Roles of Trm9- and ALKBH8-like proteins in the formation of modified wobble uridines in Arabidopsis tRNA

Uridine at the wobble position of tRNA is usually modified, and modification is required for accurate and efficient protein translation. In eukaryotes, wobble uridines are modified into 5-methoxycarbonylmethyluridine (mcm5U), 5-carbamoylmethyluridine (ncm5U) or derivatives thereof. Here, we demonstrate, both by in vitro and in vivo studies, that the Arabidopsis thaliana methyltransferase AT1G31600, denoted by us AtTRM9, is responsible for the final step in mcm5U formation, thus representing a functional homologue of the Saccharomyces cerevisiae Trm9 protein. We also show that the enzymatic activity of AtTRM9 depends on either one of two closely related proteins, AtTRM112a and AtTRM112b. Moreover, we demonstrate that AT1G36310, denoted AtALKBH8, is required for hydroxylation of mcm5U to (S)-mchm5U in tRNAGlyUCC, and has a function similar to the mammalian dioxygenase ALKBH8. Interestingly, atalkbh8 mutant plants displayed strongly increased levels of mcm5U, and also of mcm5Um, its 2′-O-ribose methylated derivative. This suggests that accumulated mcm5U is prone to further ribose methylation by a non-specialized mechanism, and may challenge the notion that the existence of mcm5U- and mcm5Um-containing forms of the selenocysteine-specific tRNASec in mammals reflects an important regulatory process. The present study reveals a role in for several hitherto uncharacterized Arabidopsis proteins in the formation of modified wobble uridines

The health Oriented pedagogical project (HOPP) - a controlled longitudinal school-based physical activity intervention program

Abstract Background The prevalence of non-communicable diseases (NCDs) is increasing worldwide, also among children. Information about primary prevention of NCD’s is increasing; however, convincing strategies among children is needed. The present paper describes the design and methods in the Health Oriented Pedagogical Project (HOPP) study. The main objective is to evaluate the effects of a school-based physical activity intervention program on cardio-metabolic risk factors. Secondary objectives include assessment of physical, psychological and academic performance variables. Methods The HOPP study is a 7 years longitudinal large-scale controlled intervention in seven elementary schools (n = 1545) with two control schools (n = 752); all aged 6–11 years at baseline. The school-based physical activity intervention program includes an increase in physical activity (PA) of 225 min/week as an integrated part of theoretical learning, in addition to the curriculum based 90 min/week of ordinary PA. Primary outcomes include cardio-metabolic risk factors measured as PA level, BMI status, waist circumference, muscle mass, percent fat, endurance test performance, total serum cholesterol, high-density lipoprotein (HDL), non-HDL, micro C-reactive protein (mCRP) and long-term blood sugar (HbA1c). In addition, secondary outcomes include anthropometric growth measures, physical fitness, quality of life (QoL), mental health, executive functions, diet and academic performance. Discussion HOPP will provide evidence of effects on cardio-metabolic risk factors after a long-term PA intervention program in elementary schoolchildren. School-based PA intervention programs may be an effective arena for health promotion and disease prevention. Trial registration The study is registered in Clinical trials (ClinicalTrials.gov Identifier: NCT02495714 ) as of June 20th – 2015, retrospectively registered. The collection of baseline values was initiated in mid-January 2015

Ten members of the Arabidopsis gene family encoding methyl-CpG-binding domain proteins are transcriptionally active and at least one, AtMBD11, is crucial for normal development

Animal proteins that contain a methyl-CpG-binding domain (MBD) are suggested to provide a link between DNA methylation, chromatin remodelling and gene silencing. However, some MBD proteins reside in chromatin remodelling complexes, but do not have specific affinity for methylated DNA. It has recently been shown that the Arabidopsis genome contains 12 putative genes encoding proteins with domains similar to MBD, of which at least three bind symmetrically methylated DNA. Using a bioinformatics approach, we have identified additional domains in a number of these proteins and, on this basis and extended sequence similarity, divided the proteins into subgroups. Using RT–PCR we show that 10 of the AtMBD genes are active and differentially expressed in diverse tissues. To investigate the biological significance of AtMBD proteins, we have transformed Arabidopsis with a construct aimed at RNA interference with expression of the AtMBD11 gene, normally active in most tissues. The resulting 35S::AtMBD11-RNAi plants displayed a variety of phenotypic effects, including aerial rosettes, serrated leaves, abnormal position of flowers, fertility problems and late flowering. Arabidopsis lines with reduced expression of genes involved in chromatin remodelling and transgene silencing show similar phenotypes. Our results suggest an important role for AtMBD proteins in plant development

Tolerated wobble mutations in siRNAs decrease specificity, but can enhance activity in vivo

in viv

Ectopic ALKBH4 expression does not change H3K79 methylation levels <i>in vivo</i>.

<p>As analyzed by Western blotting, all three methylation states (mono- di- and tri-methylation) of the H3K79 residue remained similar in histones purified from stable HEK293 transfectants after doxycycline-dependent over-expression of either ALKBH4 or an enzymatically inactive mutant (ALKBH4^H169A/D171A), compared to the equivalent, non-induced cells. Signal intensities of bands corresponding to methylated histones were quantified using ImageJ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049045#pone.0049045-Schneider1" target="_blank">[69]</a>, and normalized to the total histone H3 load, but no effect of ALKBH4 overexpression was detected (not shown).</p

Schematic representation of yeast two-hybrid high confidence hits involved in transcription.

<p>Individual prey fragment clones and the resulting selected interaction domains (SIDs) reported to bind ALKBH4 are indicated above each protein; black lines, placenta library; orange lines, fetal brain library; green lines, placenta library screened with ALKBH4^H169A/D171A; red dashed lines, SIDs. Grey boxes indicate protein domains. Proteins and domains are drawn to scale according to the InterPro (version 4.8) and PROSITE (release 20.68) databases <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049045#pone.0049045-Hunter1" target="_blank">[73]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049045#pone.0049045-Sigrist1" target="_blank">[74]</a>.//indicates regions omitted for simplicity. ZnF, C₂H₂ zinc finger; HD, homeodomain; YEATS, Yaf9 ENL AF9 Taf14 Sas5; TAZ, transcription adaptor putative zinc finger; BRD, bromodomain; PHD plant homeodomain; HAT histone acetyl transferase; DBD, DNA binding domain. Bar, 100 aa.</p

The genes most differentially expressed in either direction identified in ALKBH7 over-expressing cells compared to parental non over-expressing cells.

<p>The genes most differentially expressed in either direction identified in ALKBH7 over-expressing cells compared to parental non over-expressing cells.</p

High confidence^a ALKBH4 interacting proteins identified by the yeast two-hybrid system.

a<p>PBS (predicted biological score) of A or B. PBS (calculated according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049045#pone.0049045-Formstecher1" target="_blank">[75]</a>) represents the probability of an interaction to be non-specific, and refers to an e-value with defined thresholds to rank the results in the high-to-low confidence categories A–D.</p>b<p>SID (selected interaction domain) refers to the amino acid region shared by all prey fragments matching the same reference protein,</p>c<p>(+) Low confidence hit (PBS D),</p>d<p>EIF3CL. Grey shade, transcription related interactants. YEATS, Yaf9 ENL AF9 TAF14 Sas5; DBD, DNA binding domain; PHD, plant homeodomain; PET, Prickle Espinas Testin; LIM, Lin-11 Isl-1 Mec-3; PCI, Proteasome, COP9, Initiation factor-3; PTP, protein tyrosine phosphatase.</p

Differentially expressed genes (q-value <5, fold change >1.35) identified in ALKBH4 over-expressing cells compared to parental non over-expressing cells.

1<p>The <i>ALKBH4</i> probe was not detected due to annealing to the UTR of the gene. Sorted by fold change.</p