A fungal anticodon nuclease ribotoxin exploits a secondary cleavage site to evade tRNA repair

PaOrf2 and γ-toxin subunits of Pichia acaciae toxin (PaT) and Kluyveromyces lactis zymocin are tRNA anticodon nucleases. These secreted ribotoxins are assimilated by Saccharomyces cerevisiae, wherein they arrest growth by depleting specific tRNAs. Toxicity can be recapitulated by induced intracellular expression of PaOrf2 or γ-toxin in S. cerevisiae. Mutational analysis of γ-toxin has identified amino acids required for ribotoxicity in vivo and RNA transesterification in vitro. Here, we report that PaOrf2 residues Glu9 and His287 (putative counterparts of γ-toxin Glu9 and His209) are essential for toxicity. Our results suggest a similar basis for RNA transesterification by PaOrf2 and γ-toxin, despite their dissimilar primary structures and distinctive tRNA target specificities. PaOrf2 makes two sequential incisions in tRNA, the first of which occurs 3' from the mcm(5)s(2)U wobble nucleoside and depends on mcm(5). A second incision two nucleotides upstream results in the net excision of a di-nucleotide. Expression of phage and plant tRNA repair systems can relieve PaOrf2 toxicity when tRNA cleavage is restricted to the secondary site in elp3 cells that lack the mcm(5) wobble U modification. Whereas the endogenous yeast tRNA ligase Trl1 can heal tRNA halves produced by PaOrf2 cleavage in elp3 cells, its RNA sealing activity is inadequate to complete the repair. Compatible sealing activity can be provided in trans by plant tRNA ligase. The damage-rescuing ability of tRNA repair systems is lost when PaOrf2 can break tRNA at both sites. These results highlight the logic of a two-incision mechanism of tRNA anticodon damage that evades productive repair by tRNA ligases.     

Function of yeast and amphioxus tRNA ligase in IRE1alpha-dependent XBP1 mRNA splicing

During their maturation step, transfer RNAs (tRNAs) undergo excision of their introns by specific splicing. Although tRNA splicing is a molecular event observed in all domains of life, the machinery of the ligation reaction has diverged during evolution. Yeast tRNA ligase 1 (TRL1) is a multifunctional protein that alone catalyzes RNA ligation in tRNA splicing, whereas three molecules [RNA ligase (RNL), Clp1, and PNK/CPDase] are necessary for RNA ligation in tRNA splicing in amphioxi. RNA ligation not only occurs in tRNA splicing, but also in yeast HAC1 mRNA splicing and in animal X-box binding protein 1 (XBP1) mRNA splicing under conditions of endoplasmic reticulum (ER) stress. Yeast TRL1 is known to function as an RNA ligase for HAC1 mRNA splicing, whereas the RNA ligase for XBP1 mRNA splicing is unknown in animals. We examined whether yeast and amphioxus RNA ligases for tRNA splicing function in RNA ligation in mammalian XBP1 splicing. Both RNA ligases functioned in RNA ligation in mammalian XBP1 splicing in vitro. Interestingly, Clp1, and PNK/CPDase were not necessary for exon–exon ligation in XBP1 mRNA by amphioxus RNL. These results suggest that RNA ligase for tRNA splicing might therefore commonly function as an RNA ligase for XBP1 mRNA splicing.     

Jailbreak: Oncogene-induced senescence and its evasion

Aberrant oncogenic signals are typically counteracted by anti-proliferative mechanisms governed principally by the p53 and Rb tumour-suppressor proteins. Apoptosis is firmly established as a potent anti-proliferative mechanism to prevent tumour growth but it is only in recent years that oncogene-induced senescence has achieved similar recognition. Senescence is defined as an irreversible cell-cycle arrest suggesting that entry of oncogene-expressing cells into this static yet viable state is permanent. However, tumours do develop and express the very same oncogenes that landed them in jail. We ask whether this is because rogue incipient cancer cells find ways to escape this imposed imprisonment or otherwise entirely avoid capture by senescence gate-keepers.     

Mutational analysis reveals two independent molecular requirements during transfer RNA selection on the ribosome

Accurate discrimination between cognate and near-cognate aminoacyl-tRNAs during translation relies on the specific acceleration of forward rate constants for cognate tRNAs. Such specific rate enhancement correlates with conformational changes in the tRNA and small ribosomal subunit that depend on an RNA-specific type of interaction, the A-minor motif, between universally conserved 16S ribosomal RNA nucleotides and the cognate codon-anticodon helix. We show that perturbations of these two components of the A-minor motif, the conserved rRNA bases and the codon-anticodon helix, result in distinct outcomes. Although both cause decreases in the rates of tRNA selection that are rescued by aminoglycoside antibiotics, only disruption of the codon-anticodon helix is overcome by a miscoding tRNA variant. On this basis, we propose that two independent molecular requirements must be met to allow tRNAs to proceed through the selection pathway, providing a mechanism for exquisite control of fidelity during this step in gene expression.     

Extracellular calmodulin regulates growth and cAMP-mediated chemotaxis in Dictyostelium discoideum

Zymocin and PaT are killer toxins that induce cell cycle arrest of sensitive yeast cells in G1 and S phase, respectively. Recent studies have revealed that these two toxins cleave specific tRNAs, indicating that the cell growth impairment is due to the tRNA cleavage. Additionally, we have previously shown that the active domain of colicin D (D-CRD), which also cleaves specific Escherichia coli tRNAs, statically impairs growth when expressed in yeast cells. To verify that phase-specific cell cycle arrest is also induced by the expression of D-CRD, D-CRD and the subunits of zymocin and PaT that have tRNA cleaving activity were expressed in yeast cells and cell cycle status was analyzed. Our results indicate that phase-specific arrest does not commonly occur by tRNA cleavage, and it saves the cell viability. Furthermore, the extent of protein synthesis impairment may determine the phase specificity of cell cycle arrest.