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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.

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.

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.

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.

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 β-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.

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.

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.

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.

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 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-tRNAUAUIle and pre-tRNAUUULys 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-tRNAUAUIle. An aberrant pre-tRNATyr 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.

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.

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 β-importin complex coimmunoprecipitation (co-IP) assays using budding yeast. Our studies provide the first in vivo biochemical evidence that two β-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, β-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.

Mod5 is the yeast tRNA isopentenyl transferase, an enzyme that is conserved from bacteria to humans. Mod5 is primarily cytoplasmic where it modifies the A37 position of a few tRNAs, and the yeast enzyme has been shown capable of forming heritable, amyloid-like aggregates that confer a selective advantage in the presence of specific antifungal agents. A subpopulation of Mod5 is also found associated with nuclear tRNA genes, where it contributes tRNA-gene mediated (tgm) silencing of local transcription by RNA polymerase II. The tgm-silencing function of Mod5 has been observed in yeast and a Mod5-deletion in yeast can be complemented by the plant and human tRNA isopentenyl transferases, but not the bacterial enzymes, possibly due to the lack of an extended C-terminal domain found in eukaryotes. In light of this additional nuclear role for Mod5 we discuss the proposed role of the human homologue of Mod5, TRIT1, as a tumor suppressor protein.

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.

The subcellular distribution of yeast β-importins inverts upon acute glucose deprivation, likely due to collapse of the RanGTP nuclear–cytoplasmic gradient. This redistribution of β-importins likely results in rapid widespread alterations of the traffic of macromolecules between the nucleus and cytoplasm in response to glucose limitation., The importin-β family members (karyopherins) mediate the majority of nucleocytoplasmic transport. Msn5 and Los1, members of the importin-β family, function in tRNA nuclear export. tRNAs move bidirectionally between the nucleus and the cytoplasm. Nuclear tRNA accumulation occurs upon amino acid (aa) or glucose deprivation. To understand the mechanisms regulating tRNA subcellular trafficking, we investigated whether Msn5 and Los1 are regulated in response to nutrient availability. We provide evidence that tRNA subcellular trafficking is regulated by distinct aa-sensitive and glucose-sensitive mechanisms. Subcellular distributions of Msn5 and Los1 are altered upon glucose deprivation but not aa deprivation. Redistribution of tRNA exportins from the nucleus to the cytoplasm likely provides one mechanism for tRNA nuclear distribution upon glucose deprivation. We extended our studies to other members of the importin-β family and found that all tested karyopherins invert their subcellular distributions upon glucose deprivation but not aa deprivation. Glucose availability regulates the subcellular distributions of karyopherins likely due to alteration of the RanGTP gradient since glucose deprivation causes redistribution of Ran. Thus nuclear–cytoplasmic distribution of macromolecules is likely generally altered upon glucose deprivation due to collapse of the RanGTP gradient and redistribution of karyopherins between the nucleus and the cytoplasm.

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.

In eukaryotes, transfer RNAs (tRNAs) are transcribed in the nucleus yet function in the cytoplasm; thus, tRNA movement within the cell was believed to be unidirectional--from the nucleus to the cytoplasm. It is now known that mature tRNAs also move in a retrograde direction from the cytoplasm to the nucleus via retrograde tRNA nuclear import, a process that is conserved from yeast to vertebrates. The biological significance of this tRNA nuclear import is not entirely clear. We hypothesized that retrograde tRNA nuclear import might function in proofreading tRNAs to ensure that only proper tRNAs reside in the cytoplasm and interact with the translational machinery. Here we identify two major types of aberrant tRNAs in yeast: a 5', 3' end-extended, spliced tRNA and hypomodified tRNAs. We show that both types of aberrant tRNAs accumulate in mutant cells that are defective in tRNA nuclear traffic, suggesting that they are normally imported into the nucleus and are repaired or degraded. The retrograde pathway functions in parallel with the cytoplasmic rapid tRNA decay pathway previously demonstrated to monitor tRNA quality, and cells are not viable if they lack both pathways. Our data support the hypothesis that the retrograde process provides a newly discovered level of tRNA quality control as a pathway that monitors both end processing of pre-tRNAs and the modification state of mature tRNAs.

In eukaryotic cells, tRNAs are transcribed and partially processed in the nucleus before they are exported to the cytoplasm, where they have an essential role in protein synthesis. Surprisingly, mature cytoplasmic tRNAs shuttle between nucleus and cytoplasm, and tRNA subcellular distribution is nutrient dependent. At least three members of the β-importin family, Los1, Mtr10, and Msn5, function in tRNA nuclear-cytoplasmic intracellular movement. To test the hypothesis that the tRNA retrograde pathway regulates the translation of particular transcripts, we compared the expression profiles from nontranslating mRNAs and polyribosome-associated translating mRNAs collected from msn5Δ, mtr10Δ, and wild-type cells under fed or acute amino acid depletion conditions. Our microarray data revealed that the methionine, arginine, and leucine biosynthesis pathways are targets of the tRNA retrograde process. We confirmed the microarray data by Northern and Western blot analyses. The levels of some of the particular target mRNAs were reduced, while others appeared not to be affected. However, the protein levels of all tested targets in these pathways were greatly decreased when tRNA nuclear import or reexport to the cytoplasm was disrupted. This study provides information that tRNA nuclear-cytoplasmic dynamics is connected to the biogenesis of proteins involved in amino acid biosynthesis.

Transfer RNAs (tRNAs) are essential for protein synthesis. In eukaryotes, tRNA biosynthesis employs a specialized RNA polymerase that generates initial transcripts that must be subsequently altered via a multitude of post-transcriptional steps before the tRNAs beome mature molecules that function in protein synthesis. Genetic, genomic, biochemical, and cell biological approaches possible in the powerful Saccharomyces cerevisiae system have led to exciting advances in our understandings of tRNA post-transcriptional processing as well as to novel insights into tRNA turnover and tRNA subcellular dynamics. tRNA processing steps include removal of transcribed leader and trailer sequences, addition of CCA to the 3′ mature sequence and, for tRNAHis, addition of a 5′ G. About 20% of yeast tRNAs are encoded by intron-containing genes. The three-step splicing process to remove the introns surprisingly occurs in the cytoplasm in yeast and each of the splicing enzymes appears to moonlight in functions in addition to tRNA splicing. There are 25 different nucleoside modifications that are added post-transcriptionally, creating tRNAs in which ∼15% of the residues are nucleosides other than A, G, U, or C. These modified nucleosides serve numerous important functions including tRNA discrimination, translation fidelity, and tRNA quality control. Mature tRNAs are very stable, but nevertheless yeast cells possess multiple pathways to degrade inappropriately processed or folded tRNAs. Mature tRNAs are also dynamic in cells, moving from the cytoplasm to the nucleus and back again to the cytoplasm; the mechanism and function of this retrograde process is poorly understood. Here, the state of knowledge for tRNA post-transcriptional processing, turnover, and subcellular dynamics is addressed, highlighting the questions that remain.

Conventional isolation and detection methods for small RNAs from yeast cells have been designed for a limited number of samples. In order to be able to conduct a genome-wide assessment of how each gene product impacts upon small RNAs, we developed a rapid method for analysing small RNAs from Saccharomyces cerevisiae wild-type (wt) and mutants cells in the deletion and temperature-sensitive (ts) collections. Our method implements three optimized techniques: a procedure for growing small yeast cultures in 96-deepwell plates, a fast procedure for small RNA isolation from the plates, and a sensitive non-radioactive northern method for RNA detection. The RNA isolation procedure requires only 4 h for processing 96 samples, is highly reproducible and yields RNA of good quality and quantity. The non-radioactive northern method employs digoxigenin (DIG)-labelled DNA probes and chemiluminescence. It detects femtomole levels of small RNAs within 1 min exposure time. We minimized the processing time for large-scale analysis and optimized the stripping and reprobing procedures for analyses of multiple RNAs from a single membrane. The method described is rapid, sensitive, safe and cost-effective for genome-wide screens of novel genes involved in the biogenesis, subcellular trafficking and stability of small RNAs. Moreover, it will be useful to educational laboratory class venues and to research institutions with limited access to radioisotopes or robots. Copyright © 2013 John Wiley & Sons, Ltd.

Post-transcriptional modification of the tRNA anticodon loop is critical for translation. Yeast Trm7 is required for 2′-O-methylation of C32 and N34 of tRNAPhe, tRNATrp, and tRNALeu(UAA) to form Cm32 and Nm34, and trm7-Δ mutants have severe growth and translation defects, but the reasons for these defects are not known. We show here that overproduction of tRNAPhe suppresses the growth defect of trm7-Δ mutants, suggesting that the crucial biological role of Trm7 is the modification of tRNAPhe. We also provide in vivo and in vitro evidence that Trm7 interacts with ORF YMR259c (now named Trm732) for 2′-O-methylation of C32, and with Rtt10 (named Trm734) for 2′-O-methylation of N34 of substrate tRNAs and provide evidence for a complex circuitry of anticodon loop modification of tRNAPhe, in which formation of Cm32 and Gm34 drives modification of m1G37 (1-methylguanosine) to yW (wyebutosine). Further genetic analysis shows that the slow growth of trm7-Δ mutants is due to the lack of both Cm32 and Nm34, and the accompanying loss of yW, because trm732-Δ trm734-Δ mutants phenocopy trm7-Δ mutants, whereas each single mutant is healthy; nonetheless, TRM732 and TRM734 each have distinct roles, since mutations in these genes have different genetic interactions with trm1-Δ mutants, which lack m2,2G26 in their tRNAs. We speculate that 2′-O-methylation of the anticodon loop may be important throughout eukaryotes because of the widespread conservation of Trm7, Trm732, and Trm734 proteins, and the corresponding modifications, and because the putative human TRM7 ortholog FTSJ1 is implicated in nonsyndromic X-linked mental retardation.

tRNA research was such a small field in 1995 that we used to quip that we could have a meeting of the entire American community interested in tRNA processing and modification in one of our living rooms. Research since the 1960's had yielded the crucial role of tRNAs in translation, and contributed heavily to our understanding of RNA structure and chemistry. We knew the structure of three tRNA species, for many years the only structures of functional RNAs, and we knew the identity, position, and physical properties of many of the myriad of tRNA modifications in several experimental organisms. Moreover, tRNA research from the late 1970's and early 1980's had ushered in the era of RNA splicing and RNA catalysis, had unraveled large parts of the puzzle of the second genetic code controlling how tRNAs are specifically charged by their cognate synthetases, and had uncovered some of the important mechanisms of charging fidelity. To most observers, tRNA was mostly a solved problem.

The Rsp5 ubiquitin ligase regulates numerous cellular processes. Rsp5 is mainly localized to the cytoplasm but nuclear localization was also reported. A potential nuclear export signal was tested for activity by using a GFP(2) reporter. The 687-LIGGIAEIDI-696 sequence located in the Hect domain was identified as a nuclear export signal active in a Crm1-dependent manner, and its importance for the localization of Rsp5 was documented by using fluorescence microscopy and a lacZ-based reporter system. Analysis of the cellular location of other Rsp5 fragments fused with GFP(2) indicated two independent potential nuclear localization signals, both located in the Hect domain. We also uncovered Rsp5 fragments that are important to targeting/tethering Rsp5 to various regions in the cytoplasm. The presented data indicate that Rsp5 ligase is a shuttling protein whose distribution within the cytoplasm and partitioning between cytoplasmic and nuclear locations is determined by a balance between the actions of several targeting sequences and domains.

Transfer RNAs (tRNAs) encoded by the nuclear genome are surprisingly dynamic. Although tRNAs function in protein synthesis occurring on cytoplasmic ribosomes, tRNAs can transit from the cytoplasm to the nucleus and then again return to the cytoplasm by a process known as the tRNA retrograde process. Subsets of the cytoplasmic tRNAs are also imported into mitochondria and function in mitochondrial protein synthesis. The numbers of tRNA species that are imported into mitochondria differ among organisms, ranging from just a few to the entire set needed to decode mitochondrially encoded mRNAs. For some tRNAs, import is dependent on the mitochondrial protein import machinery, whereas the majority of tRNA mitochondrial import is independent of this machinery. Although cytoplasmic proteins and proteins located on the mitochondrial surface participating in the tRNA import process have been described for several organisms, the identity of these proteins differ among organisms. Likewise, the tRNA determinants required for mitochondrial import differ among tRNA species and organisms. Here, we present an overview and discuss the current state of knowledge regarding the mechanisms involved in the tRNA retrograde process and continue with an overview of tRNA import into mitochondria. Finally, we highlight areas of future research to understand the function and regulation of movement of tRNAs between the cytoplasm and organelles.

The nuclear-cytoplasmic distribution of tRNA depends on the balance between tRNA nuclear export/re-export and retrograde tRNA nuclear import in Saccharomyces cerevisiae. The distribution of tRNA is sensitive to nutrient availability as cells deprived of various nutrients exhibit tRNA nuclear accumulation. Starvation induces numerous events that result in translational repression and P-body formation. This study investigated the possible coordination of these responses with tRNA nuclear-cytoplasmic distribution. Dhh1 and Pat1 function in parallel to promote translation repression and P-body formation in response to starvation. Loss of both, Dhh1 and Pat1, results in a failure to repress translation and to induce P-body formation in response to glucose starvation. This study reports that nutrient deprived dhh1 pat1 cells also fail to accumulate tRNA within nuclei. Conversely, inhibition of translation initiation and induction of P-body formation by overproduction of Dhh1 or Pat1 cause tRNA nuclear accumulation in nutrient-replete conditions. Also, loss of the mRNA decapping activator, Lsm1, causes tRNA nuclear accumulation. However, the coordination between P-body formation, translation repression, and tRNA distribution is limited to the early part of the P-body formation/translation repression pathway as loss of mRNA decapping or 5′ to 3′ degradation does not influence tRNA nuclear-cytoplasmic dynamics. The data provide the first link between P-body formation/translation initiation and tRNA nuclear-cytoplasmic dynamics. The current model is that Dhh1 and Pat1 function in parallel to promote starvation-induced tRNA nuclear accumulation.

tRNA biology has come of age, revealing an unprecedented level of understanding and many unexpected discoveries along the way. This review highlights new findings on the diverse pathways of tRNA maturation, and on the formation and function of a number of modifications. Topics of special focus include the regulation of tRNA biosynthesis, quality control tRNA turnover mechanisms, widespread tRNA cleavage pathways activated in response to stress and other growth conditions, emerging evidence of signaling pathways involving tRNA and cleavage fragments, and the sophisticated intracellular tRNA trafficking that occurs during and after biosynthesis.

tRNAs traffic between the nucleus and the cytoplasm in response to nutrient availability. Using a new assay to track tRNA within cells, we show that tRNA nuclear import is constitutive, whereas tRNA reexport to the cytoplasm is regulated. Msn5 functions only in tRNA re-export, whereas Los1 functions in both the primary and reexport steps., tRNAs in yeast and vertebrate cells move bidirectionally and reversibly between the nucleus and the cytoplasm. We investigated roles of members of the β-importin family in tRNA subcellular dynamics. Retrograde import of tRNA into the nucleus is dependent, directly or indirectly, upon Mtr10. tRNA nuclear export utilizes at least two members of the β-importin family. The β-importins involved in nuclear export have shared and exclusive functions. Los1 functions in both the tRNA primary export and the tRNA reexport processes. Msn5 is unable to export tRNAs in the primary round of export if the tRNAs are encoded by intron-containing genes, and for these tRNAs Msn5 functions primarily in their reexport to the cytoplasm. The data support a model in which tRNA retrograde import to the nucleus is a constitutive process; in contrast, reexport of the imported tRNAs back to the cytoplasm is regulated by the availability of nutrients to cells and by tRNA aminoacylation in the nucleus. Finally, we implicate Tef1, the yeast orthologue of translation elongation factor eEF1A, in the tRNA reexport process and show that its subcellular distribution between the nucleus and cytoplasm is dependent upon Mtr10 and Msn5.

Trm1 is a tRNA specific m(2)(2)G methyltransferase shared by nuclei and mitochondria in Saccharomyces cerevisiae. In nuclei, Trm1 is peripherally associated with the inner nuclear membrane (INM). We investigated the mechanism delivering/tethering Trm1 to the INM. Analyses of mutations of the Ran pathway and nuclear pore components showed that Trm1 accesses the nucleoplasm via the classical nuclear import pathway. We identified a Trm1 cis-acting sequence sufficient to target passenger proteins to the INM. Detailed mutagenesis of this region uncovered specific amino acids necessary for authentic Trm1 to locate at the INM. The INM information is contained within a sequence of less than 20 amino acids, defining the first motif for addressing a peripheral protein to this important subnuclear location. The combined studies provide a multi-step process to direct Trm1 to the INM: (i) translation in the cytoplasm; (ii) Ran-dependent import into the nucleoplasm; and (iii) redistribution from the nucleoplasm to the INM via the INM motif. Furthermore, we demonstrate that the Trm1 mitochondrial targeting and nuclear localization signals are in competition with each other, as Trm1 becomes mitochondrial if prevented from entering the nucleus.

The biosynthesis of tRNA was previously thought to occur solely in the nucleus, with tRNA functioning only in the cytoplasm of eukaryotic cells. However, recent publications have reported that pre-tRNA splicing can occur in the cytoplasm, that aminoacylation can occur in the nucleus and that tRNA can travel in a retrograde direction from the cytoplasm to the nucleus. Moreover, the subcellular distribution of tRNA seems to serve unanticipated functions in diverse processes, including response to nutrient availability, DNA repair and HIV replication.

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.

Cytoplasmic tRNAs have recently been found to accumulate in the nucleus during amino acid starvation in yeast. The mechanism and regulation by which tRNAs return to the nucleus are unclear. Here, we show accumulation of cytoplasmic tRNA in the nucleus also occurs during glucose starvation. Nuclear accumulation of tRNA in response to acute glucose or amino acid starvation is rapid, reversible, requires no new transcription, and is independent of the aminoacylation status of tRNA. Gradual depletion of nutrients also results in the accrual of tRNA in the nucleus. Distinct signal transduction pathways seem to be involved in the accumulation of cytoplasmic tRNA in the nucleus in response to amino acid versus glucose starvation. These findings suggest tRNA nucleocytoplasmic distribution may play a role in gene expression in response to nutritional stress.

Until recently, transport of tRNA was presumed to be unidirectional, from the nucleus to the cytoplasm. Our published findings, however, revealed that cytoplasmic tRNAs move retrograde to the nucleus in Saccharomyces cerevisiae and that nuclear accumulation of cytoplasmic tRNAs occurs when cells are nutrient deprived. The findings led us to examine whether retrograde nuclear accumulation of cytoplasmic tRNAs occurs in higher eukaryotes. Using RNA FISH and Northern and Western analyses we show that tRNAs accumulate in nuclei of a hepatoma cell line in response to amino acid deprivation. To discern whether tRNA nuclear accumulation results from nuclear import of cytoplasmic tRNAs, transcription of new RNAs was inhibited, and the location of “old” tRNAs in response to nutrient stress was determined. Even in the absence of new RNA synthesis, there were significant tRNA nuclear pools after amino acid depletion, providing strong evidence that retrograde traffic is responsible for the tRNA nuclear pools. Further analyses showed that retrograde tRNA nuclear accumulation in hepatoma cells is a reversible and energy-dependent process. The data provide evidence for retrograde tRNA nuclear accumulation in intact mammalian cells and support the hypothesis that nuclear accumulation of cytoplasmic tRNA and tRNA re-export to the cytoplasm may constitute a universal mechanism for posttranscriptional regulation of global gene expression in response to nutrient availability.

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.

The retroviral Gag polyprotein orchestrates the assembly and release of virus particles from infected cells. We previously reported that nuclear transport of the Rous sarcoma virus (RSV) Gag protein is intrinsic to the virus assembly pathway. To identify cis- and trans- acting factors governing nucleocytoplasmic trafficking, we developed novel vectors to express regions of Gag in Saccharomyces cerevisiae . The localization of Gag proteins was examined in the wild type and in mutant strains deficient in members of the importin-β family. We confirmed the Crm1p dependence of the previously identified Gag p10 nuclear export signal. The known nuclear localization signal (NLS) in MA (matrix) was also functional in S. cerevisiae , and additionally we discovered a novel NLS within the NC (nucleocapsid) domain of Gag. MA utilizes Kap120p and Mtr10p import receptors while nuclear entry of NC involves the classical importin-α/β (Kap60p/95p) pathway. NC also possesses nuclear targeting activity in avian cells and contains the primary signal for the import of the Gag polyprotein. Thus, the nucleocytoplasmic dynamics of RSV Gag depend upon the counterbalance of Crm1p-mediated export with two independent NLSs, each interacting with distinct nuclear import factors.

This review highlights the unexpectedly complicated nuclear egress and nuclear import of small RNAs. Although nucleus/cytoplasm trafficking was thought to be restricted to snRNAs of many, but not all, eukaryotes, recent data indicate that such traffic may be more common than previously appreciated. First, in conflict with numerous previous reports, new information indicates that Saccharomyces cerevisiae snRNAs may cycle between the nucleus and the cytoplasm. Second, recent studies also provide evidence that other small RNAs that function exclusively in the nucleus-the budding yeast telomerase RNA and possibly small nucleolar RNAs-may exit to the cytoplasm, only to return to the nucleus. Third, nucleus/cytoplasm cycling of RNAs also occurs for RNAs that function solely in the cytoplasm, as it has been discovered that cytoplasmic tRNAs of budding yeast travel "retrograde" to the nucleus and, perhaps, back again to the cytoplasm to function in protein synthesis. Fourth, there is at least one example in ciliates of small double-stranded RNAs traveling multiple cycles between the cytoplasm and distinct nuclei to direct genome structure. This report discusses data that support or argue against nucleus/cytoplasm bidirectional movement for each category of small RNA and the possible roles that such movement may serve.

Appropriate nuclear membrane structure is important for all eukaryotic organisms as evidenced by the numerous human diseases and alterations in gene expression caused by inappropriate targeting of proteins to the inner nuclear membrane (INM). We report here the first genome-wide screen to identify proteins functioning in INM targeting. We transformed to near completion the 4850 members of the Saccharomyces cerevisiae deletion collection of unessential genes in the 96-well format with a plasmid encoding a reporter protein, Trm1-II-GFP, which normally resides at the INM. We found that deletion of genes encoding subunits of the N-terminal acetyltransferase, NatC, cause mislocation of Trm1-II-GFP from the INM to the nucleoplasm. Mass spectroscopic analysis indicates that Trm1-II-GFP is N-acetylated. N-terminal mutations of Trm1-II-GFP predicted to ablate N-acetylation cause nucleoplasmic location, whereas a variant with an N-terminal alteration predicted to allow N-acetylation by NatC is located at the INM, providing genetic support that Trm1p-II N-acetylation is necessary for its subnuclear INM location. However, because N-acetylation appears not to be sufficient for INM targeting, it may provide a necessary role for INM targeting by affecting Trm1-II-GFP structure and exposure of cis-acting INM targeting motifs. We also discovered that YIL090W/Ice2p, an integral membrane protein located in the endoplasmic reticulum, is necessary for efficient targeting of Trm1-II-GFP to the INM. YIL090W/Ice2p may serve as a tether for INM proteins or as a regulator of INM tethers. Our methodology can be extrapolated to obtain genome-wide perspectives of mechanisms necessary to achieve appropriate subcellular and/or suborganellar location for any resident protein.

In eukaryotes, tRNAs transcribed in the nucleus function in cytoplasmic protein synthesis. The Ran-GTP-binding exportin, Los1p/Xpo-t, and additional pathway(s) mediate tRNA transport to the cytoplasm. Although tRNA movement was thought to be unidirectional, recent reports that yeast precursor tRNA splicing occurs in the cytoplasm, whereas fully spliced tRNAs can reside in the nucleus, require that either the precursor tRNA splicing machinery or mature tRNAs move from the cytoplasm to the nucleus. Our data argue against the first possibility and strongly support the second. Combining heterokaryon analysis with fluorescence in situ hybridization, we show that a foreign tRNA encoded by one nucleus can move from the cytoplasm to a second nucleus that does not encode the tRNA. We also discovered nuclear accumulation of endogenous cytoplasmic tRNAs in haploid yeast cells in response to nutritional deprivation. Nuclear accumulation of cytoplasmic tRNA requires Ran and the Mtr10/Kap111 member of the importin-β family. Retrograde tRNA nuclear import may provide a novel mechanism to regulate gene expression in eukaryotes.

SOL1, the founding member of the S. cerevisiae SOL family, was previously identified as a multi-copy suppressor of the los1 defect in tRNA-mediated nonsense suppression. Here we report that the four-member SOL family is not essential and that individual family members appear to have distinct functions. SOL1–SOL4 are homologous to genes encoding 6-phosphogluconolactonase (6Pgl) involved in the pentose phosphate pathway. Both Sol3p and Sol4p affect this activity. However, Sol4p does not act as a los1 multi-copy suppressor. In contrast, neither Sol1p nor Sol2p, both of which correct the los1 defect in nonsense suppression, possess detectable 6Pgl activity. Rather, Sol1p and Sol2p appear to function in tRNA nuclear export as sol1 and sol2 mutants possess elevated levels of nuclear tRNA. Members of the Sol protein family appear to have different subcellular distributions. Thus, Sol3p and Sol4p likely function in carbohydrate metabolism, while Sol1p and Sol2p appear to have roles in tRNA function and nuclear export, thereby defining an unusual protein family whose individual members are biochemically distinct and spatially dispersed.

tRNA has a central role in biology as the adaptor between mRNA and protein, in which anticodon interactions with the mRNA codon at one end of the L-shaped tRNA allow an attached amino acid at the other end of the tRNA to condense with the peptide chain on a second tRNA through the action of the ribosome. This familiar role of tRNA requires mature tRNAs to be recognized by the aminoacyl tRNA synthetase to add the amino acid to its 3 end, by EF1 to form a ternary complex with the aminoacylated tRNA and GTP, and by elements of the ribosome and associated components to allow binding, peptide synthesis, and translocation. Before fulfilling this role, all tRNA transcripts have to be extensively processed. Maturation of tRNAs requires five major steps: (1) removal of the 5 leader by RNase P

In eukaryotes and archaea, tRNA splicing generates free intron molecules. Although ∼600,000 introns are produced per generation in yeast, they are barely detectable in cells, indicating efficient turnover of introns. Through a genome-wide search for genes involved in tRNA biology in yeast, we uncovered the mechanism for intron turnover. This process requires healing of the 5′ termini of linear introns by the tRNA ligase Rlg1 and destruction by the cytoplasmic tRNA quality control 5′-to-3′ exonuclease Xrn1, which has specificity for RNAs with 5′ monophosphate.

Sorting isozymes are encoded by single genes, but the encoded proteins are distributed to multiple subcellular compartments. We surveyed the predicted protein sequences of several nucleic acid interacting sorting isozymes from the eukaryotic taxonomic domain and compared them with their homologs in the archaeal and eubacterial domains. Here, we summarize the data showing that the eukaryotic sorting isozymes often possess sequences not present in the archaeal and eubacterial counterparts and that the additional sequences can act to target the eukaryotic proteins to their appropriate subcellular locations. Therefore, we have named these protein domains ADEPTs (Additional Domains for Eukaryotic Protein Targeting). Identification of additional domains by phylogenetic comparisons should be generally useful for locating candidate sequences important for subcellular distribution of eukaryotic proteins.

Cells can respond to extracellular signals by redistributing transcription regulators between the nucleus and cytosol. Recent findings in budding yeast indicate that the nuclear transport receptor Msn5p mediates the nuclear export of several transcription regulators after their phosphorylation in the nucleus.

WIP, the Wiskott-Aldrich syndrome protein-interacting protein, is a human protein involved in actin polymerization and redistribution in lymphoid cells. The mechanism by which WIP reorganizes actin cytoskeleton is unknown. WIP is similar to yeast verprolin, an actin- and myosin-interacting protein required for polarized morphogenesis. To determine whether WIP and verprolin are functional homologues, we analyzed the function of WIP in yeast. WIP suppresses the growth defects of VRP1 missense and null mutations as well as the defects in cytoskeletal organization and endocytosis observed in vrp1-1 cells. The ability of WIP to replace verprolin is dependent on its WH2 actin binding domain and a putative profilin binding domain. Immunofluorescence localization of WIP in yeast cells reveals a pattern consistent with its function at the cortical sites of growth. Thus, like verprolin, WIP functions in yeast to link the polarity development pathway and the actin cytoskeleton to generate cytoskeletal asymmetry. A role for WIP in cell polarity provides a framework for unifying, under a common paradigm, distinct molecular defects associated with immunodeficiencies like Wiskott-Aldrich syndrome.