A highly specific phosphatase that acts on ADP-ribose 1″-phosphate, a metabolite of tRNA splicing in Saccharomyces cerevisiae

One molecule of ADP-ribose 1″,2″-cyclic phosphate (Appr>p) is formed during each of the approximately 500 000 tRNA splicing events per Saccharomyces cerevisiae generation. The metabolism of Appr>p remains poorly defined. A cyclic phosphodiesterase (Cpd1p) has been shown to convert Appr>p to ADP-ribose-1″-phosphate (Appr1p). We used a biochemical genomics approach to identify two yeast phosphatases that can convert Appr1p to ADP-ribose: the product of ORF YBR022w (now Poa1p), which is completely unrelated to other known phosphatases; and Hal2p, a known 3′-phosphatase of 5′,3′-pAp. Poa1p is highly specific for Appr1p, and thus likely acts on this molecule in vivo. Poa1 has a relatively low K(M) for Appr1p (2.8 μM) and a modest k(cat) (1.7 min(−1)), but no detectable activity on several other substrates. Furthermore, Poa1p is strongly inhibited by ADP-ribose (K(I), 17 μM), modestly inhibited by other nucleotides containing an ADP-ribose moiety and not inhibited at all by other tested molecules. In contrast, Hal2p is much more active on pAp than on Appr1p, and several other tested molecules were Hal2p substrates or inhibitors. poa1-Δ mutants have no obvious growth defect at different temperatures in rich media, and analysis of yeast extracts suggests that ∼90% of Appr1p processing activity originates from Poa1p

Structural conservation of an ancient tRNA sensor in eukaryotic glutaminyl-tRNA synthetase

In all organisms, aminoacyl tRNA synthetases covalently attach amino acids to their cognate tRNAs. Many eukaryotic tRNA synthetases have acquired appended domains, whose origin, structure and function are poorly understood. The N-terminal appended domain (NTD) of glutaminyl-tRNA synthetase (GlnRS) is intriguing since GlnRS is primarily a eukaryotic enzyme, whereas in other kingdoms Gln-tRNAGln is primarily synthesized by first forming Glu-tRNAGln, followed by conversion to Gln-tRNAGln by a tRNA-dependent amidotransferase. We report a functional and structural analysis of the NTD of Saccharomyces cerevisiae GlnRS, Gln4. Yeast mutants lacking the NTD exhibit growth defects, and Gln4 lacking the NTD has reduced complementarity for tRNAGln and glutamine. The 187-amino acid Gln4 NTD, crystallized and solved at 2.3 Å resolution, consists of two subdomains, each exhibiting an extraordinary structural resemblance to adjacent tRNA specificity-determining domains in the GatB subunit of the GatCAB amidotransferase, which forms Gln-tRNAGln. These subdomains are connected by an apparent hinge comprised of conserved residues. Mutation of these amino acids produces Gln4 variants with reduced affinity for tRNAGln, consistent with a hinge-closing mechanism proposed for GatB recognition of tRNA. Our results suggest a possible origin and function of the NTD that would link the phylogenetically diverse mechanisms of Gln-tRNAGln synthesis

A genome wide dosage suppressor network reveals genomic robustness

Genomic robustness is the extent to which an organism has evolved to withstand the effects of deleterious mutations. We explored the extent of genomic robustness in budding yeast by genome wide dosage suppressor analysis of 53 conditional lethal mutations in cell division cycle and RNA synthesis related genes, revealing 660 suppressor interactions of which 642 are novel. This collection has several distinctive features, including high co-occurrence of mutant-suppressor pairs within protein modules, highly correlated functions between the pairs and higher diversity of functions among the co-suppressors than previously observed. Dosage suppression of essential genes encoding RNA polymerase subunits and chromosome cohesion complex suggests a surprising degree of functional plasticity of macromolecular complexes, and the existence of numerous degenerate pathways for circumventing the effects of potentially lethal mutations. These results imply that organisms and cancer are likely able to exploit the genomic robustness properties, due the persistence of cryptic gene and pathway functions, to generate variation and adapt to selective pressures

The Biology of Anticodon Stem-Loop Modifications of Yeast tRNAs

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biochemistry and Biophysics, 2018.tRNAs play an essential role in translation by decoding mRNA codons to add the correct amino acids to the growing polypeptide chain. During biogenesis, tRNAs undergo extensive post-transcriptional modifications to ensure correct folding and high stability, and to ensure the accuracy and efficiency of translation. Modifications in the anticodon stem-loop (ASL) are highly conserved, and their lack is often associated with growth defects in the yeast Saccharomyces cerevisiae, and with human neurological or mitochondrial disorders. However, the precise function of many ASL modifications and the specificity of the corresponding modifying enzymes remain to be determined. This thesis focused on the biology and specificity of three sets of ASL modifications in yeast tRNAs. One project focused on the importance of pseudouridine (Ψ) at N38 and N39, introduced by Pus3, a modification that is found in all kingdoms of life. This work showed that the temperature sensitivity of pus3Δ mutants was primarily due to reduced function of tRNAGln(UUG), caused by the absence of Ψ38, coupled with the temperature-dependent loss of 2-thiolation at U34 that was found to occur in commonly used yeast strains. Additional experiments showed that tRNAGln(UUG) function was compromised in pus3Δ mutants at temperatures where U34 modification was intact, and showed that Pus3 also had a role in other tRNAs, including tRNATrp(CCA) and tRNALeu(CAA). A second project focused on the substrate specificity of Trm140 for 3-methylcytidine modification. Biochemical and in vivo analysis demonstrated that Trm140 recognized its substrates by two distinct modes: tRNAThr substrates were recognized based on recognition of specific residues N35-N36-N37 in the anticodon loop; whereas tRNASer substrates were recognized by interaction with seryl-tRNA synthetase, and through the variable loop of the tRNA. A third project focused on why lack of 2'-O-methylation at C32 and N34, introduced by Trm7/FTSJ1, causes a severe growth defect in yeast and X-linked intellectual disability in humans. Several lines of genetic evidence were presented indicating that the trm7Δ growth defect in S. cerevisiae was caused by reduced tRNAPhe charging and the consequent activation of the general amino acid control response, which appeared to be conserved in distantly related organisms, including Schizosaccharomyces pombe

Hypomodified tRNA in evolutionarily distant yeasts can trigger rapid tRNA decay to activate the general amino acid control response, but with different consequences.

All tRNAs are extensively modified, and modification deficiency often results in growth defects in the budding yeast Saccharomyces cerevisiae and neurological or other disorders in humans. In S. cerevisiae, lack of any of several tRNA body modifications results in rapid tRNA decay (RTD) of certain mature tRNAs by the 5'-3' exonucleases Rat1 and Xrn1. As tRNA quality control decay mechanisms are not extensively studied in other eukaryotes, we studied trm8Δ mutants in the evolutionarily distant fission yeast Schizosaccharomyces pombe, which lack 7-methylguanosine at G46 (m7G46) of their tRNAs. We report here that S. pombe trm8Δ mutants are temperature sensitive primarily due to decay of tRNATyr(GUA) and that spontaneous mutations in the RAT1 ortholog dhp1+ restored temperature resistance and prevented tRNA decay, demonstrating conservation of the RTD pathway. We also report for the first time evidence linking the RTD and the general amino acid control (GAAC) pathways, which we show in both S. pombe and S. cerevisiae. In S. pombe trm8Δ mutants, spontaneous GAAC mutations restored temperature resistance and tRNA levels, and the trm8Δ temperature sensitivity was precisely linked to GAAC activation due to tRNATyr(GUA) decay. Similarly, in the well-studied S. cerevisiae trm8Δ trm4Δ RTD mutant, temperature sensitivity was closely linked to GAAC activation due to tRNAVal(AAC) decay; however, in S. cerevisiae, GAAC mutations increased tRNA loss and exacerbated temperature sensitivity. A similar exacerbated growth defect occurred upon GAAC mutation in S. cerevisiae trm8Δ and other single modification mutants that triggered RTD. Thus, these results demonstrate a conserved GAAC activation coincident with RTD in S. pombe and S. cerevisiae, but an opposite impact of the GAAC response in the two organisms. We speculate that the RTD pathway and its regulation of the GAAC pathway is widely conserved in eukaryotes, extending to other mutants affecting tRNA body modifications

Characterizing the Role of Elongation Factor 1A and Other Cellular Proteins in the Yeast Rapid tRNA Decay Pathway

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biochemistry and Biophysics, 2014.The structural and functional integrity of tRNA is crucial for translation, and is in part maintained by post-transcriptional modifications to the tRNA. In the yeast Saccharomyces cerevisiae, certain mature tRNAs are subject to rapid tRNA decay (RTD) if they are appropriately hypomodified or bear specific destabilizing mutations, leading to 5'-3' exonucleolytic degradation by Rat1 or Xrn1. Thus, trm8- trm4- strains are temperature sensitive due to lack of m7G46 and m5C and the consequent RTD of tRNAVal(AAC), and tan1- trm44- strains are temperature sensitive due to lack of ac4C12 and Um44 and RTD of tRNASer(CGA) and tRNASer(UGA). It was unknown how generally the RTD pathway acts on hypomodified tRNAs, and how this pathway interacts with translation and other cellular components. It is shown here that RTD acts widely on tRNAs lacking modifications, since trm1- trm4- mutants are subject to RTD of tRNASer(CGA) and tRNASer(UGA) due to lack of m2,2G26 and m5C, and since trm8-, tan1-, and trm1- single mutants are each subject to RTD. Evidence is also presented that elongation factor 1A (eEF1A) competes with the RTD pathway for substrate tRNAs, since reduced eEF1A exacerbates the growth defect of all known RTD-susceptible strains, and exacerbates tRNA degradation in all RTD-susceptible strains tested. Conversely, eEF1A overexpression suppresses RTD. To further explore the effect of eEF1A on tRNA binding and RTD, I have set up a genetic screen and a biochemical assay. My work also provides evidence for a nuclear sub-branch of RTD mediated by Rat1, and a cytoplasmic sub-branch mediated by Xrn1. Thus, the temperature sensitivity of certain RTD-susceptible strains is suppressed fully by XRN1 deletion and only mildly by rat1 mutation, whereas for other strains the opposite is true. Furthermore, the exonucleases degrade substrate tRNAs based mainly on location, since, for example, Xrn1 degrades tRNAVal(AAC) only when engineered to be nuclear. Some suppressors act upstream of the two RTD sub-branches. Thus, genetic manipulation shows that variants of Rpo31 and of Bud27 suppress both RTD sub-branches, although they were initially identified as suppressors of the Rat1 sub-branch. These results demonstrate that multiple factors influence RTD, and set the stage for further analysis

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

Investigation of the Roles of the Highly Conserved G-1 Residue of tRNAHis, and Analysis of Unexpected Modification Changes in tRNAHis Variants in Saccharomyces cerevisiae

Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Biochemistry and Biophysics, 2009.tRNAs are highly processed molecules that are crucial for protein synthesis in the cell. During tRNA processing, about 13 post-transcriptional modifications are added to each tRNA, many of which are highly conserved across all kingdoms of life. The extra guanine nucleotide at the 5’ end of tRNAHis (G-1) is virtually universally conserved, and tRNAHis is unique since it is the only tRNA to have this G-1 residue. In prokaryotes and some archaea, G-1 is encoded in the genome and retained after 5’ end processing. However, in eukaryotes and other archaea, G-1 is added posttranscriptionally by tRNAHis guanylyltransferase (Thg1), which is highly conserved and essential in Saccharomyces cerevisiae. Although it is known that G-1 is important for tRNAHis aminoacylation, no other function has been ascribed to this almost universally conserved tRNA modification. Thg1 may have other important roles in the cell beyond G-1 addition activity. Thg1 orthologs in yeast and humans are implicated in the cell cycle, particularly in mitosis. Interestingly, Thg1 can also catalyze a 3’ to 5’ (reverse) polymerization reaction in vitro, in which Thg1 adds guanine and cytidine residues to the 5’ end of RNA substrates, in the opposite direction of all other known polymerases. Because of the different possible functions of Thg1, the essential role of Thg1 in the cell is unclear. To study the functions of Thg1 and G-1 of tRNAHis in yeast, nonsense suppression assays were utilized to optimize tRNAHis function in a condition where Thg1 cannot efficiently recognize tRNAHis. Overexpression of both the tRNAHis amber suppressor and histidyl-tRNA synthetase (HTS1) drastically improve nonsense suppression. Surprisingly, the lethality of a thg1-Δ strain can be bypassed by overexpression of both HTS1 and wild-type tRNAHis A73 or tRNAHis C73. Since tRNAHis species from thg1-Δ strains lack G-1, these results demonstrate that the G-1 residue itself is not essential in yeast; however, since thg1-Δ strains are viable under these conditions, the essential function of Thg1 is G-1 addition to tRNAHis. Also surprising is that thg1-Δ strains are healthier with tRNAHis C73 than with tRNAHis A73, despite the fact that eukaryotes have largely conserved A73 on tRNAHis. However, since Thg1 can catalyze reverse polymerization on tRNAHis C73 in vivo, A73 may have been conserved to prevent this activity. Interestingly, thg1-Δ strains are sensitive to paromomycin, an aminoglycoside that induces misreading in the decoding center of the ribosome. Thus, G-1 may be important for mRNA decoding during translation. Further analysis of tRNAHis from thg1-Δ strains demonstrates that 2’-Omethylguanosine (Gm) is reduced in both tRNAHis A73 and tRNAHis C73, and additional 5-methylcytidine (m5C) is present in tRNAHis C73. Upon additional examination, Gm levels are reduced in several conditions where Thg1 activity, and thus tRNAHis function, is compromised: when THG1 is deleted; when the tRNAHis GUG anticodon is mutated to amber; and when A73 is mutated to C73. Furthermore, Gm levels are restored to wild-type levels in the absence of Thg1 when tRNAHis function is improved by mutating residues in the acceptor stem, suggesting that tRNAHis function is important for Gm modification. Analysis of the factors influencing m5C levels reveals that m5C accumulates on tRNAHis C73 variants expressed in either wild-type or thg1-Δ strains, but m5C accumulates on tRNAHis A73 only in conditions where Thg1 activity is depleted. These data are intriguing because they indicate that tRNAHis modifications are sensitive to tRNA function, tRNA sequence, and Thg1 activity. To study other roles of Thg1 in vivo, temperature-sensitive thg1 mutants were generated. These mutants are likely defective in different Thg1 activities since several thg1ts strains display intragenic complementation. These strains will be valuable in the future to dissect the mechanistic steps of both G-1 addition and reverse polymerization