Analysis of genomic tRNA sets from Bacteria, Archaea, and Eukarya points to anticodon–codon hydrogen bonds as a major determinant of tRNA compositional variations

Analysis of 100 complete sets of the cytoplasmic elongator tRNA genes from Bacteria, Archaea, and Eukarya pointed to correspondences between types of anticodon and composition of the rest of the tRNA body. The number of the hydrogen bonds formed between the complementary nucleotides in the anticodon–codon duplex appeared as a major quantitative parameter determining covariations in all three domains of life. Our analysis has supported and advanced the “extended anticodon” concept that is based on the argument that the decoding performance of the anticodon is enhanced by selection of a matching anticodon stem–loop sequence, as reported by Yarus in 1982. In addition to the anticodon stem–loop, we have found covariations between the anticodon nucleotides and the composition of the distant regions of their respective tRNAs that include dihydrouridine (D) and thymidyl (T) stem–loops. The majority of the covariable tRNA positions were found at the regions with the increased dynamic potential—such as stem–loop and stem–stem junctions. The consistent occurrences of the covariations on the multigenomic level suggest that the number and pattern of the hydrogen bonds in the anticodon–codon duplex constitute a major factor in the course of translation that is reflected in the fine-tuning of the tRNA composition and structure

Translational control by TOR and TAP42 through dephosphorylation of eIF2α kinase GCN2

Yeast protein kinase GCN2 stimulates the translation of transcriptional activator GCN4 by phosphorylating eIF2α in response to amino acid starvation. Kinase activation requires binding of uncharged tRNA to a histidyl tRNA synthetase-related domain in GCN2. Phosphorylation of serine 577 (Ser 577) in GCN2 by another kinase in vivo inhibits GCN2 function in rich medium by reducing tRNA binding activity. We show that rapamycin stimulates eIF2α phosphorylation by GCN2, with attendant induction of GCN4 translation, while reducing Ser 577 phosphorylation in nonstarved cells. The alanine 577 (Ala 577) mutation in GCN2 (S577A) dampened the effects of rapamycin on eIF2α phosphorylation and GCN4 translation, suggesting that GCN2 activation by rapamycin involves Ser 577 dephosphorylation. Rapamycin regulates the phosphorylation of Ser 577 and eIF2α by inhibiting the TOR pathway. Rapamycin-induced dephosphorylation of Ser 577, eIF2α phosphorylation, and induction of GCN4 all involve TAP42, a regulator of type 2A-related protein phosphatases. Our results add a new dimension to the regulation of protein synthesis by TOR proteins and demonstrate cross-talk between two major pathways for nutrient control of gene expression in yeast

Activity of the adrenocortical system in rats with experimental diabetes

Aim. To study the functional state of the adrenocortical system in experimental animals depending on severity of alloxan diabetes. Materials and methods. Diabetes in rats was induced by administering alloxan tetrahydrate at a dose of 17 mg/100 g b.w. Corticosteroids in plasma,adrenals, and 24-hr urine were measured by RIA, immunoenzyme assay, and HPLC. Hepatic aminotransferase activities were determined. Results. Durng the first week after induction of diabetes, the animals suffered metabolic disturbances and hypoinsulinemia the severity of which didnot significantly change up to day 30 Activation of adrenal glucocorticoid function (a rise in plasma corticosteron, urine and adrenal corticosteronand progesteron) occurred starting from days 8-9. Enhanced activity of hepatic aminotransferases confirmed physiological significance of elevatedblood corticosteron level. Conclusion. Physiological effects of glucocorticoids in the liver decreased by day 30 of experimental diabetes despite persisting disturbances in carbohydratemetabolism, probably due to reduced synthesis of corticosteron in adrenals and its concentration in blood

RNA Polymerase III Output Is Functionally Linked to tRNA Dimethyl-G26 Modification

<div><p>Control of the differential abundance or activity of tRNAs can be important determinants of gene regulation. RNA polymerase (RNAP) III synthesizes all tRNAs in eukaryotes and it derepression is associated with cancer. Maf1 is a conserved general repressor of RNAP III under the control of the target of rapamycin (TOR) that acts to integrate transcriptional output and protein synthetic demand toward metabolic economy. Studies in budding yeast have indicated that the global tRNA gene activation that occurs with derepression of RNAP III via <i>maf1-</i>deletion is accompanied by a paradoxical loss of tRNA-mediated nonsense suppressor activity, manifested as an antisuppression phenotype, by an unknown mechanism. We show that <i>maf1</i>-antisuppression also occurs in the fission yeast <i>S</i>. <i>pombe</i> amidst general activation of RNAP III. We used tRNA-HydroSeq to document that little changes occurred in the relative levels of different tRNAs in <i>maf1Δ</i> cells. By contrast, the efficiency of <i>N2</i>,<i>N2</i>-dimethyl G26 (m^<b>2</b>_<b>2</b>G26) modification on certain tRNAs was decreased in response to <i>maf1</i>-deletion and associated with antisuppression, and was validated by other methods. Over-expression of Trm1, which produces m^<b>2</b>_<b>2</b>G26, reversed <i>maf1-</i>antisuppression. A model that emerges is that competition by increased tRNA levels in <i>maf1Δ</i> cells leads to m^<b>2</b>_<b>2</b>G26 hypomodification due to limiting Trm1, reducing the activity of suppressor-tRNASerUCA and accounting for antisuppression. Consistent with this, we show that RNAP III mutations associated with hypomyelinating leukodystrophy decrease tRNA transcription, increase m^<b>2</b>_<b>2</b>G26 efficiency and reverse antisuppression. Extending this more broadly, we show that a decrease in tRNA synthesis by treatment with rapamycin leads to increased m^<b>2</b>_<b>2</b>G26 modification and that this response is conserved among highly divergent yeasts and human cells.</p></div

M²₂G26 hypomodification is responsible for the <i>maf1-</i>antisuppression paradox.

<p><b>A)</b> Box plots showing misincorporations in <i>maf1Δ</i>, WT and <i>maf1Δ+maf1</i>^{<b><i>+</i></b>} strains; G26 box shows misincorporations for 36 tRNAs with G at position 26 (*paired student t test p value <0.001); the G9, A34 and A58 box plots show misincorporations for the tRNA subsets with the corresponding nucleotide identities. <b>B)</b> G26 misincorporations for unique reads mapping to sup-tRNASerUCA in <i>maf1Δ</i>, WT and <i>maf1Δ+maf1</i>^{<b><i>+</i></b>} strains. <b>C)</b> Western blot analysis of Trm1 levels in the strains indicated above the lanes; tubulin served as a loading control. <b>D)</b> Cartoon showing G26 as red asterisk and the two probes used for PHA26 (<u>p</u>ositive <u>h</u>ybridization in the <u>a</u>bsence of G<u>26</u> modification) assay. <b>E</b>) PHA26 northern blot assay showing sequential probings with oligos to the two different tRNAsLeu indicated to the left; strains are indicated above the lanes and over-expression plasmids are indicated as <i>+trm1</i>^{<b><i>+</i></b>} <i>+maf1</i>^{<b><i>+</i></b>} or the control, empty vector. Quantification of T-loop/D-AC stem probe signal is expressed as a modification index where the value of the control, in this case lane 1, set to a value of 1.0, is shown below the lanes of each tRNA panel. <b>F</b>) tRNA-mediated suppression (TMS) for WT, <i>maf1Δ</i>, <i>trm1Δ</i>, and <i>maf1Δ+trm1</i>^{<b><i>+</i></b>} cells.</p

Lack of i6A37 is not responsible for <i>maf1-</i>antisuppression phenotype.

<p><b>A)</b> tRNA mediated suppression (TMS) in WT (wild-type, i.e., <i>maf1</i>^{<b><i>+</i></b>}), <i>tit1Δ</i> (lacking the tRNA A37-isopentenyltransferase-1 gene, <i>tit1</i>^{<b><i>+</i></b>}, see text) and <i>maf1Δ</i> cells in excess or limiting adenine (Ade200 vs. Ade10; 200 vs. 10 mg/L, respectively); transformed with empty vector (+ev) or expression vector for <i>maf1</i>^{<b><i>+</i></b>}. <b>B)</b> Midwestern blotting of RNA from <i>maf1</i>^{<b><i>+</i></b>} and <i>maf1Δ</i> cells using anti-i6A antibody, and subsequent probing for U5 snRNA as loading control. <b>C)</b> Monitoring <i>in vivo</i> i6A37 level by PHA6 (<u>p</u>ositive <u>h</u>ybridization in the <u>a</u>bsence of i<u>6</u>A modification, see text) assay in sup-tRNASerUCA (sup-tRNA) and other RNA as indicated. 1X, 2X = 5, 10 ug total RNA. <b>D)</b> Graphic plot of quantification efficiencies in the three <i>S</i>. <i>pombe</i> strains: % modification = [1− (ACL<i>tit1</i>^{<b><i>+</i></b>}/BP<i>tit1</i>^{<b><i>+</i></b>})/(ACL<i>tit1Δ/</i>BP<i>tit1Δ</i>)] X 100. ACL, anticodon loop probe; BP, body probe. <b>E)</b> Quantification of steady state levels of the sup-tRNASerUCA and tRNASerUGA examined in panel C. <b>D & E:</b> Error bars reflect standard deviations for three experiments.</p

Maf1 is a rapamycin-sensitive regulator of RNAP III-mediated tRNA transcription in <i>S</i>. <i>pombe</i>.

<p>A) Northern blot probed for <i>maf1</i>^<i>+</i> mRNA in parent wild type (WT) strain, <i>maf1Δ</i> strain and <i>maf1Δ</i> in which <i>maf1</i>^<i>+</i> is over expressed from a multicopy plasmid. The <i>rpl8</i>^<i>+</i> mRNA serves as a loading control. Each sample was loaded in duplicate at 2X and 1X. B) Northern blot analysis of tRNASerGCU, tRNAAlaUGC and U5 snRNA loading control on the same blot from the three strains indicated above the lanes. C) Quantitation of the tRNAAlaUGC (white bar) and tRNASerGCU (grey) transcripts from three northern blots, including from panel B, using U5 snRNA on the same blots for calibration. Error bars indicate standard deviations of three experiments. D) Spot assay showing growth of <i>S</i>. <i>pombe</i> strains in minimal media (EMM) with or without rapamycin at 100 ng/ml. E) Northern blot comparing the tRNA transcripts indicated in WT and <i>maf1Δ</i> cells one hour after the addition of rapamycin or DMSO carrier alone to the liquid culture media.</p

The m²₂G26 modification efficiency response is conserved in <i>S</i>. <i>cerevisiae</i> and human cells.

<p><b>A)</b> PHA26 assay of <i>S</i>. <i>cerevisiae maf1Δ</i> and WT (<i>MAF1</i>) cells. <b>B)</b> TMS assay shows that over-expression of <i>TRM1</i> reverses antisuppression phenotype of <i>S</i>. <i>cerevisiae maf1Δ</i> cells. <b>C)</b> PHA26 assay of human embryonic kidney (HEK) 293 cells grown for a period of serum starvation or after serum replenishment as indicated above the lanes. <b>D)</b> PHA26 assay of HEK293 cells in the presence or absence of rapamycin. Quantitative modification indices are shown for panels A, C and D, described as for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005671#pgen.1005671.g004" target="_blank">Fig 4D</a>.</p