Schizosaccharomyces pombe pus1 mutants are temperature sensitive due to decay of tRNAIle(UAU) by the 5'-3' exonuclease Dhp1, primarily targeting the unspliced pre-tRNA.

The pseudouridylase Pus1 catalyzes pseudouridine (Ψ) formation at multiple uridine residues in tRNAs, and in some snRNAs and mRNAs. Although Pus1 is highly conserved, and mutations are associated with human disease, little is known about eukaryotic Pus1 biology. Here, we show that Schizosaccharomyces pombe pus1Δ mutants are temperature sensitive due to decay of tRNAIle(UAU), as tRNAIle(UAU) levels are reduced, and its overexpression suppresses the defect.

A connection between the ribosome and two S. pombe tRNA modification mutants subject to rapid tRNA decay.

tRNA modifications are crucial in all organisms to ensure tRNA folding and stability, and accurate translation. In both the yeast Saccharomyces cerevisiae and the evolutionarily distant yeast Schizosaccharomyces pombe, mutants lacking certain tRNA body modifications (outside the anticodon loop) are temperature sensitive due to rapid tRNA decay (RTD) of a subset of hypomodified tRNAs. Here we show that for each of two S.

A connection between the ribosome and two tRNA modification mutants subject to rapid tRNA decay.

tRNA modifications are crucial in all organisms to ensure tRNA folding and stability, and accurate translation in the ribosome. In both the yeast and the evolutionarily distant yeast , mutants lacking certain tRNA body modifications (outside the anticodon loop) are temperature sensitive due to rapid tRNA decay (RTD) of a subset of hypomodified tRNAs. Here we show that for each of two mutants subject to RTD, mutations in ribosomal protein genes suppress the temperature sensitivity without altering tRNA levels.

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 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.

Initiator tRNA lacking 1-methyladenosine is targeted by the rapid tRNA decay pathway in evolutionarily distant yeast species.

All tRNAs have numerous modifications, lack of which often results in growth defects in the budding yeast Saccharomyces cerevisiae and neurological or other disorders in humans. In S. cerevisiae, lack of tRNA body modifications can lead to impaired tRNA stability and decay of a subset of the hypomodified tRNAs.

Identification of a Trm732 Motif Required for 2'--methylation of the tRNA Anticodon Loop by Trm7.

Posttranscriptional tRNA modifications are essential for proper gene expression, and defects in the enzymes that perform tRNA modifications are associated with numerous human disorders. Throughout eukaryotes, 2'--methylation of residues 32 and 34 of the anticodon loop of tRNA is important for proper translation, and in humans, a lack of these modifications results in non-syndromic X-linked intellectual disability. In yeast, the methyltransferase Trm7 forms a complex with Trm732 to 2'--methylate tRNA residue 32 and with Trm734 to 2'--methylate tRNA residue 34.

A semi-quantitative pull-down assay to study tRNA substrate specificity of modification enzymes.

A tRNA interacts with numerous proteins throughout its biogenesis and during translation, and a significant portion of these interacting proteins are involved in post-transcriptional modifications. While some of the modifying enzymes use relatively simple recognition elements for substrate recognition, many enzymes selectively modify a specific subset of tRNA species without obvious recognition rules. In this chapter we describe a semi-quantitative pull-down assay to study tRNA substrate specificity of modification enzymes, by using the yeast Saccharomyces cerevisiae mC methyltransferase Trm140 as an example.

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.

Mutations in the anticodon stem of tRNA cause accumulation and Met22-dependent decay of pre-tRNA in yeast.

During tRNA maturation in yeast, aberrant pre-tRNAs are targeted for 3'-5' degradation by the nuclear surveillance pathway, and aberrant mature tRNAs are targeted for 5'-3' degradation by the rapid tRNA decay (RTD) pathway. RTD is catalyzed by the 5'-3' exonucleases Xrn1 and Rat1, which act on tRNAs with an exposed 5' end due to the lack of certain body modifications or the presence of destabilizing mutations in the acceptor stem, T-stem, or tRNA fold. RTD is inhibited by mutation of , likely due to accumulation of the Met22 substrate adenosine 3',5' bis-phosphate, which inhibits 5'-3' exonucleases.

Formation of tRNA Wobble Inosine in Humans Is Disrupted by a Millennia-Old Mutation Causing Intellectual Disability.

The formation of inosine at the wobble position of eukaryotic tRNAs is an essential modification catalyzed by the ADAT2/ADAT3 complex. In humans, a valine-to-methionine mutation (V144M) in ADAT3 that originated ∼1,600 years ago is the most common cause of autosomal recessive intellectual disability (ID) in Arabia. While the mutation is predicted to affect protein structure, the molecular and cellular effects of the V144M mutation are unknown.

PUS7 mutations impair pseudouridylation in humans and cause intellectual disability and microcephaly.

Pseudouridylation is the most common post-transcriptional modification, wherein uridine is isomerized into 5-ribosyluracil (pseudouridine, Ψ). The resulting increase in base stacking and creation of additional hydrogen bonds are thought to enhance RNA stability. Pseudouridine synthases are encoded in humans by 13 genes, two of which are linked to Mendelian diseases: PUS1 and PUS3.

A rationale for tRNA modification circuits in the anticodon loop.

The numerous post-transcriptional modifications of tRNA play a crucial role in tRNA function. While most modifications are introduced to tRNA independently, several sets of modifications are found to be interconnected such that the presence of one set of modifications drives the formation of another modification. The vast majority of these modification circuits are found in the anticodon loop (ACL) region where the largest variety and highest density of modifications occur compared to the other parts of the tRNA and where there is relatively limited sequence and structural information.

Conditional accumulation of toxic tRNAs to cause amino acid misincorporation.

To develop a system for conditional amino acid misincorporation, we engineered tRNAs in the yeast Saccharomyces cerevisiae to be substrates of the rapid tRNA decay (RTD) pathway, such that they accumulate when RTD is turned off. We used this system to test the effects on growth of a library of tRNASer variants with all possible anticodons, and show that many are lethal when RTD is inhibited and the tRNA accumulates. Using mass spectrometry, we measured serine misincorporation in yeast containing each of six tRNA variants, and for five of them identified hundreds of peptides with serine substitutions at the targeted amino acid sites.

Lack of 2'-O-methylation in the tRNA anticodon loop of two phylogenetically distant yeast species activates the general amino acid control pathway.

Modification defects in the tRNA anticodon loop often impair yeast growth and cause human disease. In the budding yeast Saccharomyces cerevisiae and the phylogenetically distant fission yeast Schizosaccharomyces pombe, trm7Δ mutants grow poorly due to lack of 2'-O-methylation of C32 and G34 in the tRNAPhe anticodon loop, and lesions in the human TRM7 homolog FTSJ1 cause non-syndromic X-linked intellectual disability (NSXLID). However, it is unclear why trm7Δ mutants grow poorly.

Widespread temperature sensitivity and tRNA decay due to mutations in a yeast tRNA.

Microorganisms have universally adapted their RNAs and proteins to survive at a broad range of temperatures and growth conditions. However, for RNAs, there is little quantitative understanding of the effects of mutations on function at high temperatures. To understand how variant tRNA function is affected by temperature change, we used the tRNA nonsense suppressor of the yeast to perform a high-throughput quantitative screen of tRNA function at two different growth temperatures.

High-Throughput Small RNA Sequencing Enhanced by AlkB-Facilitated RNA de-Methylation (ARM-Seq).

N -methyladenosine (mA), N -methylcytidine (mC), and N -methylguanosine (mG) are common in transfer RNA (tRNA) and tRNA-derived fragments. These modifications alter Watson-Crick base-pairing, and cause pauses or stops during reverse transcription required for most high-throughput RNA sequencing protocols, resulting in inefficient detection of methyl-modified RNAs. Here, we describe a procedure to demethylate RNAs containing mA, mC, or mG using the Escherichia coli dealkylating enzyme AlkB, along with instructions for subsequent processing with widely used protocols for small RNA sequencing.

Trm140 has two recognition modes for 3-methylcytidine modification of the anticodon loop of tRNA substrates.

The 3-methylcytidine (mC) modification is ubiquitous in eukaryotic tRNA, widely found at C in the anticodon loop of tRNA, tRNA, and some tRNA species, as well as in the variable loop (V-loop) of certain tRNA species. In the yeast , formation of mC requires Trm140 for six tRNA substrates, including three tRNA species and three tRNA species, whereas in , two Trm140 homologs are used, one for tRNA and one for tRNA The occurrence of a single Trm140 homolog is conserved broadly among Ascomycota, whereas multiple Trm140-related homologs are found in metazoans and other fungi. We investigate here how Trm140 protein recognizes its six tRNA substrates.

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.

Mutation in WDR4 impairs tRNA m(7)G46 methylation and causes a distinct form of microcephalic primordial dwarfism.

Background: Primordial dwarfism is a state of extreme prenatal and postnatal growth deficiency, and is characterized by marked clinical and genetic heterogeneity.

Results: Two presumably unrelated consanguineous families presented with an apparently novel form of primordial dwarfism in which severe growth deficiency is accompanied by distinct facial dysmorphism, brain malformation (microcephaly, agenesis of corpus callosum, and simplified gyration), and severe encephalopathy with seizures. Combined autozygome/exome analysis revealed a novel missense mutation in WDR4 as the likely causal variant.

Defects in tRNA Anticodon Loop 2'-O-Methylation Are Implicated in Nonsyndromic X-Linked Intellectual Disability due to Mutations in FTSJ1.

tRNA modifications are crucial for efficient and accurate protein synthesis, and modification defects are frequently associated with disease. Yeast trm7Δ mutants grow poorly due to lack of 2'-O-methylated C32 (Cm32 ) and Gm34 on tRNA(Phe) , catalyzed by Trm7-Trm732 and Trm7-Trm734, respectively, which in turn results in loss of wybutosine at G37 . Mutations in human FTSJ1, the likely TRM7 homolog, cause nonsyndromic X-linked intellectual disability (NSXLID), but the role of FTSJ1 in tRNA modification is unknown.

Methodology for the High-Throughput Identification and Characterization of tRNA Variants That Are Substrates for a tRNA Decay Pathway.

The rapid tRNA decay (RTD) pathway is a tRNA quality control pathway known to degrade several specific hypomodified or destabilized tRNAs in the yeast Saccharomyces cerevisiae. In this chapter, we describe seven methods for identifying RTD substrates, with a focus on two new approaches: a high-throughput approach that utilizes a suppressor tRNA library, fluorescence-activated cell sorting, and deep sequencing, and has greatly expanded the known range of RTD substrates; and a poison primer extension assay that allows for the measurement of levels of suppressor tRNA variants, even in the presence of highly similar endogenous tRNAs. We also discuss different applications of the use of the high-throughput and poison primer extension methodologies for different problems in tRNA biology.

ARM-seq: AlkB-facilitated RNA methylation sequencing reveals a complex landscape of modified tRNA fragments.

High-throughput RNA sequencing has accelerated discovery of the complex regulatory roles of small RNAs, but RNAs containing modified nucleosides may escape detection when those modifications interfere with reverse transcription during RNA-seq library preparation. Here we describe AlkB-facilitated RNA methylation sequencing (ARM-seq), which uses pretreatment with Escherichia coli AlkB to demethylate N(1)-methyladenosine (m(1)A), N(3)-methylcytidine (m(3)C) and N(1)-methylguanosine (m(1)G), all commonly found in tRNAs. Comparative methylation analysis using ARM-seq provides the first detailed, transcriptome-scale map of these modifications and reveals an abundance of previously undetected, methylated small RNAs derived from tRNAs.

Two-subunit enzymes involved in eukaryotic post-transcriptional tRNA modification.

tRNA modifications are crucial for efficient and accurate protein translation, with defects often linked to disease. There are 7 cytoplasmic tRNA modifications in the yeast Saccharomyces cerevisiae that are formed by an enzyme consisting of a catalytic subunit and an auxiliary protein, 5 of which require only a single subunit in bacteria, and 2 of which are not found in bacteria. These enzymes include the deaminase Tad2-Tad3, and the methyltransferases Trm6-Trm61, Trm8-Trm82, Trm7-Trm732, and Trm7-Trm734, Trm9-Trm112, and Trm11-Trm112.