A Simple Method for the Detection of Wybutosine-Modified tRNAPheGAA as a Readout of Retrograde tRNA Nuclear Import and Re-export: HCl/Aniline Cleavage and Nonradioactive Northern Hybridization.

tRNAs are highly mobile molecules that are trafficked back and forth between the nucleus and cytoplasm by several proteins. However, characterization of the movement of tRNAs and the proteins mediating these movements can be difficult. Here, we describe an easy and cost-effective assay to discover genes that are involved in two specific tRNA trafficking events, retrograde nuclear import and nuclear re-export for yeast, Saccharomyces cerevisiae. This assay, referred to as the hydrochloric acid (HCl)/aniline assay, identifies the presence or absence of a unique modification on tRNAPheGAA called wybutosine (yW) that requires mature, spliced tRNAPheGAA to undergo retrograde nuclear import and subsequent nuclear re-export for its addition. Therefore, the presence/absence of yW-modified tRNAPheGAA serves as a readout of retrograde nuclear import and nuclear re-export. This simple assay can be used to determine the role of any gene product in these previously elusive tRNA trafficking events.

In Vivo Cross-Linking and Co-Immunoprecipitation Procedure to Analyze Nuclear tRNA Export Complexes in Yeast Cells.

tRNAs are small noncoding RNAs that are predominantly known for their roles in protein synthesis and also participate in numerous other functions ranging from retroviral replication to apoptosis. In eukaryotic cells, all tRNAs move bidirectionally, shuttling between the nucleus and the cytoplasm. Bidirectional nuclear-cytoplasmic tRNA trafficking requires a complex set of conserved proteins. Here, we describe an in vivo biochemical methodology in Saccharomyces cerevisiae to assess the ability of proteins implicated in tRNA nuclear export to form nuclear export complexes with tRNAs. This method employs tagged putative tRNA nuclear exporter proteins and co-immunoprecipitation of tRNA-exporter complexes using antibody-conjugated magnetic beads. Because the interaction between nuclear exporters and tRNAs may be transient, this methodology employs strategies to effectively trap tRNA-protein complexes in vivo. This pull-down method can be used to verify and characterize candidate proteins and their potential interactors implicated in tRNA nuclear-cytoplasmic trafficking.

The life and times of a tRNA.

The study of eukaryotic tRNA processing has given rise to an explosion of new information and insights in the last several years. We now have unprecedented knowledge of each step in the tRNA processing pathway, revealing unexpected twists in biochemical pathways, multiple new connections with regulatory pathways, and numerous biological effects of defects in processing steps that have profound consequences throughout eukaryotes, leading to growth phenotypes in the yeast Saccharomyces cerevisiae and to neurological and other disorders in humans. This review highlights seminal new results within the pathways that comprise the life of a tRNA, from its birth after transcription until its death by decay. We focus on new findings and revelations in each step of the pathway including the end-processing and splicing steps, many of the numerous modifications throughout the main body and anticodon loop of tRNA that are so crucial for tRNA function, the intricate tRNA trafficking pathways, and the quality control decay pathways, as well as the biogenesis and biology of tRNA-derived fragments. We also describe the many interactions of these pathways with signaling and other pathways in the cell.

Three tRNA nuclear exporters in S. cerevisiae: parallel pathways, preferences, and precision.

tRNAs that are transcribed in the nucleus are exported to the cytoplasm to perform their iterative essential function in translation. However, the complex set of tRNA post-transcriptional processing and subcellular trafficking steps are not completely understood. In particular, proteins involved in tRNA nuclear export remain unknown since the canonical tRNA nuclear exportin, Los1/Exportin-t, is unessential in all tested organisms. We previously reported that budding yeast Mex67-Mtr2, a mRNA nuclear exporter, co-functions with Los1 in tRNA nuclear export. Here we employed in vivo co-purification of tRNAs with endogenously expressed nuclear exporters to document that Crm1 also is a bona fide tRNA nuclear exporter. We document that Los1, Mex67-Mtr2 and Crm1 possess individual tRNA preferences for forming nuclear export complexes with members of the 10 families of intron-containing pre-tRNAs. Remarkably, Mex67-Mtr2, but not Los1 or Crm1, is error-prone, delivering tRNAs to the cytoplasm prior to 5' leader removal. tRNA retrograde nuclear import functions to monitor the aberrant leader-containing spliced tRNAs, returning them to the nucleus where they are degraded by 3' to 5' exonucleases. Overall, our work identifies a new tRNA nuclear exporter, uncovers exporter preferences for specific tRNA families, and documents contribution of tRNA nuclear import to tRNA quality control.

A novel assay provides insight into tRNAPhe retrograde nuclear import and re-export in S. cerevisiae.

In eukaryotes, tRNAs are transcribed in the nucleus and subsequently exported to the cytoplasm where they serve as essential adaptor molecules in translation. However, tRNAs can be returned to the nucleus by the evolutionarily conserved process called tRNA retrograde nuclear import, before relocalization back to the cytoplasm via a nuclear re-export step. Several important functions of these latter two trafficking events have been identified, yet the pathways are largely unknown. Therefore, we developed an assay in Saccharomyces cerevisiae to identify proteins mediating tRNA retrograde nuclear import and re-export using the unique wybutosine modification of mature tRNAPhe. Our hydrochloric acid/aniline assay revealed that the karyopherin Mtr10 mediates retrograde import of tRNAPhe, constitutively and in response to amino acid deprivation, whereas the Hsp70 protein Ssa2 mediates import specifically in the latter. Furthermore, tRNAPhe is re-exported by Crm1 and Mex67, but not by the canonical tRNA exporters Los1 or Msn5. These findings indicate that the re-export process occurs in a tRNA family-specific manner. Together, this assay provides insights into the pathways for tRNAPhe retrograde import and re-export and is a tool that can be used on a genome-wide level to identify additional gene products involved in these tRNA trafficking events.

Beyond rRNA and snRNA: tRNA as a 2'-O-methylation target for nucleolar and Cajal body box C/D RNPs.

Box C/D small nucleolar RNAs (snoRNAs) and small Cajal body (CB) RNAs (scaRNAs) form ribonucleoprotein (RNP) complexes to mediate 2'-O-methylation of rRNAs and small nuclear RNAs (snRNAs), respectively. The site of methylation is determined by antisense elements in the box C/D RNAs that are complementary to sequences in target RNAs. However, numerous box C/D RNAs in mammalian cells lack antisense elements to rRNAs or snRNAs; thus, their targets remain unknown. In this issue of Genes & Development, Vitali and Kiss (pp. 741-746) demonstrate that "orphan" nucleolar box C/D snoRNA SNORD97 and CB box C/D scaRNA SCARNA97 contain antisense elements that target the wobble cytidine at position 34 of human elongator tRNAMet(CAT) for 2'-O-methylation (C34m). C34m is jointly mediated by SNORD97 and SCARNA97 despite their apparently different intranuclear locations. Furthermore, the investigators demonstrate that C34m prohibits site-specific cleavage of tRNAMet (CAT) into tRNA fragments (tRFs) by the stress-responsive endoribonuclease angiogenin, thereby uncovering a role for SNORD97 and SCARNA97 in the biogenesis of tRFs, which modulate a diverse set of cellular functions in human health and disease.

Molecular determinants of metazoan tricRNA biogenesis.

Mature tRNAs are generated by multiple post-transcriptional processing steps, which can include intron removal. Recently, we discovered a new class of circular non-coding RNAs in metazoans, called tRNA intronic circular (tric)RNAs. To investigate the mechanism of tricRNA biogenesis, we generated constructs that replace native introns of human and fruit fly tRNA genes with the Broccoli fluorescent RNA aptamer. Using these reporters, we identified cis-acting elements required for tricRNA formation in vivo. Disrupting a conserved base pair in the anticodon-intron helix dramatically reduces tricRNA levels. Although the integrity of this base pair is necessary for proper splicing, it is not sufficient. In contrast, strengthening weak bases in the helix also interferes with splicing and tricRNA production. Furthermore, we identified trans-acting factors important for tricRNA biogenesis, including several known tRNA processing enzymes such as the RtcB ligase and components of the TSEN endonuclease complex. Depletion of these factors inhibits Drosophila tRNA intron circularization. Notably, RtcB is missing from fungal genomes and these organisms normally produce linear tRNA introns. Here, we show that in the presence of ectopic RtcB, yeast lacking the tRNA ligase Rlg1/Trl1 are converted into producing tricRNAs. In summary, our work characterizes the major players in eukaryotic tricRNA biogenesis.

tRNA Processing and Subcellular Trafficking Proteins Multitask in Pathways for Other RNAs.

This article focuses upon gene products that are involved in tRNA biology, with particular emphasis upon post-transcriptional RNA processing and nuclear-cytoplasmic subcellular trafficking. Rather than analyzing these proteins solely from a tRNA perspective, we explore the many overlapping functions of the processing enzymes and proteins involved in subcellular traffic. Remarkably, there are numerous examples of conserved gene products and RNP complexes involved in tRNA biology that multitask in a similar fashion in the production and/or subcellular trafficking of other RNAs, including small structured RNAs such as snRNA, snoRNA, 5S RNA, telomerase RNA, and SRP RNA as well as larger unstructured RNAs such as mRNAs and RNA-protein complexes such as ribosomes. Here, we provide examples of steps in tRNA biology that are shared with other RNAs including those catalyzed by enzymes functioning in 5' end-processing, pseudoU nucleoside modification, and intron splicing as well as steps regulated by proteins functioning in subcellular trafficking. Such multitasking highlights the clever mechanisms cells employ for maximizing their genomes.

From powerhouse to processing plant: conserved roles of mitochondrial outer membrane proteins in tRNA splicing.

The mitochondrial cytoplasmic surface serves as a processing site for numerous RNAs from budding yeast to metazoans. We report that budding yeast mitochondrial outer membrane (MOM) proteins that are subunits of the translocase of the outer mitochondrial membrane (Tom70 and Tom 22) and sorting and assembly machinery (Sam37) are required for efficient pretransfer RNA (pre-tRNA) splicing. Defective pre-tRNA splicing in MOM mutants is due not to loss of respiratory metabolism but instead inefficient targeting/tethering of tRNA splicing endonuclease (SEN) subunits to mitochondria. Schizosaccharomyces pombe SEN subunits also localize to mitochondria, and Tom70 is required for this localization and pre-tRNA splicing. Thus, the role of MOM protein in targeting/tethering SEN subunits to mitochondria has been conserved for >500 million years.

Size matters: conserved proteins function in length-dependent nuclear export of circular RNAs.

Circular RNAs (circRNAs) comprise a recently appreciated category of RNAs that are in high abundance and serve important biological functions. Although several discoveries have been made regarding the biogenesis and functions of circRNAs, their subcellular trafficking has remained largely unknown. In this issue of Genes & Development, Huang and colleagues (pp. 639-644) reported the first study of the nuclear export of circRNAs. Drosophila Hel25E and its human homologs, UAP56 and URH49, are required for nuclear export of circRNAs. Nuclear export of circRNAs is surprisingly length-dependent, and the length measurement mechanism was shown to be controlled by motifs in Hel25E and its homologs consisting of four amino acids.

tRNA dynamics between the nucleus, cytoplasm and mitochondrial surface: Location, location, location.

Although tRNAs participate in the essential function of protein translation in the cytoplasm, tRNA transcription and numerous processing steps occur in the nucleus. This subcellular separation between tRNA biogenesis and function requires that tRNAs be efficiently delivered to the cytoplasm in a step termed "primary tRNA nuclear export". Surprisingly, tRNA nuclear-cytoplasmic traffic is not unidirectional, but, rather, movement is bidirectional. Cytoplasmic tRNAs are imported back to the nucleus by the "tRNA retrograde nuclear import" step which is conserved from budding yeast to vertebrate cells and has been hijacked by viruses, such as HIV, for nuclear import of the viral reverse transcription complex in human cells. Under appropriate environmental conditions cytoplasmic tRNAs that have been imported into the nucleus return to the cytoplasm via the 3rd nuclear-cytoplasmic shuttling step termed "tRNA nuclear re-export", that again is conserved from budding yeast to vertebrate cells. We describe the 3 steps of tRNA nuclear-cytoplasmic movements and their regulation. There are multiple tRNA nuclear export and import pathways. The different tRNA nuclear exporters appear to possess substrate specificity leading to the tantalizing possibility that the cellular proteome may be regulated at the level of tRNA nuclear export. Moreover, in some organisms, such as budding yeast, the pre-tRNA splicing heterotetrameric endonuclease (SEN), which removes introns from pre-tRNAs, resides on the cytoplasmic surface of the mitochondria. Therefore, we also describe the localization of the SEN complex to mitochondria and splicing of pre-tRNA on mitochondria, which occurs prior to the participation of tRNAs in protein translation. This article is part of a Special Issue entitled: SI: Regulation of tRNA synthesis and modification in physiological conditions and disease edited by Dr. Boguta Magdalena.

Sharing the load: Mex67-Mtr2 cofunctions with Los1 in primary tRNA nuclear export.

Eukaryotic transfer RNAs (tRNAs) are exported from the nucleus, their site of synthesis, to the cytoplasm, their site of function for protein synthesis. The evolutionarily conserved ß-importin family member Los1 (Exportin-t) has been the only exporter known to execute nuclear export of newly transcribed intron-containing pre-tRNAs. Interestingly, LOS1 is unessential in all tested organisms. As tRNA nuclear export is essential, we previously interrogated the budding yeast proteome to identify candidates that function in tRNA nuclear export. Here, we provide molecular, genetic, cytological, and biochemical evidence that the Mex67-Mtr2 (TAP-p15) heterodimer, best characterized for its essential role in mRNA nuclear export, cofunctions with Los1 in tRNA nuclear export. Inactivation of Mex67 or Mtr2 leads to rapid accumulation of end-matured unspliced tRNAs in the nucleus. Remarkably, merely fivefold overexpression of Mex67-Mtr2 can substitute for Los1 in los1Δ cells. Moreover, in vivo coimmunoprecipitation assays with tagged Mex67 document that the Mex67 binds tRNAs. Our data also show that tRNA exporters surprisingly exhibit differential tRNA substrate preferences. The existence of multiple tRNA exporters, each with different tRNA preferences, may indicate that the proteome can be regulated by tRNA nuclear export. Thus, our data show that Mex67-Mtr2 functions in primary nuclear export for a subset of yeast tRNAs.

Multiple Layers of Stress-Induced Regulation in tRNA Biology.

tRNAs are the fundamental components of the translation machinery as they deliver amino acids to the ribosomes during protein synthesis. Beyond their essential function in translation, tRNAs also function in regulating gene expression, modulating apoptosis and several other biological processes. There are multiple layers of regulatory mechanisms in each step of tRNA biogenesis. For example, tRNA 3' trailer processing is altered upon nutrient stress; tRNA modification is reprogrammed under various stresses; nuclear accumulation of tRNAs occurs upon nutrient deprivation; tRNA halves accumulate upon oxidative stress. Here we address how environmental stresses can affect nearly every step of tRNA biology and we describe the possible regulatory mechanisms that influence the function or expression of tRNAs under stress conditions.

Control of Saccharomyces cerevisiae pre-tRNA processing by environmental conditions.

tRNA is essential for translation and decoding of the proteome. The yeast proteome responds to stress and tRNA biosynthesis contributes in this response by repression of tRNA transcription and alterations of tRNA modification. Here we report that the stress response also involves processing of pre-tRNA 3' termini. By a combination of Northern analyses and RNA sequencing, we show that upon shift to elevated temperatures and/or to glycerol-containing medium, aberrant pre-tRNAs accumulate in yeast cells. For pre-tRNAUAU(Ile) and pre-tRNAUUU Lys) these aberrant forms are unprocessed at the 5' ends, but they possess extended 3' termini. Sequencing analyses showed that partial 3' processing precedes 5' processing for pre-tRNAUAU(Ile). An aberrant pre-tRNA(Tyr) that accumulates also possesses extended 3' termini, but it is processed at the 5' terminus. Similar forms of these aberrant pre-tRNAs are detected in the rex1Δ strain that is defective in 3' exonucleolytic trimming of pre-tRNAs but are absent in the lhp1Δ mutant lacking 3' end protection. We further show direct correlation between the inhibition of 3' end processing rate and the stringency of growth conditions. Moreover, under stress conditions Rex1 nuclease seems to be limiting for 3' end processing, by decreased availability linked to increased protection by Lhp1. Thus, our data document complex 3' processing that is inhibited by stress in a tRNA-type and condition-specific manner. This stress-responsive tRNA 3' end maturation process presumably contributes to fine-tune the levels of functional tRNA in budding yeast in response to environmental conditions.

Genome-wide screen uncovers novel pathways for tRNA processing and nuclear-cytoplasmic dynamics.

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.

Quality Control Pathways for Nucleus-Encoded Eukaryotic tRNA Biosynthesis and Subcellular Trafficking.

tRNAs perform an essential role in translating the genetic code. They are long-lived RNAs that are generated via numerous posttranscriptional steps. Eukaryotic cells have evolved numerous layers of quality control mechanisms to ensure that the tRNAs are appropriately structured, processed, and modified. We describe the known tRNA quality control processes that check tRNAs and correct or destroy aberrant tRNAs. These mechanisms employ two types of exonucleases, CCA end addition, tRNA nuclear aminoacylation, and tRNA subcellular traffic. We arrange these processes in order of the steps that occur from generation of precursor tRNAs by RNA polymerase (Pol) III transcription to end maturation and modification in the nucleus to splicing and additional modifications in the cytoplasm. Finally, we discuss the tRNA retrograde pathway, which allows tRNA reimport into the nucleus for degradation or repair.

In vivo biochemical analyses reveal distinct roles of ß-importins and eEF1A in tRNA subcellular traffic.

Bidirectional tRNA movement between the nucleus and the cytoplasm serves multiple biological functions. To gain a biochemical understanding of the mechanisms for tRNA subcellular dynamics, we developed in vivo ß-importin complex coimmunoprecipitation (co-IP) assays using budding yeast. Our studies provide the first in vivo biochemical evidence that two ß-importin family members, Los1 (exportin-t) and Msn5 (exportin-5), serve overlapping but distinct roles in tRNA nuclear export. Los1 assembles complexes with RanGTP and tRNA. Both intron-containing pre-tRNAs and spliced tRNAs, regardless of whether they are aminoacylated, assemble into Los1-RanGTP complexes, documenting that Los1 participates in both primary nuclear export and re-export of tRNAs to the cytoplasm. In contrast, ß-importin Msn5 preferentially assembles with RanGTP and spliced, aminoacylated tRNAs, documenting its role in tRNA nuclear re-export. Tef1/2 (the yeast form of translation elongation factor 1α [eEF1A]) aids the specificity of Msn5 for aminoacylated tRNAs to form a quaternary complex consisting of Msn5, RanGTP, aminoacylated tRNA, and Tef1/2. Assembly and/or stability of this quaternary complex requires Tef1/2, thereby facilitating efficient re-export of aminoacylated tRNAs to the cytoplasm.

Requirement of the spindle pole body for targeting and/or tethering proteins to the inner nuclear membrane.

Appropriate targeting of inner nuclear membrane (INM) proteins is important for nuclear function and architecture. To gain new insights into the mechanism(s) for targeting and/or tethering peripherally associated proteins to the INM, we screened a collection of temperature sensitive S. cerevisiae yeast mutants for defects in INM location of the peripheral protein, Trm1-II-GFP. We uncovered numerous genes encoding components of the Spindle Pole Body (SPB), the yeast centrosome. SPB alterations affect the localization of both an integral (Heh2) and a peripheral INM protein (Trm1-II-GFP), but not a nucleoplasmic protein (Pus1). In wild-type cells Trm1-II-GFP is evenly distributed around the INM, but in SPB mutants, Trm1-II-GFP mislocalizes as a spot(s) near ER-nucleus junctions, perhaps its initial contact site with the nuclear envelope. Employing live cell imaging over time in a microfluidic perfusion system to study protein dynamics, we show that both Trm1-II-GFP INM targeting and maintenance depend upon the SPB. We propose a novel targeting and/or tethering model for a peripherally associated INM protein that combines mechanisms of both integral and soluble nuclear proteins, and describe a role of the SPB in nuclear envelope dynamics that affects this process.