Yeast-like mRNA Capping Apparatus in Giardia lamblia

Hausmann, Stéphane; Altura, Melissa A.; Witmer, Matthew T.; Singer, Steven M.; Elmendorf, Heidi G.; Shuman, Stewart

doi:10.1074/jbc.m412063200

Cited by 32 publications

(32 citation statements)

References 36 publications

(38 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Note that the mRNAs of two eukaryotic RNA viruses (Sindbis virus and Semliki Forest virus) have been reported to contain a significant fraction of 2,7-dimethyguanosine caps (27,28). Whereas indirect assays reveal that Giardia mRNAs contained blocked 5Ј-terminal structures that are resistant to phosphatase but sensitive to pyrophosphatase (15), the structure of the blocking nucleoside is unknown. Also, whereas Giardia contains small RNAs that can be recovered using anti-TMG antibody (18), the structures of those caps have not been determined directly.…”

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

“…Analysis of the Giardia genome is providing important insights to the early origins of RNA processing mechanisms that are regarded as uniquely eukaryotic (12). Although there had been some debate whether Giardia mRNAs even have a 5Ј-cap structure (13,14), recent studies show that Giardia does possess the enzymatic machinery for m 7 G cap synthesis (15), caps the 5Ј ends of its mRNAs, and exploits the m 7 G cap for enhanced translation of a reporter mRNA in vivo (15)(16)(17).The fact that Giardia encodes two homologs of the cap-binding translation initiation factor eIF4E (17) suggests that Giardia is not an exception to the general reliance on the cap structure for optimal gene expression in eukaryotes. However, characterization of the Giardia eIF4E proteins revealed that one paralog, eIF4E2, binds to m 7 G caps, whereas the other paralog, eIF4E1, binds to m 2,2,7 G caps (17).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Giardia lamblia RNA Cap Guanine-N2 Methyltransferase (Tgs2)

Hausmann

Shuman

2005

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Tgs1 is the enzyme responsible for converting 7-methylguanosine RNA caps to the 2,2,7-trimethylguanosine cap structures of small nuclear and small nucleolar RNAs. but is unreactive with GDP, GTP, GpppA, ATP, CTP, or UTP. We find that the conserved residues Asp-68, Glu-91, and Trp-143 are essential for Tgs2 methyltransferase activity in vitro. The m 2,7 GDP product formed by Tgs2 can be converted to m 2,2,7 GDP by S. pombe Tgs1 in the presence of excess AdoMet. However, Giardia Tgs2 itself is apparently unable to add a second methyl group at guanine-N2. This result implies that 2,2,7-trimethylguanosine caps in Giardia are either synthesized by Tgs1 alone or by the sequential action of Tgs2 and Tgs1. The specificity of Tgs2 raises the prospect that some Giardia mRNAs might contain dimethylguanosine caps.Many small noncoding eukaryotic RNAs contain a hypermodified 2,2,7-trimethylguanosine (TMG) 2 5Ј-cap structure (1, 2). TMG caps are also found on nematode mRNAs generated via trans-splicing (3). TMG cap formation in Saccharomyces cerevisiae depends on the Tgs1 protein (4). The presence of a putative AdoMet binding motif in the Tgs1 polypeptide, the mutation of which affects TMG formation in vivo (4, 5), suggested that Tgs1 might be directly involved in TMG formation. Biochemical studies of Schizosaccharomyces pombe Tgs1 showed that it is indeed a catalyst of TMG synthesis (6). Methylation of guanine-N2 by S. pombe Tgs1 in vitro is strictly dependent on the prior methylation of guanine-N7, indicating that TMG caps are formed by post-transcriptional methylation of standard m 7 G caps (6). Guanine-N2 methylation by S. pombe Tgs1 in vitro requires no RNA component and no protein cofactor (6). Although early models suggested that the TMG synthase reaction might require cis-acting RNA signals or the assembly of specific ribonucleoprotein structures (7-10), the recent work on S. pombe Tgs1 instates a more conservative model in which ribonucleoprotein components might simply target Tgs1 to a particular subset of cellular RNAs that already have an m 7 G cap. Given the ubiquity of TMG caps in eukaryotic species, it is surprising that an S. cerevisiae tgs1 deletion mutant is viable, even though the small nuclear RNAs and small nucleolar RNAs in the tgs1⌬ strain lack TMG caps (4). Genetic analysis indicates that Tgs1 is also nonessential for growth of S. pombe.3 In contrast, TMG synthesis is essential in Drosophila, where mutations in the putative Tgs1 active site cause lethality at the early pupal stage of development that correlates with depletion of TMG-containing RNAs (11).The protozoan parasite Giardia lamblia is posited to occupy a deeply branching position in eukaryotic phylogeny. Analysis of the Giardia genome is providing important insights to the early origins of RNA processing mechanisms that are regarded as uniquely eukaryotic (12). Although there had been some debate whether Giardia mRNAs even have a 5Ј-cap structure (13,14), recent studies show that Giardia does possess the enzymatic machinery for m 7 G cap synth...

show abstract

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Giardia lamblia RNA Cap Guanine-N2 Methyltransferase (Tgs2)

Hausmann

Shuman

2005

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Members of this family that have been characterized biochemically include the triphosphatase components of the cellular capping systems of Saccharomyces cerevisiae (Bisaillon and Shuman 2001), Schizosaccharomyces pombe (Pei et al 2001), Candida albicans (Pei et al 2000), Plasmodium falciparum (Ho and Shuman 2001a;Takagi and Buratowski 2001), Trypanosoma brucei (Ho and Shuman 2001b;, Encephalitozoon cuniculi (Hausmann et al 2002), and Giardia lamblia (Hausmann et al 2005), plus the triphosphatases of the Chlorella virus, poxvirus, and baculovirus mRNA capping systems (Myette and Niles 1996;Jin et al 1998;Shuman 2001, 2003;Shuman 2002, 2003). The signature biochemical property of this enzyme family is the ability to hydrolyze NTPs to NDPs and P i in the presence of manganese or cobalt.…”

Section: Introductionmentioning

confidence: 99%

Structure–function analysis of Plasmodium RNA triphosphatase and description of a triphosphate tunnel metalloenzyme superfamily that includes Cet1-like RNA triphosphatases and CYTH proteins

Gong¹,

Smith²,

Shuman³

2006

RNA

Self Cite

View full text Add to dashboard Cite

RNA triphosphatase catalyzes the first step in mRNA capping. The RNA triphosphatases of fungi and protozoa are structurally and mechanistically unrelated to the analogous mammalian enzyme, a situation that recommends RNA triphosphatase as an anti-infective target. Fungal and protozoan RNA triphosphatases belong to a family of metal-dependent phosphohydrolases exemplified by yeast Cet1. The Cet1 active site is unusually complex and located within a topologically closed hydrophilic b-barrel (the triphosphate tunnel). Here we probe the active site of Plasmodium falciparum RNA triphosphatase by targeted mutagenesis and thereby identify eight residues essential for catalysis. The functional data engender an improved structural alignment in which the Plasmodium counterparts of the Cet1 tunnel strands and active-site functional groups are located with confidence. We gain insight into the evolution of the Cet1-like triphosphatase family by noting that the heretofore unique tertiary structure and active site of Cet1 are recapitulated in recently deposited structures of proteins from Pyrococcus (PBD 1YEM) and Vibrio (PDB 2ACA). The latter proteins exemplify a CYTH domain found in CyaB-like adenylate cyclases and mammalian thiamine triphosphatase. We conclude that the tunnel fold first described for Cet1 is the prototype of a larger enzyme superfamily that includes the CYTH branch. This superfamily, which we name ''triphosphate tunnel metalloenzyme,'' is distributed widely among bacterial, archaeal, and eukaryal taxa. It is now clear that Cet1-like RNA triphosphatases did not arise de novo in unicellular eukarya in tandem with the emergence of caps as the defining feature of eukaryotic mRNA. They likely evolved by incremental changes in an ancestral tunnel enzyme that conferred specificity for RNA 59-end processing.

show abstract

“…Members of this family that have been characterized biochemically include the triphosphatase components of the cellular mRNA capping systems of Saccharomyces cerevisiae (2,3), Schizosaccharomyces pombe (4), Candida albicans (5), Plasmodium falciparum (1,6,7), Trypanosoma brucei (8,9), Encephalitozoon cuniculi (10), and Giardia lamblia (11) plus the triphosphatases of the Chlorella virus, poxvirus, and baculovirus mRNA capping systems (12)(13)(14)(15)(16)(17). The signature biochemical property of this branch of the TTM superfamily is the ability to hydrolyze NTPs to nucleoside diphosphates and P i in the presence of manganese (2).…”

mentioning

confidence: 99%

Novel Triphosphate Phosphohydrolase Activity of Clostridium thermocellum TTM, a Member of the Triphosphate Tunnel Metalloenzyme Superfamily

Keppetipola

Jain

Shuman

2007

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Triphosphate tunnel metalloenzymes (TTMs) are a newly recognized superfamily of phosphotransferases defined by a unique active site residing within an eight-stranded ␤ barrel. The prototypical members are the eukaryal metal-dependent RNA triphosphatases, which catalyze the initial step in mRNA capping. Little is known about the activities and substrate specificities of the scores of TTM homologs present in bacterial and archaeal proteomes, nearly all of which are annotated as adenylate cyclases. Here we have conducted a biochemical and structure-function analysis of a TTM protein (CthTTM) from the bacterium Clostridium thermocellum. CthTTM is a metal-dependent tripolyphosphatase and nucleoside triphosphatase; it is not an adenylate cyclase. We have identified 11 conserved amino acids in the tunnel that are critical for tripolyphosphatase and ATPase activity. The most salient findings are that (i) CthTTM is 150-fold more active in cleaving tripolyphosphate than ATP and (ii) the substrate specificity of CthTTM can be transformed by a single mutation (K8A) that abolishes tripolyphosphatase activity while strongly stimulating ATP hydrolysis. Our results underscore the plasticity of CthTTM substrate choice and suggest how novel specificities within the TTM superfamily might evolve through changes in the residues that line the tunnel walls.

show abstract

Yeast-like mRNA Capping Apparatus in Giardia lamblia

Cited by 32 publications

References 36 publications

Giardia lamblia RNA Cap Guanine-N2 Methyltransferase (Tgs2)

Giardia lamblia RNA Cap Guanine-N2 Methyltransferase (Tgs2)

Structure–function analysis of Plasmodium RNA triphosphatase and description of a triphosphate tunnel metalloenzyme superfamily that includes Cet1-like RNA triphosphatases and CYTH proteins

Novel Triphosphate Phosphohydrolase Activity of Clostridium thermocellum TTM, a Member of the Triphosphate Tunnel Metalloenzyme Superfamily

Contact Info

Product

Resources

About