 was identified via its ability to interact with Z-DNA (Zhang et al, 1992), it also interacts tightly with tRNA (Wilhelm et al, 1994) and recently was shown to bind to a small inhibitor RNA (Raychaudhuri et al, 2006). The mouse homolog MIDA1 interacts with DNA that forms small stem loop structures (Inoue et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

Ribosome-associated Complex Binds to Ribosomes in Close Proximity of Rpl31 at the Exit of the Polypeptide Tunnel in Yeast

Peisker

Braun

Wölfle

et al. 2008

MBoC

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Ribosome-associated complex (RAC) consists of the Hsp40 homolog Zuo1 and the Hsp70 homolog Ssz1. The chaperone participates in the biogenesis of newly synthesized polypeptides. Here we have identified yeast Rpl31, a component of the large ribosomal subunit, as a contact point of RAC at the polypeptide tunnel exit. Rpl31 is encoded by RPL31a and RPL31b, two closely related genes. ⌬rpl31a⌬rpl31b displayed slow growth and sensitivity to low as well as high temperatures. In addition, ⌬rpl31a⌬rpl31b was highly sensitive toward aminoglycoside antibiotics and suffered from defects in translational fidelity. With the exception of sensitivity at elevated temperature, the phenotype resembled yeast strains lacking one of the RAC subunits or Rpl39, another protein localized at the tunnel exit. Defects of ⌬rpl31a⌬rpl31b⌬zuo1 did not exceed that of ⌬rpl31a⌬rpl31b or ⌬zuo1. However, the combined deletion of RPL31a, RPL31b, and RPL39 was lethal. Moreover, RPL39 was a multicopy suppressor, whereas overexpression of RAC failed to rescue growth defects of ⌬rpl31a⌬rpl31b. The findings are consistent with a model in that Rpl31 and Rpl39 independently affect a common ribosome function, whereas Rpl31 and RAC are functionally interdependent. Rpl31, while not essential for binding of RAC to the ribosome, might be involved in proper function of the chaperone complex. INTRODUCTIONTwo Hsp70 family members Ssb1/2 (Ssb1 and Ssb2 differ by only four amino acids) and Ssz1 and one J-domain protein (Zuo1) are abundant components of the translation machinery of Saccharomyces cerevisiae (Raue et al., 2007). The three chaperones are genetically linked and form a functional triad. Lack of either SSB1/2, SSZ1, or ZUO1 results in slow growth, cold sensitivity, and pronounced hypersensitivity against aminoglycosides such as paromomycin (Gautschi et al., 2002;Hundley et al., 2002). Ssz1 and Zuo1 assemble into a stable heterodimeric complex termed RAC (ribosome-associated complex). RAC acts as a cochaperone for Ssb1/2 and stimulates its ATP hydrolysis. The function requires both RAC subunits (Huang et al., 2005;Conz et al., 2007).RAC is anchored to the ribosome via Zuo1 (Gautschi et al., 2001). The idea is that positioning of RAC on the ribosome is required for its interaction with Ssb1/2 (Yan et al., 1998). However, the function of Ssz1 does not strictly depend on stable interaction with Zuo1 or ribosomes (Conz et al., 2007). How exactly Zuo1 anchors RAC is currently unclear. It was proposed that Zuo1 binds to ribosomes, in part, by interaction with rRNA (Yan et al., 1998). However, purified Zuo1 unspecifically interacts with a variety of nucleic acids. Initially, Zuo1 was identified via its ability to interact with Z-DNA (Zhang et al., 1992), it also interacts tightly with tRNA (Wilhelm et al., 1994) and recently was shown to bind to a small inhibitor RNA (Raychaudhuri et al., 2006). The mouse homolog MIDA1 interacts with DNA that forms small stem loop structures (Inoue et al., 2000). The diversity of nucleic acids that interact with Zuo1 raises the qu...

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Zuotin, a DnaJ molecular chaperone, stimulates cap-independent translation in yeast

Cited by 5 publications

References 29 publications

The heat shock protein inhibitor Quercetin attenuates hepatitis C virus production

The heat shock protein inhibitor Quercetin attenuates hepatitis C virus production

A Small Stem Loop Element Directs Internal Initiation of the URE2 Internal Ribosome Entry Site in Saccharomyces cerevisiae

Ribosome-associated Complex Binds to Ribosomes in Close Proximity of Rpl31 at the Exit of the Polypeptide Tunnel in Yeast

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