Protein universe containing a <scp>PUA RNA</scp>‐binding domain

Cerrudo, Carolina Susana; Ghiringhelli, Pablo Daniel; Gomez, Daniel E.

doi:10.1111/febs.12602

Cited by 21 publications

(23 citation statements)

References 70 publications

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“…Our biochemical studies demonstrate that TruB's PUA domain binds RNA (Fig. 3B), as expected for this domain (25). Superimposing the TruB-T arm structure (16,17) with a full-length tRNA structure suggests a large binding interface between the PUA domain and the acceptor arm of tRNA, which is supported by our results with tRNA labeled at the 3′ end (Fig.…”

Section: Discussionsupporting

confidence: 86%

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RNA modification enzyme TruB is a tRNA chaperone

Keffer-Wilkes

Veerareddygari

Kothe

2016

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

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Cellular RNAs are chemically modified by many RNA modification enzymes; however, often the functions of modifications remain unclear, such as for pseudouridine formation in the tRNA TΨC arm by the bacterial tRNA pseudouridine synthase TruB. Here we test the hypothesis that RNA modification enzymes also act as RNA chaperones. Using TruB as a model, we demonstrate that TruB folds tRNA independent of its catalytic activity, thus increasing the fraction of tRNA that can be aminoacylated. By rapid kinetic stopped-flow analysis, we identified the molecular mechanism of TruB's RNA chaperone activity: TruB binds and unfolds both misfolded and folded tRNAs thereby providing misfolded tRNAs a second chance at folding. Previously, it has been shown that a catalytically inactive TruB variant has no phenotype when expressed in an Escherichia coli truB KO strain [Gutgsell N, et al. (2000) RNA 6(12):1870-1881]. However, here we uncover that E. coli strains expressing a TruB variant impaired in tRNA binding and in in vitro tRNA folding cannot compete with WT E. coli. Consequently, the tRNA chaperone activity of TruB is critical for bacterial fitness. In conclusion, we prove the tRNA chaperone activity of the pseudouridine synthase TruB, reveal its molecular mechanism, and demonstrate its importance for cellular fitness. We discuss the likelihood that other RNA modification enzymes are also RNA chaperones.lthough there is a wealth of information on RNA structure, we are just beginning to understand the RNA folding process that is often assisted by RNA chaperones (1). In contrast to many protein chaperones, RNA chaperones are not ATPases, but instead facilitate unfolding and folding of RNA directly through their interactions with RNA. In addition, the vast majority of all RNAs, including mRNAs, are posttranscriptionally modified by a plethora of RNA modification enzymes (2). Very little is known about the interplay of RNA folding and modification, although it has been speculated that RNA modification enzymes may also act as RNA chaperones (3).Despite the abundance of RNA modifications, their cellular functions are often unclear, including their possible contributions to RNA structure and stability (4). Interestingly, very few RNA modification enzymes are essential for the cell; however, many of these enzymes are conserved. The most abundant RNA modification is the conversion of uridines to pseudouridines that are found in almost all cellular RNAs (4-6). Pseudouridine formation is catalyzed by stand-alone pseudouridine synthases in all domains of life and in addition by H/ACA small ribonucleoproteins (H/ACA sRNPs) in eukaryotes and archaea (7). Remarkably, the only essential pseudouridine synthase is the eukaryotic enzyme Cbf5, the catalytic component of H/ACA sRNPs, whereas all known stand-alone pseudouridine synthases are nonessential (8). Indeed, deletion of most stand-alone pseudouridine synthases in Escherichia coli (9, 10) or Saccharomyces cerevisiae (11) does not impact cell growth under optimal conditions. Surprisingly, ev...

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Section: Discussionsupporting

confidence: 86%

“…The TruB family of pseudouridine synthases contains a conserved catalytic domain and a C-terminal pseudouridine synthase and archeosine transglycosylase (PUA) domain that is predicted to interact with RNA (16,25). We hypothesized that the PUA domain is critical for tRNA binding and folding and in turn for cellular fitness.…”

Section: Significancementioning

confidence: 99%

RNA modification enzyme TruB is a tRNA chaperone

Keffer-Wilkes

Veerareddygari

Kothe

2016

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

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“…The PUA domain is present in many RNA modification enzymes (Cerrudo et al, 2014). It binds tRNA or rRNA through a structurally conserved motif, which comprises α5-β7 loop and β10 strand in the human MCT-1 (Figure 1B).…”

Section: Resultsmentioning

confidence: 99%

Crystal Structure of the Human Ribosome in Complex with DENR-MCT-1

et al. 2017

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SUMMARY The repertoire of the density regulated protein (DENR) and the malignant T-cell- amplified sequence 1 (MCT-1/MCTS1) oncoprotein was recently expanded to include translational control of a specific set of cancer-related mRNAs. DENR and MCT-1 form the heterodimer, which binds to the ribosome and operates at both translation initiation and reinitiation steps, though by a mechanism that is yet unclear. Here, we determined the first crystal structure of the human small ribosomal subunit in complex with DENR-MCT-1. The structure reveals the previously unknown location of the DENR-MCT-1 dimer bound to the small ribosomal subunit. The binding site of the C-terminal domain of DENR on the ribosome has a striking similarity with those of canonical initiation factor 1 (eIF1), which controls the fidelity of translation initiation and scanning. Our findings elucidate how the DENR-MCT-1 dimer interacts with the ribosome and have functional implications for the mechanism of unconventional translation initiation and reinitiation.

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“…1A; Cerrudo et al 2014). Proteins that contain PUA domains function in enzymatic RNA modification like pseudouridine synthases or tRNA modifying enzymes (Cerrudo et al 2014).…”

Section: Methylation Of Trnamentioning

confidence: 99%

“…1A; Cerrudo et al 2014). Proteins that contain PUA domains function in enzymatic RNA modification like pseudouridine synthases or tRNA modifying enzymes (Cerrudo et al 2014). The crystal structure of the Pyrococcus horikoshii archaeosine tRNA-Guanine transglycosylase (ArcTGT) shows that the PUA domain directly contacts the RNA substrate and contributes to the specificity of the protein (Ishitani et al 2002).…”

Section: Methylation Of Trnamentioning

confidence: 99%

NSUN6 is a human RNA methyltransferase that catalyzes formation of m⁵C72 in specific tRNAs

Haag

Warda

Kretschmer

et al. 2015

RNA

165

151

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Many cellular RNAs require modification of specific residues for their biogenesis, structure, and function. 5-methylcytosine (m 5 C) is a common chemical modification in DNA and RNA but in contrast to the DNA modifying enzymes, only little is known about the methyltransferases that establish m 5 C modifications in RNA. The putative RNA methyltransferase NSUN6 belongs to the family of Nol1/Nop2/SUN domain (NSUN) proteins, but so far its cellular function has remained unknown. To reveal the target spectrum of human NSUN6, we applied UV crosslinking and analysis of cDNA (CRAC) as well as chemical crosslinking with 5-azacytidine. We found that human NSUN6 is associated with tRNAs and acts as a tRNA methyltransferase. Furthermore, we uncovered tRNA Cys and tRNA Thr as RNA substrates of NSUN6 and identified the cytosine C72 at the 3 ′ end of the tRNA acceptor stem as the target nucleoside. Interestingly, target recognition in vitro depends on the presence of the 3 ′ -CCA tail. Together with the finding that NSUN6 localizes to the cytoplasm and largely colocalizes with marker proteins for the Golgi apparatus and pericentriolar matrix, our data suggest that NSUN6 modifies tRNAs in a late step in their biogenesis.

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Protein universe containing a PUA RNA‐binding domain

Cited by 21 publications

References 70 publications

RNA modification enzyme TruB is a tRNA chaperone

RNA modification enzyme TruB is a tRNA chaperone

Crystal Structure of the Human Ribosome in Complex with DENR-MCT-1

NSUN6 is a human RNA methyltransferase that catalyzes formation of m⁵C72 in specific tRNAs

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Protein universe containing a PUA RNA‐binding domain

Cited by 21 publications

References 70 publications

RNA modification enzyme TruB is a tRNA chaperone

RNA modification enzyme TruB is a tRNA chaperone

Crystal Structure of the Human Ribosome in Complex with DENR-MCT-1

NSUN6 is a human RNA methyltransferase that catalyzes formation of m5C72 in specific tRNAs

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Product

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About

NSUN6 is a human RNA methyltransferase that catalyzes formation of m⁵C72 in specific tRNAs