Bacterial Hen1 is a 3′ terminal RNA ribose 2′-O-methyltransferase component of a bacterial RNA repair cassette

Jain, Ruchi; Shuman, Stewart

doi:10.1261/rna.1926510

Cited by 30 publications

(53 citation statements)

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“…Recent studies suggest a capacity for this type of ''intelligent'' RNA repair mechanism in the many bacterial species that encode the repair enzymes Pnkp (polynucleotide 59-kinase/39-phosphatase) and Hen1 in an operon-like gene cassette (Chan et al 2009a;Jain and Shuman 2010). Bacterial Pnkp was first discovered in Clostridium thermocellum and the CthPnkp protein has been characterized extensively as the exemplary bacterial end-healing enzyme (Martins and Shuman 2005;Shuman 2006a,b, 2007;.…”

Section: Introductionmentioning

confidence: 99%

“…The N-terminal half is conserved among the bacterial Hen1 homologs only. Our physical and functional characterization of Clostridium thermocellum Hen1 (CthHen1) has revealed the following (Jain and Shuman 2010). (1 Raven Huang's lab has studied Anabaena variabilis Hen1 methyltransferase and its connection to Anabaena Pnkp; they showed that the AvaHen1-AvaPnkp complex has endhealing and sealing activities on a broken tRNA substrate and that ribose 29O-methylation of the 39 end prior to sealing can protect the repair junction from further damage by a transesterifying ribotoxin (Chan et al 2009a).…”

Section: Introductionmentioning

confidence: 99%

“…A salient finding from our initial characterization of CthHen1 was that its activity was strictly dependent on manganese (Jain and Shuman 2010). We proposed, by analogy to catechol O-methyltransferase (Vidgren et al 1994), that Hen1 uses Mn 2+ to engage the vicinal 29 and 39 OH of the terminal ribose and to lower the pK a of the O29 to allow its attack on the AdoMet methyl group (Jain and Shuman 2010).…”

Section: Introductionmentioning

confidence: 99%

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Active site mapping and substrate specificity of bacterial Hen1, a manganese-dependent 3′ terminal RNA ribose 2′O-methyltransferase

Jain¹,

Shuman²

2011

RNA

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The RNA methyltransferase Hen1 and the RNA end-healing/sealing enzyme Pnkp comprise an RNA repair system encoded by an operon-like cassette present in bacteria from eight different phyla. Clostridium thermocellum Hen1 (CthHen1) is a manganesedependent RNA ribose 29O-methyltransferase that marks the 39 terminal nucleoside of broken RNAs and protects repair junctions from iterative damage by transesterifying endonucleases. Here we used the crystal structure of the homologous plant Hen1 to guide a mutational analysis of CthHen1, the results of which provide new insights to RNA end recognition and catalysis. We illuminated structure-activity relations at eight essential constituents of the active site implicated in binding the 39 dinucleotide of the RNA methyl acceptor (Arg273, Arg414), the manganese cofactor (Glu366, Glu369, His370, His418), and the AdoMet methyl donor (Asp291, Asp316). We investigated the effects of varying the terminal nucleobase, RNA size, RNA content, and RNA secondary structure on methyl acceptor activity. Key findings are as follows. CthHen1 displayed a fourfold preference for guanosine as the terminal nucleoside. RNA size had little impact in the range of 12-24 nucleotides, but activity declined sharply with a 9-mer. CthHen1 was adept at methylating a polynucleotide composed of 23 deoxyribonucleotides and one 39 terminal ribonucleotide, signifying that it has no strict RNA specificity beyond the 39 nucleoside. CthHen1 methylated RNA ends in the context of duplex secondary structures. These properties distinguish bacterial Hen1 from plant and metazoan homologs.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Active site mapping and substrate specificity of bacterial Hen1, a manganese-dependent 3′ terminal RNA ribose 2′O-methyltransferase

Jain¹,

Shuman²

2011

RNA

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“…The capacity for protective immunity during RNA repair is illustrated by the many bacterial species that encode the repair enzymes Pnkp (polynucleotide 5′-kinase/3′-phosphatase) and Hen1 in an operon-like gene cassette (6,7) (Fig. 1A).…”

mentioning

confidence: 99%

“…C. thermocellum Hen1 is a a 465-aa polypeptide. The C-terminal half of CthHen1 is an autonomous manganesedependent 3′-terminal ribose 2′O-methyltransferase (7,12,13). The N-terminal half of CthHen1 has no known enzymatic activity.…”

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confidence: 99%

The adenylyltransferase domain of bacterial Pnkp defines a unique RNA ligase family

Smith

Wang²,

Nair³

et al. 2012

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

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Pnkp is the end-healing and end-sealing component of an RNA repair system present in diverse bacteria from ten different phyla. To gain insight to the mechanism and evolution of this repair system, we determined the crystal structures of the ligase domain of Clostridium thermocellum Pnkp in three functional states along the reaction pathway: apoenzyme, ligase•ATP substrate complex, and covalent ligase-AMP intermediate. The tertiary structure is composed of a classical ligase nucleotidyltransferase module that is embellished by a unique α-helical insert module and a unique C-terminal α-helical module. Structure-guided mutational analysis identified active site residues essential for ligase adenylylation. Pnkp defines a new RNA ligase family with signature structural and functional properties.covalent catalysis | enzyme evolution | RNA repair R NA breakage by site-specific "ribotoxins" is an ancient mechanism by which microbes respond to cellular stress and distinguish self from nonself (1-3). Ribotoxins are transesterifying endonucleases that generate 5′-OH and 2′,3′ cyclic phosphate termini. Repair of this type of RNA damage is feasible via sequential enzymatic end-healing and end-sealing steps (4, 5). In the healing phase, the 2′,3′ cyclic phosphate end is hydrolyzed to a 3′-OH and the 5′-OH end is phosphorylated. The healed 3′-OH and 5′-PO 4 termini are then suitable substrates for sealing by an RNA ligase that restores the 3′,5′ phosphodiester backbone. However, in the event that a ribotoxin is constitutively activated, the RNA repair system faces an uphill battle against relentless RNA cleavage. An ingenious way to evade the vicious cycle is to install a 2′-OCH 3 "mark" at the repair junction (as depicted in Fig. 1B), which then protects the marked RNAs from recurrent damage (6).The capacity for protective immunity during RNA repair is illustrated by the many bacterial species that encode the repair enzymes Pnkp (polynucleotide 5′-kinase/3′-phosphatase) and Hen1 in an operon-like gene cassette (6, 7) (Fig. 1A). Clostridium thermocellum Pnkp (CthPnkp) is an 870-aa polypeptide composed of three catalytic domains: N-terminal kinase, central phosphoesterase, and C-terminal adenylyltransferase (8-11). The kinase module catalyzes phosphoryl transfer from ATP to the 5′-OH RNA end. The phosphoesterase domain releases P i from 2′-PO 4 , 3′-PO 4 or 2′,3′ cyclic phosphate ribonucleotides. The adenylyltransferase domain reacts with ATP to form a covalent enzyme-AMP adduct, just as RNA ligases do during strand joining, but it is unable per se to seal RNA strands (8). The sealing function of the Pnkp ligase-like domain is activated by the Hen1 protein (6) encoded by a flanking genomic ORF. C. thermocellum Hen1 is a a 465-aa polypeptide. The C-terminal half of CthHen1 is an autonomous manganesedependent 3′-terminal ribose 2′O-methyltransferase (7, 12, 13). The N-terminal half of CthHen1 has no known enzymatic activity.Raven Huang's lab has studied Anabaena variabilis Hen1 methyltransferase and its connection to Anabaena Pn...

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RNA nucleotide methylation: 2021 update

Motorin

Helm

2021

WIREs RNA

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Among RNA modifications, transfer of methylgroups from the typical cofactor S-adenosyl-L-methionine by methyltransferases (MTases) to RNA is by far the most common reaction. Since our last review about a decade ago, the field has witnessed the re-emergence of mRNA methylation as an important mechanism in gene regulation. Attention has then spread to many other RNA species; all being included into the newly coined concept of the "epitranscriptome." The focus moved from prokaryotes and single cell eukaryotes as model organisms to higher eukaryotes, in particular to mammals. The perception of the field has dramatically changed over the past decade. A previous lack of phenotypes in knockouts in single cell organisms has been replaced by the apparition of MTases in numerous disease models and clinical investigations. Major driving forces of the field include methylation mapping techniques, as well as the characterization of the various MTases, termed "writers." The latter term has spilled over from DNA modification in the neighboring epigenetics field, along with the designations "readers," applied to mediators of biological effects upon specific binding to a methylated RNA. Furthermore "eraser" enzymes effect the newly discovered oxidative removal of methylgroups. A sense of reversibility and dynamics has replaced the older perception of RNA modification as a concrete-cast, irreversible part of RNA maturation. A related concept concerns incompletely methylated residues, which, through permutation of each site, lead to inhomogeneous populations of numerous modivariants. This review recapitulates the major developments of the past decade outlined above, and attempts a prediction of upcoming trends.

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Bacterial Hen1 is a 3′ terminal RNA ribose 2′-O-methyltransferase component of a bacterial RNA repair cassette

Cited by 30 publications

References 37 publications

Active site mapping and substrate specificity of bacterial Hen1, a manganese-dependent 3′ terminal RNA ribose 2′O-methyltransferase

Active site mapping and substrate specificity of bacterial Hen1, a manganese-dependent 3′ terminal RNA ribose 2′O-methyltransferase

The adenylyltransferase domain of bacterial Pnkp defines a unique RNA ligase family

RNA nucleotide methylation: 2021 update

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