Noncatalytic regulation of 18S rRNA methyltransferase DIMT1 in acute myeloid leukemia.

Several rRNA-modifying enzymes install rRNA modifications while participating in ribosome assembly. Here, we show that 18S rRNA methyltransferase DIMT1 is essential for acute myeloid leukemia (AML) proliferation through a noncatalytic function. We reveal that targeting a positively charged cleft of DIMT1, remote from the catalytic site, weakens the binding of DIMT1 to rRNA and mislocalizes DIMT1 to the nucleoplasm, in contrast to the primarily nucleolar localization of wild-type DIMT1. Mechanistically, rRNA binding is required for DIMT1 to undergo liquid-liquid phase separation, which explains the distinct nucleoplasm localization of the rRNA binding-deficient DIMT1. Re-expression of wild-type or a catalytically inactive mutant E85A, but not the rRNA binding-deficient DIMT1, supports AML cell proliferation. This study provides a new strategy to target DIMT1-regulated AML proliferation via targeting this essential noncatalytic region.

Human DIMT1 generates N26,6A-dimethylation-containing small RNAs.

Dimethyladenosine transferase 1 (DIMT1) is an evolutionarily conserved RNA N6,6-dimethyladenosine (m26,6A) methyltransferase. DIMT1 plays an important role in ribosome biogenesis, and the catalytic activity of DIMT1 is indispensable for cell viability and protein synthesis. A few RNA-modifying enzymes can install the same modification in multiple RNA species. However, whether DIMT1 can work on RNA species other than 18S rRNA is unclear. Here, we describe that DIMT1 generates m26,6A not only in 18S rRNA but also in small RNAs. In addition, m26,6A in small RNAs were significantly decreased in cells expressing catalytically inactive DIMT1 variants (E85A or NLPY variants) compared with cells expressing wildtype DIMT1. Both E85A and NLPY DIMT1 variant cells present decreased protein synthesis and cell viability. Furthermore, we observed that DIMT1 is highly expressed in human cancers, including acute myeloid leukemia. Our data suggest that downregulation of DIMT1 in acute myeloid leukemia cells leads to a decreased m26,6A level in small RNAs. Together, these data suggest that DIMT1 not only installs m26,6A in 18S rRNA but also generates m26,6A-containing small RNAs, both of which potentially contribute to the impact of DIMT1 on cell viability and gene expression.

Loss of the ribosomal RNA methyltransferase NSUN5 impairs global protein synthesis and normal growth.

Modifications of ribosomal RNA expand the nucleotide repertoire and thereby contribute to ribosome heterogeneity and translational regulation of gene expression. One particular m5C modification of 25S ribosomal RNA, which is introduced by Rcm1p, was previously shown to modulate stress responses and lifespan in yeast and other small organisms. Here, we report that NSUN5 is the functional orthologue of Rcm1p, introducing m5C3782 into human and m5C3438 into mouse 28S ribosomal RNA. Haploinsufficiency of the NSUN5 gene in fibroblasts from William Beuren syndrome patients causes partial loss of this modification. The N-terminal domain of NSUN5 is required for targeting to nucleoli, while two evolutionary highly conserved cysteines mediate catalysis. Phenotypic consequences of NSUN5 deficiency in mammalian cells include decreased proliferation and size, which can be attributed to a reduction in total protein synthesis by altered ribosomes. Strikingly, Nsun5 knockout in mice causes decreased body weight and lean mass without alterations in food intake, as well as a trend towards reduced protein synthesis in several tissues. Together, our findings emphasize the importance of single RNA modifications for ribosome function and normal cellular and organismal physiology.

Modulation of mammalian translation by a ribosome-associated tRNA half.

Originally considered futile degradation products, tRNA-derived RNA fragments (tdRs) have been shown over the recent past to be crucial players in orchestrating various cellular functions. Unlike other small non-coding RNA (ncRNA) classes, tdRs possess a multifaceted functional repertoire ranging from regulating transcription, apoptosis, RNA interference, ribosome biogenesis to controlling translation efficiency. A subset of the latter tdRs has been shown to directly target the ribosome, the central molecular machine of protein biosynthesis. Here we describe the function of the mammalian tRNAPro 5' half, a 35 residue long ncRNA associated with ribosomes and polysomes in several mammalian cell lines. Addition of tRNAPro halves to mammalian in vitro translation systems results in global translation inhibition and concomitantly causes the upregulation of a specific low molecular weight translational product. This tRNAPro 5' half-dependent translation product consists of both RNA and amino acids. Transfection of the tRNAPro half into HeLa cells leads to the formation of the same product in vivo. The migration of this product in acidic gels, the insensitivity to copper sulphate treatment, the resistance to 3' polyadenylation, and the association with 80S monosomes indicate that the accumulated product is peptidyl-tRNA. Our data thus suggest that binding of the tRNAPro 5' half to the ribosome leads to ribosome stalling and to the formation of peptidyl-tRNA. Our findings revealed a so far unknown functional role of a tdR thus further enlarging the functional heterogeneity of this emerging class of ribo-regulators.

A copper-induced quinone degradation pathway provides protection against combined copper/quinone stress in Lactococcus lactis IL1403.

Quinones are ubiquitous in the environment. They occur naturally but are also in widespread use in human and industrial activities. Quinones alone are relatively benign to bacteria, but in combination with copper, they become toxic by a mechanism that leads to intracellular thiol depletion. Here, it was shown that the yahCD-yaiAB operon of Lactococcus lactis IL1403 provides resistance to combined copper/quinone stress. The operon is under the control of CopR, which also regulates expression of the copRZA copper resistance operon as well as other L. lactis genes. Expression of the yahCD-yaiAB operon is induced by copper but not by quinones. Two of the proteins encoded by the operon appear to play key roles in alleviating quinone/copper stress: YaiB is a flavoprotein that converts p-benzoquinones to less toxic hydroquinones, using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as reductant; YaiA is a hydroquinone dioxygenase that converts hydroquinone putatively to 4-hydroxymuconic semialdehyde in an oxygen-consuming reaction. Hydroquinone and methylhydroquinone are both substrates of YaiA. Deletion of yaiB causes increased sensitivity of L. lactis to quinones and complete growth arrest under combined quinone and copper stress. Copper induction of the yahCD-yaiAB operon offers protection to copper/quinone toxicity and could provide a growth advantage to L. lactis in some environments.

Non-enzymic copper reduction by menaquinone enhances copper toxicity in Lactococcus lactis IL1403.

Lactococcus lactis possesses a pronounced extracellular Cu(2+)-reduction activity which leads to the accumulation of Cu(+) in the medium. The kinetics of this reaction were not saturable by increasing copper concentrations, suggesting a non-enzymic reaction. A copper-reductase-deficient mutant, isolated by random transposon mutagenesis, had an insertion in the menE gene, which encodes O-succinylbenzoic acid CoA ligase. This is a key enzyme in menaquinone biosynthesis. The ΔmenE mutant was deficient in short-chain menaquinones, and exogenously added menaquinone complemented the copper-reductase-deficient phenotype. Haem-induced respiration of wild-type L. lactis efficiently suppressed copper reduction, presumably by competition by the bd-type quinol oxidase for menaquinone. As expected, the ΔmenE mutant was respiration-deficient, but could be made respiration-proficient by supplementation with menaquinone. Growth of wild-type cells was more copper-sensitive than that of the ΔmenE mutant, due to the production of Cu(+) ions by the wild-type. This growth inhibition of the wild-type was strongly attenuated if Cu(+) was scavenged with the Cu(I) chelator bicinchoninic acid. These findings support a model whereby copper is non-enzymically reduced at the membrane by menaquinones. Respiration effectively competes for reduced quinones, which suppresses copper reduction. These findings highlight novel links between copper reduction, respiration and Cu(+) toxicity in L. lactis.

Mammalian In Vitro Translation Systems.

Under cellular stress, tight and coordinated regulation of the gene expression allows to minimize cellular damage, maintains cellular homeostasis, and ensures cell survival. Among stress-induced cellular responses, alteration of translation rates represents one of the most effective and rapid regulatory mechanisms available for cells. Here we report on detailed protocols of mammalian in vitro translation systems. While most of the available in vitro translation methods are based on bacterial or yeast components, tailor-made and robust mammalian systems are sparse. Our protocols allow measuring global translation of the total mRNA pool as well as translation of one specific reporter mRNA. Furthermore, it provides access to measuring translational activity of isolated ribosomes combined with non-ribosomal cytosolic fractions using reduced amounts of biological starting material. The herein described method can be applied to (1) investigate the effects of stress-dependent soluble factors regulating translation (such as tRNA fragments or ribosome-associated ncRNAs), (2) compare translational activity and translational fidelity of different ribosomes supplemented with the same non-ribosomal fractions, and (3) to investigate protein biosynthesis in various mammalian cell lines as well as tissue samples.

Alterations of the translation apparatus during aging and stress response.

Aging is a biological process characterized by the irreversible and time-dependent deterioration of cell functions, tissues, and organs. Accumulating studies in a wide range of species from yeast to human revealed changes associated with the aging process to be conserved throughout evolution. The main characteristics of aging are (i) genomic instability, (ii) loss of telomere function, (iii) epigenetic changes,(iv) increased cellular senescence, (v) depletion of the stem cell pool, (vi) altered intercellular communication and (vii) loss of protein homeostasis. Among the multiple molecular mechanisms underlying aging, alterations of the translation machinery affecting the rate and selectivity of protein biosynthesis seem to play a central role. At the very heart of translation is the ribosome, a multifaceted and universally conserved RNA-protein particle responsible for accurate polypeptide synthesis and co-translational protein folding. Here we summarize and discuss recent developments on the contribution of altered translation and age-dependent modifications on the ribosome structure to aging and cellular senescence.