Marcus J.O. Johansson

A primordial tRNA modification required for the evolution of life

The evolution of reading frame maintenance must have been an early event, and presumably preceded the emergence of the three domains Archaea, Bacteria and Eukarya. Features evolved early in reading frame maintenance may still exist in present‐day organisms. We show that one such feature may be the modified nucleoside 1‐methylguanosine (m1G37), which prevents frameshifting and is present adjacent to and 3′ of the anticodon (position 37) in the same subset of tRNAs from all organisms, including that with the smallest sequenced genome (Mycoplasma genitalium), and organelles. We have identified the genes encoding the enzyme tRNA(m1G37)methyltransferase from all three domains. We also show that they are orthologues, and suggest that they originated from a primordial gene. Lack of m1G37 severely impairs the growth of a bacterium and a eukaryote to a similar degree. Yeast tRNA(m1G37)methyltransferase also synthesizes 1‐methylinosine and participates in the formation of the Y‐base (yW). Our results suggest that m1G37 existed in tRNA before the divergence of the three domains, and that a tRNA(m1G37)methyltrans ferase is part of the minimal set of gene products required for life.

Eukaryotic Wobble Uridine Modifications Promote a Functionally Redundant Decoding System

ABSTRACT The translational decoding properties of tRNAs are modulated by naturally occurring modifications of their nucleosides. Uridines located at the wobble position (nucleoside 34 [U34]) in eukaryotic cytoplasmic tRNAs often harbor a 5-methoxycarbonylmethyl (mcm5) or a 5-carbamoylmethyl (ncm5) side chain and sometimes an additional 2-thio (s2) or 2′-O-methyl group. Although a variety of models explaining the role of these modifications have been put forth, their in vivo functions have not been defined. In this study, we utilized recently characterized modification-deficient Saccharomyces cerevisiae cells to test the wobble rules in vivo. We show that mcm5 and ncm5 side chains promote decoding of G-ending codons and that concurrent mcm5 and s2 groups improve reading of both A- and G-ending codons. Moreover, the observation that the mcm5U34- and some ncm5U34-containing tRNAs efficiently read G-ending codons challenges the notion that eukaryotes do not use U-G wobbling.

Association of yeast Upf1p with direct substrates of the NMD pathway

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that detects and degrades transcripts containing premature translation termination codons. Gene expression profiling experiments have shown that inactivation of the NMD pathway leads to the accumulation of both aberrant, nonsense-containing mRNAs, and many apparently wild-type transcripts. Such increases in transcript steady-state levels could arise from direct changes in the respective mRNA half-lives, or indirectly, as a consequence of the stabilization of transcripts encoding specific regulatory proteins. Here, we distinguished direct from indirect substrates by virtue of their association with the Saccharomyces cerevisiae Upf1 protein. Analyses of this dataset, and its comparison to the sets of transcripts that respectively increase or decrease in abundance when NMD is either inactivated or reactivated, indicate that the number of direct NMD substrates is larger than previously thought and that low abundance, alternatively transcribed mRNAs, i.e., mRNAs whose 5′ ends are derived from previously unannotated 5′ flanking sequences, comprise a significant class of direct substrates. Using thiamine metabolism as an example, we also show that apparent NMD-regulated cellular pathways may actually reflect the detection of low-abundance alternative transcripts under conditions where a pathway is repressed.

Dual function of the tRNA(m(5)U54)methyltransferase in tRNA maturation.

A 5-methyluridine (m(5)U) residue at position 54 is a conserved feature of bacterial and eukaryotic tRNAs. The methylation of U54 is catalyzed by the tRNA(m5U54)methyltransferase, which in Saccharomyces cerevisiae is encoded by the nonessential TRM2 gene. In this study, we identified four different strains with mutant forms of tRNA(Ser)CGA. The absence of the TRM2 gene in these strains decreased the stability of tRNA(Ser)CGA and induced lethality. Two alleles of TRM2 encoding catalytically inactive tRNA(m5U54)methyltransferases were able to stabilize tRNA(Ser)CGA in one of the mutants, revealing a role for the Trm2 protein per se in tRNA maturation. Other tRNA modification enzymes interacting with tRNA(Ser)CGA in the maturation process, such as Pus4p, Trm1 p, and Trm3p were essential or important for growth of the tRNA(Ser)CGA mutants. Moreover, Lhp1p, a protein binding RNA polymerase III transcripts, was required to stabilize the mutant tRNAs. Based on our results, we suggest that tRNA modification enzymes might have a role in tRNA maturation not necessarily linked to their known catalytic activity.

Nonsense-mediated mRNA decay maintains translational fidelity by limiting magnesium uptake

Inactivation of the yeast nonsense-mediated mRNA decay (NMD) pathway stabilizes nonsense mRNAs and promotes readthrough of premature translation termination codons. Although the latter phenotype is thought to reflect a direct role of NMD factors in translation termination, its mechanism is unknown. Here we show that the reduced termination efficiency of NMD-deficient cells is attributable to increased expression of the magnesium transporter Alr1p and the resulting effects of elevated Mg(2+) levels on termination fidelity. Alr1p levels increase because an upstream ORF in ALR1 mRNA targets the transcript for NMD. Our results demonstrate that NMD, at least in yeast, controls Mg(2+) homeostasis and, consequently, translational fidelity.

Chapter 6. Qualitative and quantitative assessment of the activity of the yeast nonsense-mediated mRNA decay pathway.

The yeast Saccharomyces cerevisiae provides an ideal model system for elucidation of the molecular mechanisms that regulate the nonsense-mediated mRNA decay (NMD) pathway. This chapter describes an array of molecular biological, genetic, and biochemical tools that facilitate the characterization of transcripts that comprise NMD substrates and provide insights into the roles of the upf/nmd proteins in mRNA decay and translation termination. Examples illustrate the use of these methods in wild-type and NMD-deficient cells to monitor the abundance, structure, and half-lives of nonsense-containing mRNAs, the read through of premature termination codons by the ribosome, and the positioning of ribosomes at or near normal and premature termination codons.

The pre-mRNA retention and splicing complex controls tRNA maturation by promoting TAN1 expression

The conserved pre-mRNA retention and splicing (RES) complex, which in yeast consists of Bud13p, Snu17p and Pml1p, is thought to promote nuclear retention of unspliced pre-mRNAs and enhance splicing of a subset of transcripts. Here, we find that the absence of Bud13p or Snu17p causes greatly reduced levels of the modified nucleoside N4-acetylcytidine (ac4C) in tRNA and that a lack of Pml1p reduces ac4C levels at elevated temperatures. The ac4C nucleoside is normally found at position 12 in the tRNA species specific for serine and leucine. We show that the tRNA modification defect in RES-deficient cells is attributable to inefficient splicing of TAN1 pre-mRNA and the effects of reduced Tan1p levels on formation of ac4C. Analyses of cis-acting elements in TAN1 pre-mRNA showed that the intron sequence between the 5′ splice site and branchpoint is necessary and sufficient to mediate RES dependency. We also show that in RES-deficient cells, the TAN1 pre-mRNA is targeted for degradation by the cytoplasmic nonsense-mediated mRNA decay pathway, indicating that poor nuclear retention may contribute to the tRNA modification defect. Our results demonstrate that TAN1 pre-mRNA processing has an unprecedented requirement for RES factors and that the complex controls the formation of ac4C in tRNA.

Determining if an mRNA is a Substrate of Nonsense-Mediated mRNA Decay in Saccharomyces cerevisiae.

Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic quality control mechanism which triggers decay of mRNAs harboring premature translation termination codons. In this chapter, I describe methods for monitoring the influence of NMD on mRNA abundance and decay rates in Saccharomyces cerevisiae. The descriptions include detailed methods for growing yeast cells, total RNA isolation, and Northern blotting. Although the chapter focuses on NMD, the methods can be easily adapted to assess the effect of other mRNA decay pathways.

The pre-mRNA retention and splicing complex controls expression of the Mediator subunit Med20

ABSTRACT The heterotrimeric pre-mRNA retention and splicing (RES) complex, consisting of Bud13p, Snu17p and Pml1p, promotes splicing and nuclear retention of a subset of intron-containing pre-mRNAs. Yeast cells deleted for individual RES genes show growth defects that are exacerbated at elevated temperatures. Although the growth phenotypes correlate to the splicing defects in the individual mutants, the underlying mechanism is unknown. Here, we show that the temperature sensitive (Ts) growth phenotype of bud13Δ and snu17Δ cells is a consequence of inefficient splicing of MED20 pre-mRNA, which codes for a subunit of the Mediator complex; a co-regulator of RNA polymerase II transcription. The MED20 pre-mRNA splicing defect is less pronounced in pml1Δ cells, explaining why they grow better than the other 2 RES mutants at elevated temperatures. Inactivation of the cytoplasmic nonsense-mediated mRNA decay (NMD) pathway in the RES mutants leads to accumulation of MED20 pre-mRNA, indicating that inefficient nuclear retention contributes to the growth defect. Further, the Ts phenotype of bud13Δ and snu17Δ cells is partially suppressed by the inactivation of NMD, showing that the growth defects are augmented by the presence of a functional NMD pathway. Collectively, our results demonstrate an important role of the RES complex in maintaining the Med20p levels.

Ribosome profiling analysis of eEF3-depleted Saccharomyces cerevisiae

In addition to the standard set of translation factors common in eukaryotic organisms, protein synthesis in the yeast Saccharomyces cerevisiae requires an ABCF ATPase factor eEF3, eukaryotic Elongation Factor 3. eEF3 is an E-site binder that was originally identified as an essential factor involved in the elongation stage of protein synthesis. Recent biochemical experiments suggest an additional function of eEF3 in ribosome recycling. We have characterised the global effects of eEF3 depletion on translation using ribosome profiling. Depletion of eEF3 results in decreased ribosome density at the stop codon, indicating that ribosome recycling does not become rate limiting when eEF3 levels are low. Consistent with a defect in translation elongation, eEF3 depletion causes a moderate redistribution of ribosomes towards the 5’ part of the open reading frames. We observed no E-site codon-or amino acid-specific ribosome stalling upon eEF3 depletion, supporting its role as a general elongation factor. Surprisingly, depletion of eEF3 leads to a relative decrease in P-site proline stalling, which we hypothesise is a secondary effect of generally decreased translation and/or decreased competition for the E-site with eIF5A.