Genome-wide screen uncovers novel pathways for tRNA processing and nuclear–cytoplasmic dynamics
2015
Transfer ribonucleic acids (tRNAs) are essential components of the protein synthesis machinery in all kingdoms of life, as they bring amino acids to ribosomes during translation. tRNAs are also involved in many critical processes, including cellular responses to stress, protein degradation, apoptosis, and retrovirus replication (Phizicky and Hopper 2010). Moreover, defects in tRNA processing are responsible for numerous human disorders (Kirchner and Ignatova 2015). The important roles of tRNAs have highlighted a need for a complete understanding of the steps that affect tRNA processing and subcellular dynamics.
To date, 94 gene products that participate in tRNA post-transcriptional processing have been identified in Saccharomyces cerevisiae. Seventy-three are involved in the 25 known tRNA nucleotide modifications, and 21 are involved in tRNA end processing and splicing (Hopper 2013; Sharma et al. 2015). Moreover, there are numerous additional genes encoding proteins that function in tRNA turnover and nuclear–cytoplasmic dynamic pathways that are shared with other types of RNA (Hopper 2013; Takano et al. 2015).
Although genes involved in tRNA biology have been studied extensively, many aspects of tRNA processing and subcellular dynamics remain unclear (Fig. 1A). For example, RNA polymerase III (Pol III) mediates tRNA transcription, but so far only a single yeast Pol III regulator, Maf1, has been identified (Pluta et al. 2001), and it is not clear whether other regulators exist. After tRNA transcription, endonucleases RNase P and RNase Z and exonucleases catalyze tRNA leader and trailer removal (Frank and Pace 1998; Skowronek et al. 2014), and tRNA nucleotidyltransferase Cca1 adds the CCA sequence to the 3′ end (Aebi et al. 1990). It is unknown whether there are additional enzymes or regulators for the end processing steps. In yeast, but not in vertebrate cells, end-processed, partially modified, intron-containing tRNAs are exported to the cytoplasm. Los1 is the only known exporter for initial nuclear export of intron-containing tRNAs (Hopper et al. 1980; Murthi et al. 2010; Huang and Hopper 2015). However, los1Δ cells are healthy (Hurt et al. 1987), indicating that there must be other unknown pathways for tRNA export. Since the tRNA splicing endonuclease (SEN) complex is located on the mitochondrial outer surface (Yoshihisa et al. 2003), intron-containing tRNAs must travel to mitochondria for intron removal. Intron-containing tRNAs are barely detectable in the cytoplasm (Sarkar and Hopper 1998), implicating efficient localization of tRNAs to mitochondria. Nonetheless, gene products that would function in tRNA cytoplasmic movement have not been discovered. Furthermore, little is known about the mechanisms for regulating the proper mitochondrial localization of the SEN complex. In addition, two tRNA degradation pathways for destroying tRNAs with defects in modification and/or stability have been uncovered (Kadaba et al. 2004; Chernyakov et al. 2008), and it is plausible that there might be other missing proteins for degradation of tRNA and their processing intermediates and byproducts.
Figure 1.
Genome-wide screen for novel gene products involved in tRNA biology. (A) Many key gene products involved in tRNA biogenesis and subcellular movement have not been identified. The steps that likely have missing gene products are highlighted by red numbers. ...
In order to identify gene products that function in tRNA production, processing, degradation, and subcellular movement, we conducted an unbiased genome-wide screen of nearly all annotated genes in S. cerevisiae. We used one yeast haploid deletion collection (Winzeler et al. 1999) and two temperature-sensitive collections (Ben-Aroya et al. 2008; Li et al. 2011) to identify mutants with defects in tRNA processing. Previously, we reported that Xrn1, found by this screen, is a key component of the mechanism for tRNA intron turnover (Wu and Hopper 2014). Here, we provide the results of the entire screen that uncovered 162 novel gene products involved in various tRNA biology processes. These include nuclear pore complex (NPC) proteins, tRNA-aminoacyl synthetases (aaRS), cytoskeleton components, mitochondrial membrane proteins, and mRNA/ribosome export machinery. Importantly, we found that the Ran GTPase-dependent karyopherin Crm1/Xpo1 may provide a novel pathway for nuclear export of intron-containing tRNAs.
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