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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"In sum, a major discovery from this work is the possibility that three pathways operate in parallel to export introncontaining pre-tRNAs from the nucleus to the cytoplasm (Fig. 1A). Targeting tRNAs and the SEN complex to mitochondria for tRNA splicing Unlike the human SEN complex that is located in the nucleus (Paushkin et al. 2004 ), the yeast SEN complex is located on the outer surface of mitochondria (Yoshihisa et al. 2003 ). Therefore, after nuclear export, intron-containing tRNAs must locate to mitochondria. "
[Show abstract][Hide abstract]ABSTRACT: Transfer ribonucleic acids (tRNAs) are essential for protein synthesis. However, key gene products involved in tRNA biogenesis and subcellular movement remain to be discovered. We conducted the first comprehensive unbiased analysis of the role of nearly an entire proteome in tRNA biology and describe 162 novel and 12 previously known Saccharomyces cerevisiae gene products that function in tRNA processing, turnover, and subcellular movement. tRNA nuclear export is of particular interest because it is essential, but the known tRNA exporters (Los1 [exportin-t] and Msn5 [exportin-5]) are unessential. We report that mutations of CRM1 (Exportin-1), MEX67/MTR2 (TAP/p15), and five nucleoporins cause accumulation of unspliced tRNA, a hallmark of defective tRNA nuclear export. CRM1 mutation genetically interacts with los1Δ and causes altered tRNA nuclear–cytoplasmic distribution. The data implicate roles for the protein and mRNA nuclear export machineries in tRNA nuclear export. Mutations of genes encoding actin cytoskeleton components and mitochondrial outer membrane proteins also cause accumulation of unspliced tRNA, likely due to defective splicing on mitochondria. Additional gene products, such as chromatin modification enzymes, have unanticipated effects on pre-tRNA end processing. Thus, this genome-wide screen uncovered putative novel pathways for tRNA nuclear export and extensive links between tRNA biology and other aspects of cell physiology.
Full-text · Article · Dec 2015 · Genes & Development
"During their nuclear biogenesis, pre-tRNAs undergo various maturation steps including cleavage of the 5′ leader sequence by RNase P, RNase Z-dependent 3′ end processing, 3′ CCA addition and introduction of a myriad of nucleotide modifications (reviewed in Ref. ). Interestingly, in yeast, splicing of intron-containing tRNAs takes place on the mitochondrial outer surface  , whereas in mammalian cells, the tRNA splicing machinery is located within the nucleus  and intron removal takes place prior to export through the NPC. The major export receptor for tRNAs, which is specific for this class of RNAs, is Los1 in yeast (EXP-t/ XpoT in mammals)    and translocation follows the general paradigm for Ran-dependent transport (Fig. 2a). "
[Show abstract][Hide abstract]ABSTRACT: RNAs and ribonucleoprotein complexes (RNPs) play key roles in mediating and regulating gene expression. In eukaryotes, most RNAs are transcribed, processed and assembled with proteins in the nucleus and then either function in the cytoplasm or also undergo a cytoplasmic phase in their biogenesis. This compartmentalisation ensures that sequential steps in gene expression and RNP production are performed in the correct order and allows important quality control mechanisms that prevent the involvement of aberrant RNAs/RNPs in these cellular pathways. The selective exchange of RNAs/RNPs between the nucleus and cytoplasm is enabled by nuclear pore complexes (NPCs), which function as gateways between these compartments. RNA/RNP transport is facilitated by a range of nuclear transport receptors and adaptors, which are specifically recruited to their cargos and mediate interactions with nucleoporins to allow directional translocation through NPCs. While some transport factors are only responsible for the export/import of a certain class of RNA/RNP, others are multifunctional and, in the case of large RNPs, several export factors appear to work together to bring about export. Recent structural studies have revealed aspects of the mechanisms employed by transport receptors to enable specific cargo recognition, and genome-wide approaches have provided the first insights into the diverse composition of pre-mRNPs during export. Furthermore, the regulation of RNA/RNP export is emerging as an important means to modulate gene expression in stress conditions and disease.
Full-text · Article · Oct 2015 · Journal of Molecular Biology
"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). "