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Identification of a Human Endonuclease Complex Reveals a Link between tRNA Splicing and Pre-mRNA 3′ End Formation

PTC Therapeutics, 100 Corporate Court, South Plainfield, NJ 07080, USA.
Cell (Impact Factor: 33.12). 05/2004; 117(3):311-21. DOI: 10.1016/S0092-8674(04)00342-3
Source: PubMed

ABSTRACT tRNA splicing is a fundamental process required for cell growth and division. The first step in tRNA splicing is the removal of introns catalyzed in yeast by the tRNA splicing endonuclease. The enzyme responsible for intron removal in mammalian cells is unknown. We present the identification and characterization of the human tRNA splicing endonuclease. This enzyme consists of HsSen2, HsSen34, HsSen15, and HsSen54, homologs of the yeast tRNA endonuclease subunits. Additionally, we identified an alternatively spliced isoform of SEN2 that is part of a complex with unique RNA endonuclease activity. Surprisingly, both human endonuclease complexes are associated with pre-mRNA 3' end processing factors. Furthermore, siRNA-mediated depletion of SEN2 exhibited defects in maturation of both pre-tRNA and pre-mRNA. These findings demonstrate a link between pre-tRNA splicing and pre-mRNA 3' end formation, suggesting that the endonuclease subunits function in multiple RNA-processing events.

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    • "ELAC2 was suggested to be involved in nuclear and mitochondrial tRNA 3 ′ end processing in vivo, whereas the cellular role of ELAC1 remains unclear (Rossmanith 2011). A subset of pre-tRNAs contains introns that are removed during nuclear tRNA splicing reactions in mammals (Paushkin et al. 2004). Prior to export to the cytoplasm, the nucleotidyltransferase Trnt1 adds a nontemplated CCA trinucleotide to the 3 ′ end of the tRNA that acts as a prerequisite for aminoacylation (Reichert et al. 2001). "
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    ABSTRACT: Many cellular RNAs require modification of specific residues for their biogenesis, structure, and function. 5-methylcytosine (m(5)C) is a common chemical modification in DNA and RNA but in contrast to the DNA modifying enzymes, only little is known about the methyltransferases that establish m(5)C modifications in RNA. The putative RNA methyltransferase NSUN6 belongs to the family of Nol1/Nop2/SUN domain (NSUN) proteins, but so far its cellular function has remained unknown. To reveal the target spectrum of human NSUN6, we applied UV crosslinking and analysis of cDNA (CRAC) as well as chemical crosslinking with 5-azacytidine. We found that human NSUN6 is associated with tRNAs and acts as a tRNA methyltransferase. Furthermore, we uncovered tRNA(Cys) and tRNA(Thr) as RNA substrates of NSUN6 and identified the cytosine C72 at the 3' end of the tRNA acceptor stem as the target nucleoside. Interestingly, target recognition in vitro depends on the presence of the 3'-CCA tail. Together with the finding that NSUN6 localizes to the cytoplasm and largely colocalizes with marker proteins for the Golgi apparatus and pericentriolar matrix, our data suggest that NSUN6 modifies tRNAs in a late step in their biogenesis. © 2015 Haag et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.
    RNA 07/2015; DOI:10.1261/rna.051524.115 · 4.62 Impact Factor
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    • "One possible function that may or may not be essential for the tRNA SEN is in pre-rRNA processing , either directly or indirectly. Our studies of the yeast tRNA splicing machinery may be applicable to vertebrate pre-tRNA splicing, since in mammalian cells, an alternative tRNA SEN complex with a Sen2 splice variant has been postulated to function in the catalysis of unknown RNA substrates (Paushkin et al. 2004). "
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    ABSTRACT: Pre-tRNA splicing is an essential process in all eukaryotes. In yeast and vertebrates, the enzyme catalyzing intron removal from pre-tRNA is a heterotetrameric complex (splicing endonuclease [SEN] complex). Although the SEN complex is conserved, the subcellular location where pre-tRNA splicing occurs is not. In yeast, the SEN complex is located at the cytoplasmic surface of mitochondria, whereas in vertebrates, pre-tRNA splicing is nuclear. We engineered yeast to mimic the vertebrate cell biology and demonstrate that all three steps of pre-tRNA splicing, as well as tRNA nuclear export and aminoacylation, occur efficiently when the SEN complex is nuclear. However, nuclear pre-tRNA splicing fails to complement growth defects of cells with defective mitochondrial-located splicing, suggesting that the yeast SEN complex surprisingly serves a novel and essential function in the cytoplasm that is unrelated to tRNA splicing. The novel function requires all four SEN complex subunits and the catalytic core. A subset of pre-rRNAs accumulates when the SEN complex is restricted to the nucleus, indicating that the SEN complex moonlights in rRNA processing. Thus, findings suggest that selection for the subcellular distribution of the SEN complex may reside not in its canonical, but rather in a novel, activity.
    Genes & development 03/2012; 26(5):503-14. DOI:10.1101/gad.183004.111 · 12.64 Impact Factor
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    • "To remove such tRNA introns, proteinaceous enzymes catalyze all of the splicing steps. tRNA splicing endonucleases, which govern cleavage of splice sites, are conserved in their amino acid sequences among eukaryotes and archaea, while their intracellular localization shows diversity in eukaryotic cells (Trotta et al. 1997; Yoshihisa et al. 2003; Paushkin et al. 2004; Englert et al. 2007; Yoshihisa et al. 2007). Furthermore, organisms have adopted several different reaction pathways to ligate tRNA exons (Filipowicz and Shatkin 1983; Phizicky et al. 1986; Culver et al. 1997; Englert and Beier 2005; Weitzer and Martinez 2007; Schwer et al. 2008; Englert et al. 2010; Popow et al. 2011). "
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    ABSTRACT: A part of eukaryotic tRNA genes harbor an intron at one nucleotide 3' to the anticodon, so that removal of the intron is an essential processing step for tRNA maturation. While some tRNA introns have important roles in modification of certain nucleotides, essentiality of the tRNA intron in eukaryotes has not been tested extensively. This is partly because most of the eukaryotic genomes have multiple genes encoding an isoacceptor tRNA. Here, we examined whether the intron of tRNA-Trp(CCA) genes, six copies of which are scattered on the genome of yeast, Saccharomyces cerevisiae, is essential for growth or translation of the yeast in vivo. We devised a procedure to remove all of the tRNA introns from the yeast genome iteratively with marker cassettes containing both positive and negative markers. Using this procedure, we removed all the introns from the six tRNA-Trp(CCA) genes, and found that the intronless strain grew normally and expressed tRNA-Trp(CCA) in an amount similar to that of the wild-type genes. Neither incorporation of (35)S-labeled amino acids into a TCA-insoluble fraction nor the major protein pattern on SDS-PAGE/2D gel were affected by complete removal of the intron, while expression levels of some proteins were marginally affected. Therefore, the tRNA-Trp(CCA) intron is dispensable for growth and bulk translation of the yeast. This raises the possibility that some mechanism other than selective pressure from translational efficiency maintains the tRNA intron on the yeast genome.
    RNA 09/2011; 17(9):1760-9. DOI:10.1261/rna.2851411 · 4.62 Impact Factor
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