Anita K. Hopper

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.

A Yeast Mutant Which Accumulates Precursor tRNAs

It has been proposed that the conditional yeast mutant ts136 is defective in the transport of mRNA from the nucleus to the cytoplasm (Hutchinson, Hartwell and McLaughlin, 1969). We have examined ts136 to determine whether it is defective in tRNA biosynthesis. At the restrictive temperature, the mutant accumulates twelve new species of RNA. These species co-migrate on polyacrylamide gels with some of the pulse-labeled precursor tRNAs. Three of the new RNAs (species 1a, 1b and 1c are large enough to contain two tandom tRNAs. Although RNAs 1a, 1b, and 1c do not contain detectable levels of modified and methylated bases, at least one of them hybridizes to DNA from an E. coli plasmid containing a yeast tRNA gene. All the remaining RNAs (2--8) contain modified and methylated bases typical of tRNA. Three of these species were tested and were found to hybridize to tRNA genes. Ribosomal RNA synthesis is also defective in ts136. It is suggested that ts136 may be defective in a nucleolytic activity, which is a prerequisite to RNA transport.

Maf1p, a Negative Effector of RNA Polymerase III in Saccharomyces cerevisiae

ABSTRACT Although yeast RNA polymerase III (Pol III) and the auxiliary factors TFIIIC and TFIIIB are well characterized, the mechanisms of class III gene regulation are poorly understood. Previous studies identified MAF1, a gene that affects tRNA suppressor efficiency and interacts genetically with Pol III. We show here that tRNA levels are elevated in maf1 mutant cells. In keeping with the higher levels of tRNA observed in vivo, the in vitro rate of Pol III RNA synthesis is significantly increased in maf1cell extracts. Mutations in the RPC160 gene encoding the largest subunit of Pol III which reduce tRNA levels were identified as suppressors of the maf1 growth defect. Interestingly, Maf1p is located in the nucleus and coimmunopurifies with epitope-tagged RNA Pol III. These results indicate that Maf1p acts as a negative effector of Pol III synthesis. This potential regulator of Pol III transcription is likely conserved since orthologs of Maf1p are present in other eukaryotes, including humans.

Processing of intervening sequences: a new yeast mutant which fails to excise intervening sequences from precursor tRNAs

By using conditional loss of suppression an an assay, we have been successful in screening for a yeast mutant which is defective in tRNA processing. The los1-1 mutation causes an accumulation of a subset of precursor tRNAs at the nonpermissive temperature. These pre-tRNAs are like those which accumulate in the yeast mutant ts 136 (rna1) in that they have transcribed intervening sequences. The mutations at los1-1 and rna1 complement and segregate independently of each other. The los1-1 mutation affects the expression of all 8 tyrosine-inserting suppressor loci, but does not seem to affect rRNA or mRNA synthesis.

Isolation and characterization of MOD5, a gene required for isopentenylation of cytoplasmic and mitochondrial tRNAs of Saccharomyces cerevisiae.

The mod5-1 mutation is a nuclear mutation in Saccharomyces cerevisiae that reduces the biosynthesis of N6-(delta 2-isopentenyl)adenosine in both cytoplasmic and mitochondrial tRNAs to less than 1.5% of wild-type levels. The tRNA modification enzyme, delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase, cannot be detected in vitro with extracts from mod5-1 cells. A characterization of the MOD5 gene would help to determine how the same enzyme activity in different cellular compartments can be abolished by a single nuclear mutation. To that end we have cloned the MOD5 gene and shown that it restores delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase activity and N6-(delta 2-isopentenyl)adenosine to tRNA in both the mitochondria and the nucleus/cytoplasm compartments of mod5-1 yeast cells. That MOD5 sequences are expressed in Escherichia coli and can complement an N6-(delta 2-isopentenyl)-2-methylthioadenosine-deficient E. coli mutant leads us to conclude that MOD5 is the structural gene for delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase.

Transfer RNA Post-Transcriptional Processing, Turnover, and Subcellular Dynamics in the Yeast Saccharomyces cerevisiae

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.

Defects in tRNA processing and nuclear export induce GCN4 translation independently of phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2.

ABSTRACT Induction of GCN4 translation in amino acid-starved cells involves the inhibition of initiator tRNAMetbinding to eukaryotic translation initiation factor 2 (eIF2) in response to eIF2 phosphorylation by protein kinase GCN2. It was shown previously that GCN4 translation could be induced independently of GCN2 by overexpressing a mutant tRNAAACVal (tRNAVal*) or the RNA component of RNase MRP encoded by NME1. Here we show that overexpression of the tRNA pseudouridine 55 synthase encoded byPUS4 also leads to translational derepression ofGCN4 (Gcd− phenotype) independently of eIF2 phosphorylation. Surprisingly, the Gcd− phenotype of high-copy-number PUS4 (hcPUS4) did not require PUS4 enzymatic activity, and several lines of evidence indicate thatPUS4 overexpression did not diminish functional initiator tRNAMet levels. The presence of hcPUS4 or hcNME1 led to the accumulation of certain tRNA precursors, and their Gcd− phenotypes were reversed by overexpressing the RNA component of RNase P (RPR1), responsible for 5′-end processing of all tRNAs. Consistently, overexpression of a mutant pre-tRNATyr that cannot be processed by RNase P had a Gcd− phenotype. Interestingly, the Gcd− phenotype of hcPUS4also was reversed by overexpressing LOS1, required for efficient nuclear export of tRNA, and los1Δ cells have a Gcd− phenotype. Overproduced PUS4 appears to impede 5′-end processing or export of certain tRNAs in the nucleus in a manner remedied by increased expression of RNase P or LOS1, respectively. The mutant tRNAVal* showed nuclear accumulation in otherwise wild-type cells, suggesting a defect in export to the cytoplasm. We propose that yeast contains a nuclear surveillance system that perceives defects in processing or export of tRNA and evokes a reduction in translation initiation at the step of initiator tRNAMet binding to the ribosome.

The Human WASP-interacting Protein, WIP, Activates the Cell Polarity Pathway in Yeast

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 VRP1missense 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.

Defects in modification of cytoplasmic and mitochondrial transfer RNAs are caused by single nuclear mutations

Many nucleus-encoded mitochondrial enzymes differ in physical and chemical properties from analogous cytoplasmic enzymes, and it is therefore generally assumed that different genes encode analogous mitochondrial and cytoplasmic enzymes. However, our genetic studies show that for at least two different tRNA modifications, mutations in nuclear genes affect cytoplasmic as well as mitochondrial tRNAs. These studies utilize two yeast genes: TRM1 and TRM2. trm1 cells do not have the enzyme activity necessary to methylate guanosine to N2,N2-dimethylguanosine. trm2 is a new mutation that we describe here. trm2 cells are deficient in tRNA-(uridine-5)methyltransferase, and hence contain tRNA lacking 5-methyluridine or ribothymidine. Other than lacking 5-methyluridine trm2 cells have no obvious physiological defect. These studies also show that the N2,N2-dimethylguanosine and 5-methyluridine modifications are not added to tRNA in an obligatory order, and that 5-methyluridine is not required for removal of intervening sequences from precursor tRNA.

Depletion of Saccharomyces cerevisiae tRNAHis Guanylyltransferase Thg1p Leads to Uncharged tRNAHis with Additional m5C

ABSTRACT The essential Saccharomyces cerevisiae tRNAHis guanylyltransferase (Thg1p) is responsible for the unusual G−1 addition to the 5′ end of cytoplasmic tRNAHis. We report here that tRNAHis from Thg1p-depleted cells is uncharged, although histidyl tRNA synthetase is active and the 3′ end of the tRNA is intact, suggesting that G−1 is a critical determinant for aminoacylation of tRNAHis in vivo. Thg1p depletion leads to activation of the GCN4 pathway, most, but not all, of which is Gcn2p dependent, and to the accumulation of tRNAHis in the nucleus. Surprisingly, tRNAHis in Thg1p-depleted cells accumulates additional m5C modifications, which are delayed relative to the loss of G−1 and aminoacylation. The additional modification is likely due to tRNA m5C methyltransferase Trm4p. We developed a new method to map m5C residues in RNA and localized the additional m5C to positions 48 and 50. This is the first documented example of the accumulation of additional modifications in a eukaryotic tRNA species.