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

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Cell (Impact Factor: 32.24). 05/2004; 117(3):311-21. DOI: 10.1016/S0092-8674(04)00342-3
Source: PubMed


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; 21(9). DOI:10.1261/rna.051524.115 · 4.94 Impact Factor
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    • "There is appreciable conservation among these eukaryotic Sen2 and Sen34 subunits, and archaeal endonucleases, indicating the same evolutional origin of splicing machinery. The human Sen complex also contains Clp1 (hClp1) (Paushkin et al., 2004; Weitzer and Martinez, 2007). Clp1 was first identified as a component of the cleavage factor II for polyadenylation of pre-mRNAs (de Vries et al., 2000), and then re-identified as an in vitro kinase for tRNA exons when searching for an enzyme phosphorylating siRNAs displaying a 5′-OH group (Figure 4) (Weitzer and Martinez, 2007). "
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    ABSTRACT: Introns are found in various tRNA genes in all the three kingdoms of life. Especially, archaeal and eukaryotic genomes are good sources of tRNA introns that are removed by proteinaceous splicing machinery. Most intron-containing tRNA genes both in archaea and eukaryotes possess an intron at a so-called canonical position, one nucleotide 3' to their anticodon, while recent bioinformatics have revealed unusual types of tRNA introns and their derivatives especially in archaeal genomes. Gain and loss of tRNA introns during various stages of evolution are obvious both in archaea and eukaryotes from analyses of comparative genomics. The splicing of tRNA molecules has been studied extensively from biochemical and cell biological points of view, and such analyses of eukaryotic systems provided interesting findings in the past years. Here, I summarize recent progresses in the analyses of tRNA introns and the splicing process, and try to clarify new and old questions to be solved in the next stages.
    Frontiers in Genetics 07/2014; 5:213. DOI:10.3389/fgene.2014.00213
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    • "In eukaryotes, each tRNA processing step occurs at a different location in the cell, and the cellular distribution of processing enzymes is not conserved among organisms. In animal cells, the tRNA-splicing endonuclease and ligase are localized to the nucleus (Westaway and Abelson, 1995; Paushkin et al., 2004). By contrast, in budding yeast, the endonuclease is present on the surface of mitochondria (Huh et al., 2003; Yoshihisa et al., 2003) and the ligase is present in the cytosol (Huh et al., 2003). "
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    ABSTRACT: A number of genome analyses and searches using programs that focus on the RNA-specific bulge-helix-bulge (BHB) motif have uncovered a wide variety of disrupted tRNA genes. The results of these analyses have shown that genetic information encoding functional RNAs is described in the genome cryptically and is retrieved using various strategies. One such strategy is represented by circularly permuted tRNA genes, in which the sequences encoding the 5'-half and 3'-half of the specific tRNA are separated and inverted on the genome. Biochemical analyses have defined a processing pathway in which the termini of tRNA precursors (pre-tRNAs) are ligated to form a characteristic circular RNA intermediate, which is then cleaved at the acceptor-stem to generate the typical cloverleaf structure with functional termini. The sequences adjacent to the processing site located between the 3'-half and the 5'-half of pre-tRNAs potentially form a BHB motif, which is the dominant recognition site for the tRNA-intron splicing endonuclease, suggesting that circularization of pre-tRNAs depends on the splicing machinery. Some permuted tRNAs contain a BHB-mediated intron in their 5'- or 3'-half, meaning that removal of an intron, as well as swapping of the 5'- and 3'-halves, are required during maturation of their pre-tRNAs. To date, 34 permuted tRNA genes have been identified from six species of unicellular algae and one archaeon. Although their physiological significance and mechanism of development remain unclear, the splicing system of BHB motifs seems to have played a key role in the formation of permuted tRNA genes. In this review, current knowledge of circularly permuted tRNA genes is presented and some unanswered questions regarding these species are discussed.
    Frontiers in Genetics 04/2014; 5:63. DOI:10.3389/fgene.2014.00063
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