scholarly journals Yeast tRNA precursor mutated at a splice junction is correctly processed in vivo.

1981 ◽  
Vol 78 (1) ◽  
pp. 415-419 ◽  
Author(s):  
D. Colby ◽  
P. S. Leboy ◽  
C. Guthrie
Keyword(s):  
Splice Junction ◽  
Yeast Trna ◽  
Trna Precursor

scholarly journals Circular RNA hsa_circ_0006401 promotes proliferation and metastasis in colorectal carcinoma

2021 ◽  
Vol 12 (5) ◽  
Author(s):  
Chenjing Zhang ◽  
Xiaolu Zhou ◽  
Xiaoge Geng ◽  
Yu Zhang ◽  
Jingya Wang ◽  
...  
Keyword(s):  
Splice Junction ◽  
Circular Rna ◽  
Host Gene ◽  
Cell Counting ◽  
Tissue Samples ◽  
Proliferation And Metastasis ◽  
And Migration ◽  
Proliferation And Migration

AbstractDysregulation of circular RNA (circRNA) expression is involved in the progression of cancer. Here, we aimed to study the potential function of hsa_circ_0006401 in colorectal cancer (CRC). CircRNA hsa_circ_0006401 expression levels in CRC and adjacent nontumor tissues were analyzed by real-time quantitative PCR (qRT-PCR) and circRNA in situ hybridization (RNA-ISH). Then, CRC cell proliferation was assessed by cell counting. Wound-healing and transwell assays were utilized to detect the effect of hsa_circ_0006401 on CRC migration. A circRNA-ORF construct was created, and a specific antibody against the splice junction of hsa_circ_0006401 was prepared. Finally, the proteins directly binding to hsa_circ_0006401 peptides were identified by immunoprecipitation combined with mass spectrometry. In our study, we found hsa_circ_0006401 was closely related to CRC metastasis and exhibited upregulated expression in metastatic CRC tissue samples. Proliferation and migration were inhibited in vitro when hsa_circ_0006401 expression was silenced. Downregulation of hsa_circ_0006401 expression decreased CRC proliferation and liver metastasis in vivo. A 198-aa peptide was encoded by sequences of the splice junction absent from col6a3. Hsa_circ_0006401 promoted CRC proliferation and migration by encoding the hsa_circ_0006401 peptide. Hsa_circ_0006401 peptides decreased the mRNA and protein level of the host gene col6a3 by promoting col6a3 mRNA stabilation. In conclusion, our study revealed that circRNAs generated from col6a3 that contain an open-reading frame (ORF) encode a novel 198-aa functional peptide and hsa_circ_0006401 peptides promote stability of the host gene col6a3 mRNA to promote CRC proliferation and metastasis.

Synthetic Riboswitches for the Analysis of tRNA Processing by eukaryotic RNase P Enzymes

RNA ◽  
2022 ◽  
pp. rna.078814.121
Author(s):  
Anna Ender ◽  
Nadine Grafl ◽  
Tim Kolberg ◽  
Sven Findeiss ◽  
Peter F. Stadler ◽  
...  
Keyword(s):  
Homo Sapiens ◽  
Rnase P ◽  
Single Stranded Region ◽  
Domains Of Life ◽  
The Individual ◽  
The Impact ◽  
Individual Enzyme ◽  
Trna Precursor

Removal of the 5' leader region is an essential step in the maturation of tRNA molecules in all domains of life. This reaction is catalyzed by various RNase P activities, ranging from ribonucleoproteins with ribozyme activity to protein-only forms. In Escherichia coli, the efficiency of RNase P mediated cleavage can be controlled by computationally designed riboswitch elements in a ligand-dependent way, where the 5' leader sequence of a tRNA precursor is either sequestered in a hairpin structure or presented as a single-stranded region accessible for maturation. In the presented work, the regulatory potential of such artificial constructs is tested on different forms of eukaryotic RNase P enzymes – two protein-only RNase P enzymes (PRORP1 and PRORP2) from Arabidopsis thaliana and the ribonucleoprotein of Homo sapiens. The PRORP enzymes were analyzed in vitro as well as in vivo in a bacterial RNase P complementation system. We also tested in HEK293T cells whether the riboswitches remain functional with human nuclear RNase P. While the regulatory principle of the synthetic riboswitches applies for all tested RNase P enzymes, the results also show differences in the substrate requirements of the individual enzyme versions. Hence, such designed RNase P riboswitches represent a novel tool to investigate the impact of the structural composition of the 5'-leader on substrate recognition by different types of RNase P enzymes.

scholarly journals Trm11p and Trm112p Are both Required for the Formation of 2-Methylguanosine at Position 10 in Yeast tRNA

2005 ◽  
Vol 25 (11) ◽  
pp. 4359-4370 ◽  
Author(s):  
Suresh K. Purushothaman ◽  
Janusz M. Bujnicki ◽  
Henri Grosjean ◽  
Bruno Lapeyre
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Enzymatic Activity ◽  
Zinc Binding ◽  
Growth Defect ◽  
Putative Protein ◽  
Yeast Trna ◽  
Protein Methyltransferase ◽  
Severe Growth Defect ◽  
Severe Growth

ABSTRACT N 2 -Monomethylguanosine-10 (m2G10) and N 2 ,N 2 -dimethylguanosine-26 (m2 2G26) are the only two guanosine modifications that have been detected in tRNA from nearly all archaea and eukaryotes but not in bacteria. In Saccharomyces cerevisiae, formation of m2 2G26 is catalyzed by Trm1p, and we report here the identification of the enzymatic activity that catalyzes the formation of m2G10 in yeast tRNA. It is composed of at least two subunits that are associated in vivo: Trm11p (Yol124c), which is the catalytic subunit, and Trm112p (Ynr046w), a putative zinc-binding protein. While deletion of TRM11 has no detectable phenotype under laboratory conditions, deletion of TRM112 leads to a severe growth defect, suggesting that it has additional functions in the cell. Indeed, Trm112p is associated with at least four proteins: two tRNA methyltransferases (Trm9p and Trm11p), one putative protein methyltransferase (Mtc6p/Ydr140w), and one protein with a Rossmann fold dehydrogenase domain (Lys9p/Ynr050c). In addition, TRM11 interacts genetically with TRM1, thus suggesting that the absence of m2G10 and m2 2G26 affects tRNA metabolism or functioning.

Turnover of the 5′-end phosphate in yeast tRNA in vivo

FEBS Letters ◽  
1978 ◽  
Vol 89 (2) ◽  
pp. 260-262
Author(s):  
T. Wasiak ◽  
M. Gniazdowski
Keyword(s):  
Yeast Trna

A highly specific phosphatase from Saccharomyces cerevisiae implicated in tRNA splicing

1990 ◽  
Vol 10 (3) ◽  
pp. 1049-1055
Author(s):  
S M McCraith ◽  
E M Phizicky
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Splice Junction ◽  
Trna Splicing ◽  
Crude Extracts ◽  
Efficient Expression

We identified and partially purified a phosphatase from crude extracts of Saccharomyces cerevisiae cells that can catalyze the last step of tRNA splicing in vitro. This phosphatase can remove the 2'-phosphate left over at the splice junction after endonuclease has removed the intron and ligase has joined together the two half-molecules. We suggest that this phosphatase is responsible for the completion of tRNA splicing in vivo, based primarily on its specificity for the 2'-phosphate of spliced tRNA and on the resistance of the splice junction 2'-phosphate to a nonspecific phosphatase. Removal of the splice junction 2'-phosphate from the residue adjacent to the anticodon is likely necessary for efficient expression of spliced tRNA. The phosphatase appears to be composed of at least two components which, together with endonuclease and ligase, can be used to reconstitute the entire tRNA-splicing reaction.

scholarly journals Intron mutations affect splicing of Saccharomyces cerevisiae SUP53 precursor tRNA.

1986 ◽  
Vol 6 (7) ◽  
pp. 2674-2683 ◽  
Author(s):  
M C Strobel ◽  
J Abelson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Splice Junction ◽  
Yeast Genome ◽  
Suppressor Trna ◽  
Trna Splicing ◽  
Intron Structure ◽  
Mature Domain ◽  
Mutant Genes

The Saccharomyces cerevisiae amber suppressor tRNA gene SUP53 (a tRNALeu3 allele) was used to investigate the role of intron structure and sequence on precursor tRNA splicing in vivo and in vitro. This gene encodes a pre-tRNA which contains a 32-base intervening sequence. Two types of SUP53 intron mutants were constructed: ones with an internal deletion of the natural SUP53 intron and ones with a novel intron. These mutant genes were transcribed in vitro, and the end-processed transcripts were analyzed for their ability to serve as substrates for the partially purified S. cerevisiae tRNA endonuclease and ligase. The in vitro phenotype of these mutant RNAs was correlated with the in vivo suppressor tRNA function of these SUP53 alleles after integration of the genes into the yeast genome. Analysis of these mutant pre-tRNAs, which exhibited no perturbation of the mature domain, clearly showed that intron structure and sequence can have profound effects on pre-tRNA splicing. All of the mutant RNAs, which were inefficiently spliced or unspliced, evidenced cleavage only at the 5' splice junction. Base changes in the intron proximal to the 3' splice junction could partially rescue the splicing defect. The implications of these data for tRNA endonuclease-substrate interactions are discussed.

scholarly journals A highly specific phosphatase from Saccharomyces cerevisiae implicated in tRNA splicing.

1990 ◽  
Vol 10 (3) ◽  
pp. 1049-1055 ◽  
Author(s):  
S M McCraith ◽  
E M Phizicky
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Splice Junction ◽  
Trna Splicing ◽  
Crude Extracts ◽  
Efficient Expression

We identified and partially purified a phosphatase from crude extracts of Saccharomyces cerevisiae cells that can catalyze the last step of tRNA splicing in vitro. This phosphatase can remove the 2'-phosphate left over at the splice junction after endonuclease has removed the intron and ligase has joined together the two half-molecules. We suggest that this phosphatase is responsible for the completion of tRNA splicing in vivo, based primarily on its specificity for the 2'-phosphate of spliced tRNA and on the resistance of the splice junction 2'-phosphate to a nonspecific phosphatase. Removal of the splice junction 2'-phosphate from the residue adjacent to the anticodon is likely necessary for efficient expression of spliced tRNA. The phosphatase appears to be composed of at least two components which, together with endonuclease and ligase, can be used to reconstitute the entire tRNA-splicing reaction.

In vivo modulation of yeast tRNA gene expression by 5′-flanking sequences.

1985 ◽  
Vol 4 (10) ◽  
pp. 2649-2656 ◽  
Author(s):  
K.C. Raymond ◽  
G.J. Raymond ◽  
J.D. Johnson
Keyword(s):  
Gene Expression ◽  
Trna Gene ◽  
Yeast Trna ◽  
Flanking Sequences

Splice junction mutations in a yeast tRNA gene which alter the rate and precision of processing

1990 ◽  
Vol 1048 (2-3) ◽  
pp. 156-164
Author(s):  
James Chambers ◽  
Gregory J. Raymond ◽  
Daemyung Kim ◽  
Kathleen C. Raymond ◽  
Colleen Nelson ◽  
...  
Keyword(s):  
Trna Gene ◽  
Splice Junction ◽  
Yeast Trna ◽  
Precision Of Processing

scholarly journals Nuclear Pnn/DRS Protein Binds to Spliced mRNPs and Participates in mRNA Processing and Export via Interaction with RNPS1

2003 ◽  
Vol 23 (20) ◽  
pp. 7363-7376 ◽  
Author(s):  
Chin Li ◽  
Ru-Inn Lin ◽  
Ming-Chih Lai ◽  
Pin Ouyang ◽  
Woan-Yuh Tarn
Keyword(s):  
Splice Junction ◽  
Potential Interaction ◽  
Rnase H ◽  
Mrna Processing ◽  
Mrna Splicing ◽  
Nuclear Accumulation ◽  
Exon Junction Complex ◽  
Amino Terminal

ABSTRACT Pnn/DRS protein is associated with desmosomes and colocalizes with splicing factors in nuclear speckled domains. The potential interaction of Pnn with RNPS1, a pre-mRNA splicing factor and a component of the exon-exon junction complex, prompted us to examine whether Pnn is involved in nuclear mRNA processing. By immunoprecipitation, we found that Pnn associates preferentially with mRNAs produced by splicing in vitro. Oligonucleotide-directed RNase H digestion revealed that Pnn binds to the spliced mRNAs at a position immediately upstream of the splice junction and that 5′ splice site utilization determines the location of Pnn in alternatively spliced mRNAs. Immunoprecipitation further showed that Pnn binds to mRNAs produced from a transiently expressed reporter in vivo. Although associated with mRNPs, Pnn is a nuclear-restricted protein as revealed by the heterokaryon assay. Overexpression of an amino-terminal fragment of Pnn that directly interacts with RNPS1 leads to blockage of pre-mRNA splicing. However, although suppression of Pnn expression shows no significant effect on splicing, it leads to some extent to nuclear accumulation of bulk poly(A)+ RNA. Therefore, Pnn may participate, via its interaction with RNPS1, in mRNA metabolism in the nucleus, including mRNA splicing and export.

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