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.

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.

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.

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.

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.

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.

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.

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.

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.

Healing for destruction: tRNA intron degradation in yeast is a two-step cytoplasmic process catalyzed by tRNA ligase Rlg1 and 5'-to-3' exonuclease Xrn1.

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.

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.

Beyond tRNA cleavage: novel essential function for yeast tRNA splicing endonuclease unrelated to tRNA processing.

Pre-tRNA splicing is an essential process in all eukaryotes. In yeast and vertebrates, the enzyme catalyzing intron removal from pre-tRNA is a heterotetrameric complex (splicing endonuclease [SEN] complex). Although the SEN complex is conserved, the subcellular location where pre-tRNA splicing occurs is not. In yeast, the SEN complex is located at the cytoplasmic surface of mitochondria, whereas in vertebrates, pre-tRNA splicing is nuclear. We engineered yeast to mimic the vertebrate cell biology and demonstrate that all three steps of pre-tRNA splicing, as well as tRNA nuclear export and aminoacylation, occur efficiently when the SEN complex is nuclear. However, nuclear pre-tRNA splicing fails to complement growth defects of cells with defective mitochondrial-located splicing, suggesting that the yeast SEN complex surprisingly serves a novel and essential function in the cytoplasm that is unrelated to tRNA splicing. The novel function requires all four SEN complex subunits and the catalytic core. A subset of pre-rRNAs accumulates when the SEN complex is restricted to the nucleus, indicating that the SEN complex moonlights in rRNA processing. Thus, findings suggest that selection for the subcellular distribution of the SEN complex may reside not in its canonical, but rather in a novel, activity.

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.

tRNA biology charges to the front.

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.

Retrograde transfer RNA nuclear import provides a new level of tRNA quality control in Saccharomyces cerevisiae.

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.

Mod5 protein binds to tRNA gene complexes and affects local transcriptional silencing.

The tRNA gene-mediated (tgm) silencing of RNA polymerase II promoters is dependent on subnuclear clustering of the tRNA genes, but genetic analysis shows that the silencing requires additional mechanisms. We have identified proteins that bind tRNA gene transcription complexes and are required for tgm silencing but not required for gene clustering. One of the proteins, Mod5, is a tRNA modifying enzyme that adds an N6-isopentenyl adenosine modification at position 37 on a small number of tRNAs in the cytoplasm, although a subpopulation of Mod5 is also found in the nucleus. Recent publications have also shown that Mod5 has tumor suppressor characteristics in humans as well as confers drug resistance through prion-like misfolding in yeast. Here, we show that a subpopulation of Mod5 associates with tRNA gene complexes in the nucleolus. This association occurs and is required for tgm silencing regardless of whether the pre-tRNA transcripts are substrates for Mod5 modification. In addition, Mod5 is bound to nuclear pre-tRNA transcripts, although they are not substrates for the A37 modification. Lastly, we show that truncation of the tRNA transcript to remove the normal tRNA structure also alleviates silencing, suggesting that synthesis of intact pre-tRNAs is required for the silencing mechanism. These results are discussed in light of recent results showing that silencing near tRNA genes also requires chromatin modification.

Yeast Trm7 interacts with distinct proteins for critical modifications of the tRNAPhe anticodon loop.

Post-transcriptional modification of the tRNA anticodon loop is critical for translation. Yeast Trm7 is required for 2'-O-methylation of C(32) and N(34) of tRNA(Phe), tRNA(Trp), and tRNA(Leu(UAA)) to form Cm(32) and Nm(34), 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 tRNA(Phe) suppresses the growth defect of trm7-Δ mutants, suggesting that the crucial biological role of Trm7 is the modification of tRNA(Phe). We also provide in vivo and in vitro evidence that Trm7 interacts with ORF YMR259c (now named Trm732) for 2'-O-methylation of C(32), and with Rtt10 (named Trm734) for 2'-O-methylation of N(34) of substrate tRNAs and provide evidence for a complex circuitry of anticodon loop modification of tRNA(Phe), in which formation of Cm(32) and Gm(34) drives modification of m(1)G(37) (1-methylguanosine) to yW (wyebutosine). Further genetic analysis shows that the slow growth of trm7-Δ mutants is due to the lack of both Cm(32) and Nm(34), 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 m(2,2)G(26) 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.

P-body components, Dhh1 and Pat1, are involved in tRNA nuclear-cytoplasmic dynamics.

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.

A rapid and sensitive non-radioactive method applicable for genome-wide analysis of Saccharomyces cerevisiae genes involved in small RNA biology.

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.

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.