Involvement of Tyrosyl-tRNA Synthetase in Splicing of Group I Introns in <i>Neurospora crassa</i> Mitochondria: Biochemical and Immunochemical Analyses of Splicing Activity

Majumder, Arun Lahiri; Akins, Robert A.; Wilkinson, Julie; Kelley, Robert; Snook, Amy J.; Lambowitz, Alan M.

doi:10.1128/mcb.9.5.2089-2104.1989

Cited by 4 publications

(8 citation statements)

References 36 publications

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“…A New Method for Purifying Bacterially Synthesized CYT-18 Protein. Previous purification protocols for CYT-18 used micrococcal nuclease to free the protein from endogenous nucleic acids prior to fractionation by phosphocellulose or heparin-Sepharose chromatography (Akins & Lambowitz, 1987;Majumder et al, 1989;Kittle et al" 1991). Irrespective of whether the purification was from Neurospora mt ribonucleoprotein (RNP) particles or E. coli, such columns typically resolved two components of CYT-18 protein, one bound to the column and the other in the column flow through.…”

Section: Resultsmentioning

confidence: 99%

“…Irrespective of whether the purification was from Neurospora mt ribonucleoprotein (RNP) particles or E. coli, such columns typically resolved two components of CYT-18 protein, one bound to the column and the other in the column flow through. The former was active in splicing and aminoacylation, whereas the latter had a decreased ratio of splicing to TyrRS activity (Akins & Lambowitz, 1987;Majumder et al, 1989;Kittle et al. 1991).…”

Section: Resultsmentioning

confidence: 99%

“…Similarly for the protein, nitrocellulose filter binding experiments indicated that one molecule of intron RNA is bound per two monomer units (Guo & Lambowitz, 1992). Gel filtration of CYT-18 protein from micrococcal nuclease-digested mt RNP particles showed that both TyrRS and RNA splicing activity coeluted with a peak of CYT-18 at approximately the position expected for a homodimer (Majumder et al, 1989). However, the mt RNP particle preparations were impure, some splicing activity trailed the major peak, and a substantial amount of inactive protein eluted at higher apparent molecular weights, possibly reflecting the presence of larger complexes or aggregation.…”

mentioning

confidence: 92%

“…1992; Mohr et al, 1992). Although additional components may be required for efficient splicing in vivo, purified protein isolated from mitochondria or synthesized via an expression plasmid in Escherichia coli is by itself sufficient to splice the Neurospora mt large rRNA intron in vitro (Majumder et al, 1989;Kittle et al, 1991). The purified CYT-18 protein also binds group I introns from yeast, other fungi, and bacteriophage (Guo & Lambowitz, 1992), and the protein expressed in E. coli can restore the splicing of mutants of the phage T4 td intron in vivo (Mohr et al, 1992).…”

mentioning

confidence: 99%

“…Such mechanisms might include the binding of substrates or cofactors of the aminoacylation or splicing reactions, the binding of other small molecules, covalent modification of the protein, or changes in quaternary structure. Two-dimensional isoelectric focusing/SDS-polyacrylamide gel electrophoresis failed to detect covalently modified forms of the CYT-18 protein in Neurospora mitochondria (Majumder et al, 1989). Although two distinct peaks of CYT-18 were resolved by phosphocellulose or heparin-Sepharose chromatography (Akins & Lambowitz, 1987;Majumder et al, 1989;Kittle et al, 1991), it remained possible that the chromatographic separation merely reflected the incomplete removal of nucleic acids that were tightly bound to some proportion of the protein.…”

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confidence: 99%

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Involvement of Neurospora mitochondrial tyrosyl-tRNA synthetase in RNA splicing. A new method for purifying the protein and characterization of physical and enzymic properties pertinent to splicing

Saldanha¹,

Patel²,

Rajendran³

et al. 1995

Biochemistry

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The Neurospora CYT-18 protein, the mitochondrial tyrosyl-tRNA synthetase, functions in the splicing of group I introns. Here, bacterially expressed CYT-18 protein, purified by a new procedure involving polyethyleneimine precipitation to remove tightly bound nucleic acids, was used to characterize properties pertinent to RNA splicing. Analytical ultracentrifugation and other methods showed that the CYT-18 protein is an asymmetric homodimer. The measured frictional ratio, f/fo = 1.55, corresponds to an axial ratio of 10 for a prolate ellipsoid or 12 for an oblate ellipsoid. Like bacterial TyrRSs, the CYT-18 protein exhibits half-sites reactivity, each homodimer having one active site for tyrosyl adenylation and RNA splicing. The splicing activity of CYT-18 was unaffected by aminoacylation substrates at concentrations used in aminoacylation reactions, whereas the TyrRS activity was inhibited by physiological concentrations of the splicing cofactor GTP, as well as CTP or UTP, or by low concentrations of a group I intron RNA. Kinetic measurements suggest that the binding of CYT-18 to a group I intron substrate is a two-step process, with an initial biomolecular step that is close to diffusion limited (3.24 +/- 0.03 x 10(7) M-1s-1) followed by a slower conformational change (0.54 +/- 0.07 s-1). After CYT-18 binding, splicing occurs at a rate of 0.0025 s-1, within 6-fold of the rate of self-splicing of the Tetrahymena large rRNA intron in vitro. The Kd for the complex between the CYT-18 protein and a group I intron substrate, calculated from koff/kon, was < 0.3 pM, substantially lower than determined by presumed equilibrium measurements [Guo, Q., & Lambowitz, A. M. (1992) Genes Dev. 6, 1357-1372]. As a result of this tight binding, the CYT-18 protein functions stoichiometrically in in vitro splicing reactions due to its extremely slow dissociation from the excised intron RNA. The very tight binding of the CYT-18 protein to the intron RNA raises the possibility that specific mechanisms exist for dissociating the protein from the excised intron in vivo.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 92%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Involvement of Neurospora mitochondrial tyrosyl-tRNA synthetase in RNA splicing. A new method for purifying the protein and characterization of physical and enzymic properties pertinent to splicing

Saldanha¹,

Patel²,

Rajendran³

et al. 1995

Biochemistry

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Nonspecific binding to structured RNA and preferential unwinding of an exposed helix by the CYT-19 protein, a DEAD-box RNA chaperone

Tijerina

Bhaskaran

Russell

2006

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

163

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We explore the interactions of CYT-19, a DExD͞H-box protein that functions in folding of group I RNAs, with a well characterized misfolded species of the Tetrahymena ribozyme. Consistent with its function, CYT-19 accelerates refolding of the misfolded RNA to its native state. Unexpectedly, CYT-19 performs another reaction much more efficiently; it unwinds the 6-bp P1 duplex formed between the ribozyme and its oligonucleotide substrate. Furthermore, CYT-19 performs this reaction 50-fold more efficiently than it unwinds the same duplex free in solution, suggesting that it forms additional interactions with the ribozyme, most likely using a distinct RNA binding site from the one responsible for unwinding. This site can apparently bind double-stranded RNA, as attachment of a simple duplex adjacent to P1 recapitulates much of the activation provided by the ribozyme. Unwinding the native P1 duplex does not accelerate refolding of the misfolded ribozyme, implying that CYT-19 can disrupt multiple contacts on the RNA, consistent with its function in folding of multiple RNAs. Further experiments showed that the P1 duplex unwinding activity is virtually the same whether the ribozyme is misfolded or native but is abrogated by formation of tertiary contacts between the P1 duplex and the body of the ribozyme. Together these results suggest a mechanism for CYT-19 and other general DExD͞H-box RNA chaperones in which the proteins bind to structured RNAs and efficiently unwind loosely associated duplexes, which biases the proteins to disrupt nonnative base pairs and gives the liberated strands an opportunity to refold.group I RNA ͉ RNA folding ͉ RNA unwinding ͉ Tetrahymena ribozyme E ssentially all cellular processes that are mediated by structured RNAs also require one or more DExD͞H-box proteins (1). These proteins use the energy from ATP binding and hydrolysis to accelerate RNA structural transitions, which can represent folding steps toward the native state or conformational switches between functional forms. The requirement for proteins presumably arises because RNA base pairs and other local structure can be highly stable even in the absence of enforcing structure, such that folding steps or rearrangements that require significant unfolding require assistance to proceed efficiently (2-4).Despite their ubiquitous presence, key questions about the functions of DExD͞H-box proteins remain largely unanswered. First, what interactions direct different DExD͞H-box proteins to their physiological substrates? All of these proteins share a core ''helicase'' domain containing a set of conserved motifs, and most have additional domains, a few of which have been shown to recognize substrate RNAs or RNA-protein complexes (reviewed in ref. 5). On this basis, targeting has been proposed as a general role for these domains, and the specific interactions that target one DExD͞H-box protein have been delineated (6-9). Nevertheless, in general, the interactions that direct DExD͞H box proteins to their substrates remain to be identified.Second, h...

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Aminoacyl‐tRNA synthetase – a molecular multitasker

Gupta,

Jani,

Vijayasurya

et al. 2023

The FASEB Journal

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Aminoacyl‐tRNA synthetases (AaRSs) are valuable “housekeeping” enzymes that ensure the accurate transmission of genetic information in living cells, where they aminoacylated tRNA molecules with their cognate amino acid and provide substrates for protein biosynthesis. In addition to their translational or canonical function, they contribute to nontranslational/moonlighting functions, which are mediated by the presence of other domains on the proteins. This was supported by several reports which claim that AaRS has a significant role in gene transcription, apoptosis, translation, and RNA splicing regulation. Noncanonical/ nontranslational functions of AaRSs also include their roles in regulating angiogenesis, inflammation, cancer, and other major physio‐pathological processes. Multiple AaRSs are also associated with a broad range of physiological and pathological processes; a few even serve as cytokines. Therefore, the multifunctional nature of AaRSs suggests their potential as viable therapeutic targets as well. Here, our discussion will encompass a range of noncanonical functions attributed to Aminoacyl‐tRNA Synthetases (AaRSs), highlighting their links with a diverse array of human diseases.

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Involvement of Tyrosyl-tRNA Synthetase in Splicing of Group I Introns in Neurospora crassa Mitochondria: Biochemical and Immunochemical Analyses of Splicing Activity

Cited by 4 publications

References 36 publications

Involvement of Neurospora mitochondrial tyrosyl-tRNA synthetase in RNA splicing. A new method for purifying the protein and characterization of physical and enzymic properties pertinent to splicing

Involvement of Neurospora mitochondrial tyrosyl-tRNA synthetase in RNA splicing. A new method for purifying the protein and characterization of physical and enzymic properties pertinent to splicing

Nonspecific binding to structured RNA and preferential unwinding of an exposed helix by the CYT-19 protein, a DEAD-box RNA chaperone

Aminoacyl‐tRNA synthetase – a molecular multitasker

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