RNA size is a critical factor for U-containing substrate selectivity and permanent pseudouridylated product release during the RNA:Ψ-synthase reaction catalyzed by box H/ACA sRNP enzyme at high temperature

Tillault, Anne‐Sophie; Fourmann, Jean-Baptiste; Loegler, Christine; Blaud, Magali; Branlant, Christiane; Charpentier, Bruno

doi:10.1016/j.biochi.2015.04.009

Cited by 2 publications

(10 citation statements)

References 41 publications

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“…Arg, His, and Ser mutation of Tyr182 of PfuCbf5 abolished enzymatic activity under in vitro conditions, but an aromatic replacement (Y182F) only reduced the activity (Zhou et al 2010). Similarly, the Y179F mutant of P. abyssi Cbf5 retained some in vitro activity and the Y179A mutant almost lost this activity (Tillault et al 2015a). Therefore, it appears that the residues of Cbf5 that are adjacent to the conserved residues of Ψ synthases are also important for the optimum activity of Cbf5.…”

Section: Discussionmentioning

confidence: 98%

“…Different mutants of Tyr of Pyrococcus proteins (Y113A, Y113H, and Y113L of PfuCbf5, and Y110A and Y110F of P. abyssi Cbf5) that correspond to Y81 of HvCbf5 also retained some in vitro activity (Zhou et al 2010;Tillault et al 2015a). Therefore, this nonessential but enhancing role of Y81 in Ψ formation is somewhat surprising, because it (or another aromatic residue) is conserved in all Ψ synthases.…”

Section: Discussionmentioning

confidence: 99%

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Structure–function relationships of archaeal Cbf5 during in vivo RNA-guided pseudouridylation

Majumder

Bosmeny

Gupta

2016

RNA

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In Eukarya and Archaea, in addition to protein-only pseudouridine (Ψ) synthases, complexes containing one guide RNA and four proteins can also produce Ψ. Cbf5 protein is the Ψ synthase in the complex. Previously, we showed that Ψ's at positions 1940, 1942, and 2605 of Haloferax volcanii 23S rRNA are absent in a cbf5-deleted strain, and a plasmid-borne copy of cbf5 can rescue the synthesis of these Ψ's. Based on published reports of the structure of archaeal Cbf5 complexed with other proteins and RNAs, we identified several potential residues and structures in H. volcanii Cbf5, which were expected to play important roles in pseudouridylation. We mutated these structures and determined their effects on Ψ production at the three rRNA positions under in vivo conditions. Mutations of several residues in the catalytic domain and certain residues in the thumb loop either abolished Ψ's or produced partial modification; the latter indicates a slower rate of Ψ formation. The universal catalytic aspartate of Ψ synthases could be replaced by glutamate in Cbf5. A conserved histidine, which is common to Cbf5 and TruB is not needed, but another conserved histidine of Cbf5 is required for the in vivo RNA-guided Ψ formation. We also identified a previously unreported novelty in the pseudouridylation activity of Cbf5 where a single stem-loop of a guide H/ACA RNA is used to produce two closely placed Ψ's and mutations of certain residues of Cbf5 abolished one of these two Ψ's. In summary, this first in vivo study identifies several structures of an archaeal Cbf5 protein that are important for its RNAguided pseudouridylation activity.

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Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

Structure–function relationships of archaeal Cbf5 during in vivo RNA-guided pseudouridylation

Majumder

Bosmeny

Gupta

2016

RNA

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“…Up to now, the in vitro specificity and turnover of the H/ACA sRNP enzymes from P . abyssi have been shown to rely on high temperatures 39 and in vivo data have not been documented.…”

Section: Discussionmentioning

confidence: 99%

“…However, Gar1 is dispensable for total modification of an excess of substrate RNA in single-turnover reaction catalyzed by H/ACA RNPs 31 . Nevertheless, under these conditions the presence of Gar1 enhances the kinetics of the reaction 30 , 31 , 38 , 39 . Interestingly, the critical contribution of Gar1 to the turnover of an H/ACA RNP is also evidenced by the activity test performed in vitro in this study on the large 23S rRNA fragment of domain V (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Cbf5 interacts together with the box H/ACA sRNA and proteins Nop10 and Gar1 31 , 32 , 35 – 37 . The Gar1 protein is dispensable for total modification of substrate RNAs in single-turnover reactions 31 , but it enhances the kinetics of the reaction 30 , 31 , 38 , 39 . By modulating the formation of open and closed states of the catalytic center, Gar1 facilitates substrate RNA loading, its efficient placement and the release of the modified RNA 38 , 40 – 42 .…”

Section: Introductionmentioning

confidence: 99%

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Contribution of protein Gar1 to the RNA-guided and RNA-independent rRNA:Ψ-synthase activities of the archaeal Cbf5 protein

Fujikane

Behm‐Ansmant

Tillault

et al. 2018

Sci Rep

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Archaeal RNA:pseudouridine-synthase (PUS) Cbf5 in complex with proteins L7Ae, Nop10 and Gar1, and guide box H/ACA sRNAs forms ribonucleoprotein (RNP) catalysts that insure the conversion of uridines into pseudouridines (Ψs) in ribosomal RNAs (rRNAs). Nonetheless, in the absence of guide RNA, Cbf5 catalyzes the in vitro formation of Ψ2603 in Pyrococcus abyssi 23S rRNA and of Ψ55 in tRNAs. Using gene-disrupted strains of the hyperthermophilic archaeon Thermococcus kodakarensis, we studied the in vivo contribution of proteins Nop10 and Gar1 to the dual RNA guide-dependent and RNA-independent activities of Cbf5 on 23S rRNA. The single-null mutants of the cbf5, nop10, and gar1 genes are viable, but display a thermosensitive slow growth phenotype. We also generated a single-null mutant of the gene encoding Pus10, which has redundant activity with Cbf5 for in vitro formation of Ψ55 in tRNA. Analysis of the presence of Ψs within the rRNA peptidyl transferase center (PTC) of the mutants demonstrated that Cbf5 but not Pus10 is required for rRNA modification. Our data reveal that, in contrast to Nop10, Gar1 is crucial for in vivo and in vitro RNA guide-independent formation of Ψ2607 (Ψ2603 in P. abyssi) by Cbf5. Furthermore, our data indicate that pseudouridylation at orphan position 2589 (2585 in P. abyssi), for which no PUS or guide sRNA has been identified so far, relies on RNA- and Gar1-dependent activity of Cbf5.

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RNA size is a critical factor for U-containing substrate selectivity and permanent pseudouridylated product release during the RNA:Ψ-synthase reaction catalyzed by box H/ACA sRNP enzyme at high temperature

Cited by 2 publications

References 41 publications

Structure–function relationships of archaeal Cbf5 during in vivo RNA-guided pseudouridylation

Structure–function relationships of archaeal Cbf5 during in vivo RNA-guided pseudouridylation

Contribution of protein Gar1 to the RNA-guided and RNA-independent rRNA:Ψ-synthase activities of the archaeal Cbf5 protein

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