scholarly journals Assembly defects of human tRNA splicing endonuclease contribute to impaired pre-tRNA processing in pontocerebellar hypoplasia

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Samoil Sekulovski ◽  
Pascal Devant ◽  
Silvia Panizza ◽  
Tasos Gogakos ◽  
Anda Pitiriciu ◽  
...  
Keyword(s):  
Transfer Rna ◽  
Endonuclease Activity ◽  
Pontocerebellar Hypoplasia ◽  
Trna Processing ◽  
X Ray ◽  
Trna Splicing ◽  
Cleavage Activity ◽  
Differential Scanning Fluorimetry ◽  
Mature Domain ◽  
Site Recognition

AbstractIntrons of human transfer RNA precursors (pre-tRNAs) are excised by the tRNA splicing endonuclease TSEN in complex with the RNA kinase CLP1. Mutations in TSEN/CLP1 occur in patients with pontocerebellar hypoplasia (PCH), however, their role in the disease is unclear. Here, we show that intron excision is catalyzed by tetrameric TSEN assembled from inactive heterodimers independently of CLP1. Splice site recognition involves the mature domain and the anticodon-intron base pair of pre-tRNAs. The 2.1-Å resolution X-ray crystal structure of a TSEN15–34 heterodimer and differential scanning fluorimetry analyses show that PCH mutations cause thermal destabilization. While endonuclease activity in recombinant mutant TSEN is unaltered, we observe assembly defects and reduced pre-tRNA cleavage activity resulting in an imbalanced pre-tRNA pool in PCH patient-derived fibroblasts. Our work defines the molecular principles of intron excision in humans and provides evidence that modulation of TSEN stability may contribute to PCH phenotypes.

scholarly journals Assembly defects of the human tRNA splicing endonuclease contribute to impaired pre-tRNA processing in pontocerebellar hypoplasia

2020 ◽  
Author(s):  
Samoil Sekulovski ◽  
Pascal Devant ◽  
Silvia Panizza ◽  
Tasos Gogakos ◽  
Anda Pitiriciu ◽  
...  
Keyword(s):  
Transfer Rna ◽  
Endonuclease Activity ◽  
Pontocerebellar Hypoplasia ◽  
Trna Processing ◽  
X Ray ◽  
Trna Splicing ◽  
Cleavage Activity ◽  
Differential Scanning Fluorimetry ◽  
Mature Domain ◽  
Site Recognition

AbstractIntrons of human transfer RNA precursors (pre-tRNAs) are excised by the tRNA splicing endonuclease TSEN in complex with the RNA kinase CLP1. Mutations in TSEN/CLP1 occur in patients with pontocerebellar hypoplasia (PCH), however, their role in the disease is unclear. Here, we show that intron excision is catalyzed by tetrameric TSEN assembled from inactive heterodimers independently of CLP1. Splice site recognition involves the mature domain and the anticodon-intron base pair of pre-tRNAs. The 2.1-Å resolution X-ray crystal structure of a TSEN15–34 heterodimer and differential scanning fluorimetry analyses show that PCH mutations cause thermal destabilization. While endonuclease activity in recombinant mutant TSEN is unaltered, we observe assembly defects and reduced pre-tRNA cleavage activity resulting in an imbalanced pre-tRNA pool in PCH patient-derived fibroblasts. Our work defines the molecular principles of intron excision in humans and provides evidence that modulation of TSEN stability may contribute to PCH phenotypes.

scholarly journals Impairment of the tRNA-splicing endonuclease subunit 54 (tsen54) gene causes neurological abnormalities and larval death in zebrafish models of pontocerebellar hypoplasia

2011 ◽  
Vol 20 (8) ◽  
pp. 1574-1584 ◽  
Author(s):  
Paul R. Kasher ◽  
Yasmin Namavar ◽  
Paula van Tijn ◽  
Kees Fluiter ◽  
Aleksander Sizarov ◽  
...  
Keyword(s):  
Pontocerebellar Hypoplasia ◽  
Trna Splicing ◽  
Neurological Abnormalities

Isolation of a yeast gene involved in species-specific pre-tRNA processing

1988 ◽  
Vol 8 (12) ◽  
pp. 5140-5149
Author(s):  
S S Wang ◽  
A K Hopper
Keyword(s):  
Dna Sequences ◽  
Genomic Library ◽  
Nonsense Suppression ◽  
Suppressor Activity ◽  
Specific Product ◽  
Trna Processing ◽  
Trna Splicing ◽  
Trna Species ◽  
Multiple Copies ◽  
Species Specific

To identify genes involved in pre-tRNA processing, we searched for yeast DNA sequences that specifically enhanced the expression of the SUP4(G37) gene. The SUP4(G37) gene possesses a point mutation at position 37 of suppressor tRNA(Tyr). This lesion results in a reduced rate of pre-tRNA splicing and a decreased level of nonsense suppression. A SUP4(G37) strain was transformed with a yeast genomic library, and the transformants were screened for increased suppressor activity. One transformant contained a plasmid that encoded an unessential gene, STP1, that in multiple copies enhanced the suppression of SUP4(G37) and caused increased production of mature SUP4(G37) product. Disruption of the genomic copy of STP1 resulted in a reduced efficiency of SUP4-mediated suppression and the accumulation of pre-tRNAs. Not all intron-containing pre-tRNAs were affected by the stp1-disruption. At least five of the nine families of pre-tRNAs were affected. Two other species, pre-tRNA(Ile) and pre-tRNA(3Leu), were not. We propose that STP1 encodes a tRNA species-specific product that functions as a helper for pre-tRNA splicing. The STP1 product may interact with pre-tRNAs to generate a structure that is efficiently recognized by splicing machinery.

scholarly journals Mutations affecting the tRNA-splicing endonuclease activity of Saccharomyces cerevisiae.

Genetics ◽  
1988 ◽  
Vol 118 (4) ◽  
pp. 609-617
Author(s):  
M Winey ◽  
M R Culbertson
Keyword(s):  
Growth Defect ◽  
Temperature Sensitive ◽  
Gene Products ◽  
Endonuclease Activity ◽  
Temperature Dependent ◽  
Direct Role ◽  
Trna Splicing

Abstract Two unlinked mutations that alter the enzyme activity of tRNA-splicing endonuclease have been identified in yeast. The sen1-1 mutation, which maps on chromosome 12, causes temperature-sensitive growth, reduced in vitro endonuclease activity, and in vivo accumulation of unspliced pre-tRNAs. The sen2-1 mutation does not confer a detectable growth defect, but causes a temperature-dependent reduction of in vitro endonuclease activity. Pre-tRNAs do not accumulate in sen2-1 strains. The in vitro enzyme activities of sen1-1 and sen2-1 complement in extracts from a heterozygous diploid, but fail to complement in mixed extracts from separate sen1-1 and sen2-1 haploid strains. These results suggest a direct role for SEN gene products in the enzymatic removal of introns from tRNA that is distinct from the role of other products known to affect tRNA splicing.

X-ray Diffraction Studies of Crystalline Transfer RNA

1969 ◽  
Vol 34 (0) ◽  
pp. 149-152 ◽  
Author(s):  
R. M. Bock ◽  
J. D. Young ◽  
M. Labanauskas ◽  
P. G. Connors
Keyword(s):  
Transfer Rna ◽  
X Ray Diffraction ◽  
X Ray

scholarly journals Isolation of a yeast gene involved in species-specific pre-tRNA processing.

1988 ◽  
Vol 8 (12) ◽  
pp. 5140-5149 ◽  
Author(s):  
S S Wang ◽  
A K Hopper
Keyword(s):  
Dna Sequences ◽  
Genomic Library ◽  
Nonsense Suppression ◽  
Suppressor Activity ◽  
Specific Product ◽  
Trna Processing ◽  
Trna Splicing ◽  
Trna Species ◽  
Multiple Copies ◽  
Species Specific

To identify genes involved in pre-tRNA processing, we searched for yeast DNA sequences that specifically enhanced the expression of the SUP4(G37) gene. The SUP4(G37) gene possesses a point mutation at position 37 of suppressor tRNA(Tyr). This lesion results in a reduced rate of pre-tRNA splicing and a decreased level of nonsense suppression. A SUP4(G37) strain was transformed with a yeast genomic library, and the transformants were screened for increased suppressor activity. One transformant contained a plasmid that encoded an unessential gene, STP1, that in multiple copies enhanced the suppression of SUP4(G37) and caused increased production of mature SUP4(G37) product. Disruption of the genomic copy of STP1 resulted in a reduced efficiency of SUP4-mediated suppression and the accumulation of pre-tRNAs. Not all intron-containing pre-tRNAs were affected by the stp1-disruption. At least five of the nine families of pre-tRNAs were affected. Two other species, pre-tRNA(Ile) and pre-tRNA(3Leu), were not. We propose that STP1 encodes a tRNA species-specific product that functions as a helper for pre-tRNA splicing. The STP1 product may interact with pre-tRNAs to generate a structure that is efficiently recognized by splicing machinery.

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