Function of the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase in RNA splicing. Role of the idiosyncratic N-terminal extension and different modes of interaction with different group I introns

2001 ◽  
Vol 307 (1) ◽  
pp. 75-92 ◽  
Author(s):  
Georg Mohr ◽  
Rachel Rennard ◽  
Andrew D Cherniack ◽  
John Stryker ◽  
Alan M Lambowitz
Keyword(s):  
Neurospora Crassa ◽  
Rna Splicing ◽  
Trna Synthetase ◽  
Group I Introns ◽  
Group I ◽  
Modes Of Interaction

Involvement of tyrosyl-tRNA synthetase in splicing of group I introns in Neurospora crassa mitochondria: biochemical and immunochemical analyses of splicing activity

1989 ◽  
Vol 9 (5) ◽  
pp. 2089-2104
Author(s):  
A L Majumder ◽  
R A Akins ◽  
J G Wilkinson ◽  
R L Kelley ◽  
A J Snook ◽  
...  
Keyword(s):  
Neurospora Crassa ◽  
Molecular Mass ◽  
Mitochondrial Protein ◽  
Gel Filtration ◽  
Nuclear Gene ◽  
Trna Synthetase ◽  
Synthetase Activity ◽  
Group I Introns ◽  
Group I

We reported previously that mitochondrial tyrosyl-tRNA synthetase, which is encoded by the nuclear gene cyt-18 in Neurospora crassa, functions in splicing several group I introns in N. crassa mitochondria (R. A. Akins and A. M. Lambowitz, Cell 50:331-345, 1987). Two mutants in the cyt-18 gene (cyt-18-1 and cyt-18-2) are defective in both mitochondrial protein synthesis and splicing, and an activity that splices the mitochondrial large rRNA intron copurifies with a component of mitochondrial tyrosyl-tRNA synthetase. Here, we used antibodies against different trpE-cyt-18 fusion proteins to identify the cyt-18 gene product as a basic protein having an apparent molecular mass of 67 kilodaltons (kDa). Both the cyt-18-1 and cyt-18-2 mutants contain relatively high amounts of inactive cyt-18 protein detected immunochemically. Biochemical experiments show that the 67-kDa cyt-18 protein copurifies with splicing and synthetase activity through a number of different column chromatographic procedures. Some fractions having splicing activity contain only one or two prominent polypeptide bands, and the cyt-18 protein is among the few, if not only, major bands in common between the different fractions that have splicing activity. Phosphocellulose columns resolve three different forms or complexes of the cyt-18 protein that have splicing or synthetase activity or both. Gel filtration experiments show that splicing activity has a relatively small molecular mass (peak at 150 kDa with activity trailing to lower molecular masses) and could correspond simply to dimers or monomers, or both, of the cyt-18 protein. Finally, antibodies against different segments of the cyt-18 protein inhibit splicing of the large rRNA intron in vitro. Our results indicate that both splicing and tyrosyl-tRNA synthetase activity are associated with the same 67-kDa protein encoded by the cyt-18 gene. This protein is a key constituent of splicing activity; it functions directly in splicing, and few, if any, additional components are required for splicing the large rRNA intron.

scholarly journals Involvement of tyrosyl-tRNA synthetase in splicing of group I introns in Neurospora crassa mitochondria: biochemical and immunochemical analyses of splicing activity.

1989 ◽  
Vol 9 (5) ◽  
pp. 2089-2104 ◽  
Author(s):  
A L Majumder ◽  
R A Akins ◽  
J G Wilkinson ◽  
R L Kelley ◽  
A J Snook ◽  
...  
Keyword(s):  
Neurospora Crassa ◽  
Molecular Mass ◽  
Mitochondrial Protein ◽  
Gel Filtration ◽  
Nuclear Gene ◽  
Trna Synthetase ◽  
Synthetase Activity ◽  
Group I Introns ◽  
Group I

We reported previously that mitochondrial tyrosyl-tRNA synthetase, which is encoded by the nuclear gene cyt-18 in Neurospora crassa, functions in splicing several group I introns in N. crassa mitochondria (R. A. Akins and A. M. Lambowitz, Cell 50:331-345, 1987). Two mutants in the cyt-18 gene (cyt-18-1 and cyt-18-2) are defective in both mitochondrial protein synthesis and splicing, and an activity that splices the mitochondrial large rRNA intron copurifies with a component of mitochondrial tyrosyl-tRNA synthetase. Here, we used antibodies against different trpE-cyt-18 fusion proteins to identify the cyt-18 gene product as a basic protein having an apparent molecular mass of 67 kilodaltons (kDa). Both the cyt-18-1 and cyt-18-2 mutants contain relatively high amounts of inactive cyt-18 protein detected immunochemically. Biochemical experiments show that the 67-kDa cyt-18 protein copurifies with splicing and synthetase activity through a number of different column chromatographic procedures. Some fractions having splicing activity contain only one or two prominent polypeptide bands, and the cyt-18 protein is among the few, if not only, major bands in common between the different fractions that have splicing activity. Phosphocellulose columns resolve three different forms or complexes of the cyt-18 protein that have splicing or synthetase activity or both. Gel filtration experiments show that splicing activity has a relatively small molecular mass (peak at 150 kDa with activity trailing to lower molecular masses) and could correspond simply to dimers or monomers, or both, of the cyt-18 protein. Finally, antibodies against different segments of the cyt-18 protein inhibit splicing of the large rRNA intron in vitro. Our results indicate that both splicing and tyrosyl-tRNA synthetase activity are associated with the same 67-kDa protein encoded by the cyt-18 gene. This protein is a key constituent of splicing activity; it functions directly in splicing, and few, if any, additional components are required for splicing the large rRNA intron.

scholarly journals Mutations in nuclear gene cyt-4 of Neurospora crassa result in pleiotropic defects in processing and splicing of mitochondrial RNAs.

Genetics ◽  
1989 ◽  
Vol 123 (1) ◽  
pp. 97-108 ◽  
Author(s):  
K F Dobinson ◽  
M Henderson ◽  
R L Kelley ◽  
R A Collins ◽  
A M Lambowitz
Keyword(s):  
Neurospora Crassa ◽  
Mitochondrial Protein ◽  
Nuclear Gene ◽  
Northern Hybridization ◽  
Rrna Gene ◽  
Mitochondrial Rna ◽  
Group I Introns ◽  
Group I ◽  
Processing Pathways ◽  
Aberrant Rna

Abstract The nuclear cyt-4 mutants of Neurospora crassa have been shown previously to be defective in splicing the group I intron in the mitochondrial large rRNA gene and in 3' end synthesis of the mitochondrial large rRNA. Here, Northern hybridization experiments show that the cyt-4-1 mutant has alterations in a number of mitochondrial RNA processing pathways, including those for cob, coI, coII and ATPase 6 mRNAs, as well as mitochondrial tRNAs. Defects in these pathways include inhibition of 5' and 3' end processing, accumulation of aberrant RNA species, and inhibition of splicing of both group I introns in the cob gene. The various defects in mitochondrial RNA synthesis in the cyt-4-1 mutant cannot be accounted for by deficiency of mitochondrial protein synthesis or energy metabolism, and they suggest that the cyt-4-1 mutant is defective in a component or components required for processing and/or turnover of a number of different mitochondrial RNAs. Defective splicing of the mitochondrial large rRNA intron in the cyt-4-1 mutant may be a secondary effect of failure to synthesize pre-rRNAs having the correct 3' end. However, a similar explanation cannot be invoked to account for defective splicing of the cob pre-mRNA introns, and the cyt-4-1 mutation may directly affect splicing of these introns.

The mitochondrial tyrosyl-tRNA synthetase of Podospora anserina is a bifunctional enzyme active in protein synthesis and RNA splicing

1992 ◽  
Vol 12 (2) ◽  
pp. 499-511
Author(s):  
U Kämper ◽  
U Kück ◽  
A D Cherniack ◽  
A M Lambowitz
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Nuclear Gene ◽  
Trna Synthetase ◽  
Podospora Anserina ◽  
The Other ◽  
Group I Introns ◽  
Hybridization Probe ◽  
Significant Similarity ◽  
Group I

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (mt tyrRS), which is encoded by the nuclear gene cyt-18, functions not only in aminoacylation but also in the splicing of group I introns. Here, we isolated the cognate Podospora anserina mt tyrRS gene, designated yts1, by using the N. crassa cyt-18 gene as a hybridization probe. DNA sequencing of the P. anserina gene revealed an open reading frame (ORF) of 641 amino acids which has significant similarity to other tyrRSs. The yts1 ORF is interrupted by two introns, one near its N terminus at the same position as the single intron in the cyt-18 gene and the other downstream in a region corresponding to the nucleotide-binding fold. The P. anserina yts1+ gene transformed the N. crassa cyt-18-2 mutant at a high frequency and rescued both the splicing and protein synthesis defects. Furthermore, the YTS1 protein synthesized in Escherichia coli was capable of splicing the N. crassa mt large rRNA intron in vitro. Together, these results indicate that YTS1 is a bifunctional protein active in both splicing and protein synthesis. The P. anserina YTS1 and N. crassa CYT-18 proteins share three blocks of amino acids that are not conserved in bacterial or yeast mt tyrRSs which do not function in splicing. One of these blocks corresponds to the idiosyncratic N-terminal domain shown previously to be required for splicing activity of the CYT-18 protein. The other two are located in the putative tRNA-binding domain toward the C terminus of the protein and also appear to be required for splicing. Since the E. coli and yeast mt tyrRSs do not function in splicing, the adaptation of the Neurospora and Podospora spp. mt tyrRSs to function in splicing most likely occurred after the divergence of their common ancestor from yeast.

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

2002 ◽  
Vol 99 (5) ◽  
pp. 2630-2635 ◽  
Author(s):  
C. A. Myers ◽  
B. Kuhla ◽  
S. Cusack ◽  
A. M. Lambowitz
Keyword(s):  
Trna Synthetase ◽  
Group I Introns ◽  
Group I

scholarly journals ATP utilization by a DEAD-box protein during refolding of a misfolded group I intron ribozyme

2020 ◽  
pp. jbc.RA120.015029
Author(s):  
Inga Jarmoskaite ◽  
Pilar Tijerina ◽  
Rick Russell
Keyword(s):  
Neurospora Crassa ◽  
Atp Hydrolysis ◽  
Group I Intron ◽  
Group I Introns ◽  
Structural Rearrangements ◽  
Rna Chaperone ◽  
Dead Box ◽  
Group I ◽  
Rna Biology ◽  
Evolutionary Convergence

DEAD-box helicase proteins perform ATP-dependent rearrangements of structured RNAs throughout RNA biology. Short RNA helices are unwound in a single ATPase cycle, but the ATP requirement for more complex RNA structural rearrangements is unknown. Here we measure the amount of ATP used for native refolding of a misfolded group I intron ribozyme by CYT-19, a Neurospora crassa DEAD-box protein that functions as a general chaperone for mitochondrial group I introns. By comparing the rates of ATP hydrolysis and ribozyme refolding, we find that several hundred ATP molecules are hydrolyzed during refolding of each ribozyme molecule. After subtracting non-productive ATP hydrolysis that occurs in the absence of ribozyme refolding, we find that approximately 100 ATPs are hydrolyzed per refolded RNA as a consequence of interactions specific to the misfolded ribozyme. This value is insensitive to changes in ATP and CYT-19 concentration and decreases with decreasing ribozyme stability. Because of earlier findings that ~90% of global ribozyme unfolding cycles lead back to the kinetically preferred misfolded conformation and are not observed, we estimate that each global unfolding cycle consumes ~10 ATPs. Our results indicate that CYT-19 functions as a general RNA chaperone by using a stochastic, energy-intensive mechanism to promote RNA unfolding and refolding, suggesting an evolutionary convergence with protein chaperones.

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