The neurospora CYT-18 protein suppresses defects in the phage T4 td intron by stabilizing the catalytically active structure of the intron core

Mohr, Georg; Zhang, Aixia; Gianelos, Janet A.; Belfort, Marlene; Lambowitz, Alan M.

doi:10.1016/0092-8674(92)90449-m

Cited by 100 publications

(97 citation statements)

References 33 publications

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“…It facilitates splicing of diverse group I introns by stabilizing the catalytically active structure (Guo and Larnbowitz 1992;Mohr et al 1992). It can also suppress defects in mutant phage T4 thymidylate synthase mRNA introns that self-splice at high but not low concentration of MgClz (Mohr et al 1992). The Neurospora pre-rRNA has not been found to undergo detectable self-splicing under any condition in vitro.…”

Section: Protein Facilitationmentioning

confidence: 99%

“…recognizing primarily the P4-P6 region (Mohr et al 1994). It facilitates splicing of diverse group I introns by stabilizing the catalytically active structure (Guo and Larnbowitz 1992;Mohr et al 1992). It can also suppress defects in mutant phage T4 thymidylate synthase mRNA introns that self-splice at high but not low concentration of MgClz (Mohr et al 1992).…”

Section: Protein Facilitationmentioning

confidence: 99%

See 1 more Smart Citation

Group I Ribozymes: Substrate Recognition, Catalytic Strategies, and Comparative Mechanistic Analysis

Cech

Herschlag

1996

Nucleic Acids and Molecular Biology

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Section: Protein Facilitationmentioning

confidence: 99%

Section: Protein Facilitationmentioning

confidence: 99%

Group I Ribozymes: Substrate Recognition, Catalytic Strategies, and Comparative Mechanistic Analysis

Cech

Herschlag

1996

Nucleic Acids and Molecular Biology

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“…Groups I and II intron splicing factors differ among organisms and are commonly host proteins that have or had some other functions. Most promote the splicing of only one or a small number of structurally related introns, but CYT-18 is an exception, because it is able to splice diverse group I introns from N. crassa and other organisms (4)(5)(6). The idiosyncratic nature of group I and II intron splicing factors suggests that they adapted to function in splicing relatively recently in evolution, after the dispersal of the introns as mobile elements (2,3).…”

mentioning

confidence: 99%

Identification and evolution of fungal mitochondrial tyrosyl-tRNA synthetases with group I intron splicing activity

Paukstelis

Lambowitz

2008

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

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The bifunctional Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) both aminoacylates mitochondrial tRNA Tyr and acts as a structure-stabilizing splicing cofactor for group I introns. Previous studies showed that CYT-18 has distinct tRNA Tyr and group I intron-binding sites, with the latter formed by three small ''insertions'' in the nucleotide-binding fold and other structural adaptations compared with nonsplicing bacterial tyrosyl-tRNA synthetases. Here, analysis of genomic sequences shows that mitochondrial tyrosyl-tRNA synthetases with structural adaptations similar to CYT-18's are uniquely characteristic of fungi belonging to the subphylum Pezizomycotina, and biochemical assays confirm group I intron splicing activity for the enzymes from several of these organisms, including Aspergillus nidulans and the human pathogens Coccidioides posadasii and Histoplasma capsulatum. By combining multiple sequence alignments with a previously determined cocrystal structure of a CYT-18/group I intron RNA complex, we identify conserved features of the Pezizomycotina enzymes related to group I intron and tRNA interactions. Our results suggest that mitochondrial tyrosyl-tRNA synthetases with group I intron splicing activity evolved during or after the divergence of the fungal subphyla Pezizomycotina and Saccharomycotina by a mechanism involving the concerted differentiation of preexisting protein loop regions. The unique group I intron splicing activity of these fungal enzymes may provide a new target for antifungal drugs.aminoacyl-tRNA synthetase ͉ medical mycology ͉ ribozyme ͉ RNA splicing ͉ protein structure function T he Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (mtTyrRS; CYT-18 protein) is one of a number of proteins that adapted to function in splicing autocatalytic group I and group II introns in different organisms (1-3). One class of these proteins, splicing factors, functions by binding specifically to the intron RNAs and stabilizing their catalytically active RNA structure for efficient splicing in vivo. Groups I and II intron splicing factors differ among organisms and are commonly host proteins that have or had some other functions. Most promote the splicing of only one or a small number of structurally related introns, but CYT-18 is an exception, because it is able to splice diverse group I introns from N. crassa and other organisms (4-6). The idiosyncratic nature of group I and II intron splicing factors suggests that they adapted to function in splicing relatively recently in evolution, after the dispersal of the introns as mobile elements (2, 3).The CYT-18 protein was identified genetically in a screen for splicing-deficient mutants and shown to function in splicing three group I introns in N. crassa mitochondria: the mitochondrial (mt) large subunit rRNA (mtLSU) intron, ND1-I1, and cob-I2 (7,8). Biochemical studies showed that purified recombinant CYT-18 by itself stimulates group I intron splicing in vitro, that it recognizes conserved secondary and tertiary structure f...

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“…With these putative interacting regions fixed, the structural models based on the T. thermophilus TyrRS͞tRNA Tyr cocrystal structure indicate a decidedly tRNA-like recognition of the group I intron catalytic core, with the acceptor-arm cognate (P9͞L9) interacting with the nucleotide-binding fold of one subunit, and the D-anticodon arm cognate (P4-P6) interacting with the C-terminal domain of the other subunit. The relatively large interface between the protein and intron RNA affords the potential for multiple contacts, as in tRNA binding, and explains how CYT-18 can suppress structural mutations throughout the group I intron catalytic core (3,9,10). The ability of CYT-18 to function in splicing many different group I introns likely reflects that the tRNA-like structural features with which it interacts are highly conserved in group I introns, presumably because they are required for the catalytic activity of the intron RNAs.…”

Section: Discussionmentioning

confidence: 99%

“…The splicing function reflects that CYT-18 recognizes conserved structural features of group I intron and promotes the formation of the catalytically active RNA structure (2)(3)(4)(5). Group I introns, similar to tRNAs, have minimal sequence conservation but share a conserved three-dimensional structure consisting of two double-helical domains (6,7).…”

mentioning

confidence: 99%

tRNA-like recognition of group I introns by a tyrosyl-tRNA synthetase

Myers

Kuhla

Cusack

et al. 2002

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

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The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns by promoting the formation of the catalytically active RNA structure. Previous work suggested that CYT-18 recognizes a conserved tRNA-like structure of the group I intron catalytic core. Here, directed hydroxyl-radical cleavage assays show that the nucleotide-binding fold and C-terminal domains of CYT-18 interact with the expected group I intron cognates of the aminoacyl-acceptor stem and Danticodon arms, respectively. Further, three-dimensional graphic modeling, supported by biochemical data, shows that conserved regions of group I introns can be superimposed over interacting regions of the tRNA in a Thermus thermophilus TyrRS͞tRNA Tyr cocrystal structure. Our results support the hypothesis that CYT-18 and other aminoacyl-tRNA synthetases interact with group I introns by recognizing conserved tRNA-like structural features of the intron RNAs.T he Neurospora crassa mitochondrial (mt) tyrosyl-tRNA synthetase (TyrRS), or CYT-18 protein, functions in both tRNA Tyr aminoacylation and group I intron splicing (1). The splicing function reflects that CYT-18 recognizes conserved structural features of group I intron and promotes the formation of the catalytically active RNA structure (2-5). Group I introns, similar to tRNAs, have minimal sequence conservation but share a conserved three-dimensional structure consisting of two double-helical domains (6, 7). The group I intron's P4-P6 domain is formed by the coaxial stacking of secondary structure elements P5, P4, P6, and P6a, and the P3-P9 domain is formed by P3, P8, P7, and P9, with the juxtaposition of the two domains creating a cleft that contains the intron's active site. RNA-footprinting experiments with the CYT-18-dependent N. crassa mt large subunit rRNA (LSU) and ND1 introns showed that the protein interacts with the phosphodiester backbone on the side opposite the active-site cleft, with most of the potential contact sites in the P4-P6 domain and a few additional sites in the P3-P9 domain (4, 5). Biochemical and genetic studies led to a model in which CYT-18 binds first to the P4-P6 domain to promote its assembly and then makes additional contacts with the P3-P9 domain to stabilize the two domains in the correct relative orientation to form the intron's active site (4,5,(8)(9)(10).Remarkably, comparison of the CYT-18 binding sites in the N. crassa mt LSU and ND1 introns with that in the N. crassa mt tRNA Tyr by graphic modeling revealed an extended threedimensional overlap between the tRNA and highly conserved regions of the group I intron catalytic core (5). In this overlap, the group I intron's P4-P6 stacked helices almost completely superimpose on the tRNA's D-anticodon arm stacked helices, P7 overlaps the variable arm, and P9 largely parallels the acceptor stem, with the discriminator base directly overlapping L9-1, a putative protein contact site in both RNAs. These structural similarities suggest that aminoacyl-tRNA synthetases (aaRSs) and othe...

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The neurospora CYT-18 protein suppresses defects in the phage T4 td intron by stabilizing the catalytically active structure of the intron core

Cited by 100 publications

References 33 publications

Group I Ribozymes: Substrate Recognition, Catalytic Strategies, and Comparative Mechanistic Analysis

Group I Ribozymes: Substrate Recognition, Catalytic Strategies, and Comparative Mechanistic Analysis

Identification and evolution of fungal mitochondrial tyrosyl-tRNA synthetases with group I intron splicing activity

tRNA-like recognition of group I introns by a tyrosyl-tRNA synthetase

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