The human ortholog of archaeal Pus10 produces pseudouridine 54 in select tRNAs where its recognition sequence contains a modified residue.

The nearly conserved U54 of tRNA is mostly converted to a version of ribothymidine (T) in Bacteria and eukaryotes and to a version of pseudouridine (Ψ) in Archaea. Conserved U55 is nearly always modified to Ψ55 in all organisms. Orthologs of TrmA and TruB that produce T54 and Ψ55, respectively, in Bacteria and eukaryotes are absent in Archaea. Pus10 produces both Ψ54 and Ψ55 in Archaea. Pus10 orthologs are found in nearly all sequenced archaeal and most eukaryal genomes, but not in yeast and bacteria. This coincides with the presence of Ψ54 in most archaeal tRNAs and some animal tRNAs, but its absence from yeast and bacteria. Moreover, Ψ54 is found in several tRNAs that function as primers for retroviral DNA synthesis. Previously, no eukaryotic tRNA Ψ54 synthase had been identified. We show here that human Pus10 can produce Ψ54 in select tRNAs, including tRN

Evolution of Eukaryal and Archaeal Pseudouridine Synthase Pus10

Fitzek E, Joardar A, Gupta R, Geisler M. Evolution of Eukaryal and Archaeal Pseudouridine Synthase Pus10. Journal of Molecular Evolution. 2018;86(1):77-89

Role of forefinger and thumb loops in production of Ψ54 and Ψ55 in tRNAs by archaeal Pus10

Pseudouridines (Ψ) are found in structurally and functionally important regions of RNAs. Six families of Ψ synthases, TruA, TruB, TruD, RsuA, RluA, and Pus10 have been identified. Pus10 is present in Archaea and Eukarya. While most archaeal Pus10 produce both tRNA Ψ54 and Ψ55, some produce only Ψ55. Interestingly, human PUS10 has been implicated in apoptosis and Crohn\u27s and Celiac diseases. Homology models of archaeal Pus10 proteins based on the crystal structure of human PUS10 reveal that there are subtle structural differences in all of these Pus10 proteins. These observations suggest that structural changes in homologous proteins may lead to loss, gain, or change of their functions, warranting the need to study the structure-function relationship of these proteins. Using comparison of structural models and a series of mutations, we identified forefinger loop (reminiscent of that of RluA) and an Arg and a Tyr residue of archaeal Pus10 as critical determinants for its Ψ54, but not for its Ψ55 activity. We also found that a Leu residue, in addition to the catalytic Asp, is essential for both activities. Since forefinger loop is needed for both rRNA and tRNA Ψ synthase activities of RluA, but only for tRNA Ψ54 activity of Pus10, archaeal Pus10 proteins must use a different mechanism of recognition for Ψ55 activity. We propose that archaeal Pus10 uses two distinct mechanisms for substrate uridine recognition and binding. However, since we did not observe any mutation that affected only Ψ55 activity, both mechanisms for archaeal Pus10 activities must share some common features

Role of forefinger and thumb loops in production of 54 and 55 in tRNAs by archaeal Pus10

Joardar A, Jana S, Fitzek E, et al. Role of forefinger and thumb loops in production of  54 and  55 in tRNAs by archaeal Pus10. RNA. 2013;19(9):1279-1294

Glycolysis Upregulation Is Neuroprotective As A Compensatory Mechanism In Als

Amyotrophic Lateral Sclerosis (ALS), is a fatal neurodegenerative disorder, with TDP-43 inclusions as a major pathological hallmark. Using a Drosophila model of TDP-43 proteinopathy we found significant alterations in glucose metabolism including increased pyruvate, suggesting that modulating glycolysis may be neuroprotective. Indeed, a high sugar diet improves locomotor and lifespan defects caused by TDP-43 proteinopathy in motor neurons or glia, but not muscle, suggesting that metabolic dysregulation occurs in the nervous system. Overexpressing human glucose transporter GLUT-3 in motor neurons mitigates TDP-43 dependent defects in synaptic vesicle recycling and improves locomotion. Furthermore, PFK mRNA, a key indicator of glycolysis, is upregulated in flies and patient derived iPSC motor neurons with TDP-43 pathology. Surprisingly, PFK overexpression rescues TDP-43 induced locomotor deficits. These findings from multiple ALS models show that mechanistically, glycolysis is upregulated in degenerating motor neurons as a compensatory mechanism and suggest that increased glucose availability is protective

Glycolysis upregulation is neuroprotective as a compensatory mechanism in ALS

Amyotrophic Lateral Sclerosis (ALS), is a fatal neurodegenerative disorder, with TDP-43 inclusions as a major pathological hallmark. Using a Drosophila model of TDP-43 proteinopathy we found significant alterations in glucose metabolism including increased pyruvate, suggesting that modulating glycolysis may be neuroprotective. Indeed, a high sugar diet improves locomotor and lifespan defects caused by TDP-43 proteinopathy in motor neurons or glia, but not muscle, suggesting that metabolic dysregulation occurs in the nervous system. Overexpressing human glucose transporter GLUT-3 in motor neurons mitigates TDP-43 dependent defects in synaptic vesicle recycling and improves locomotion. Furthermore, PFK mRNA, a key indicator of glycolysis, is upregulated in flies and patient derived iPSC motor neurons with TDP-43 pathology. Surprisingly, PFK overexpression rescues TDP-43 induced locomotor deficits. These findings from multiple ALS models show that mechanistically, glycolysis is upregulated in degenerating motor neurons as a compensatory mechanism and suggest that increased glucose availability is protective.National Institutes of Health [T32GM008659, NS091299]; Howard Hughes Medical Institute; University of Arizona; Arnold and Mabel Beckman Foundation; Association pour la Recherche sur la Sclerose Laterale Amyotrophique et autres Maladies du Motoneurone; Target ALS; Barrow Neurological Foundation; Muscular Dystrophy Association [418515]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

GUIDE RNA-DEPENDENT AND INDEPENDENT tRNA MODIFICATIONS IN ARCHAEA

Stable RNAs undergo a wide variety of post-transcriptional modifications, that add to the functional repertoire of these molecules. Some of these modifications are catalyzed by stand-alone protein enzymes, while some others are catalyzed by RNA-protein complexes. tRNAs from all domains of life contain many such modifications, that increase their structural stability and refine their decoding properties. Certain regions of tRNAs are more frequently modified than others. Two such regions are the anticodon loop, and the TψC stem. In the halophilic euryarchaeon Haloferax volcanii, tRNATrp and tRNAMet, both of which are transcribed as intron-containing pre-tRNA forms, contain Cm34 and ψ54, in addition to other modifications, in these two regions, respectively. The Cm34 modification in both cases is RNP-mediated: tRNATrp Cm34 formation being guided by its own intron, while that of tRNAMet being guided by a unique guide RNA called sR-tMet. We created genomic deletion of H. volcanii tRNATrp intron by homologous recombination based technique, and showed that this strain is viable, and does not demonstrate any observable growth phenotype. However, the corresponding modifications are absent in this intron-deleted strain. Our structural and functional characterizations of sR-tMet revealed that it is unique in its structural properties and deviates considerably from its homologs in other Archaea. We also identified a novel L7Ae (a core protein associated with archaeal methylation guide RNPs) binding motif in sR-tMet. ψ54, the near universal modification found in TψC stem-loop of archaeal tRNAs is catalyzed by the protein Pus10. An earlier study from our laboratory had shown that Pus10 from two different archaea, Methanocaldococcus jannaschii (MjPus10) and Pyrococcus furiosus (PfuPus10) have differential activities towards ψ54 formation. Using the crystal structure of Human Pus10 as template, we created homology models of MjPus10 and PfuPus10 proteins and identified several residues and motifs that might lead to this difference in activity. By a combination of both in vitro and in vivo mutational approaches, we confirmed several previously unidentified residues/motifs that serve as positive determinants of tRNA ψ54 formation. Finally, as an extension to this study, we have identified a novel tRNA ψ54 forming activity in mammalian nuclear extracts, and attributed this activity to Pus10

Box C/D RNA-Guided 2′-O Methylations and the Intron of tRNATrp Are Not Essential for the Viability of Haloferax volcanii▿

Deleting the box C/D RNA-containing intron in the Haloferax volcanii tRNATrp gene abolishes RNA-guided 2′-O methylations of C34 and U39 residues of tRNATrp. However, this deletion does not affect growth under standard conditions

Regulation of Heart Rate in Drosophila via Fragile X Mental Retardation Protein.

RNA binding proteins play a pivotal role in post-transcriptional gene expression regulation, however little is understood about their role in cardiac function. The Fragile X (FraX) family of RNA binding proteins is most commonly studied in the context of neurological disorders, as mutations in Fragile X Mental Retardation 1 (FMR1) are the leading cause of inherited mental retardation. More recently, alterations in the levels of Fragile X Related 1 protein, FXR1, the predominant FraX member expressed in vertebrate striated muscle, have been linked to structural and functional defects in mice and zebrafish models. FraX proteins are established regulators of translation and are known to regulate specific targets in different tissues. To decipher the direct role of FraX proteins in the heart in vivo, we turned to Drosophila, which harbors a sole, functionally conserved and ubiquitously expressed FraX protein, dFmr1. Using classical loss of function alleles as well as muscle specific RNAi knockdown, we show that Drosophila FMRP, dFmr1, is required for proper heart rate during development. Functional analyses in the context of cardiac-specific dFmr1 knockdown by RNAi demonstrate that dFmr1 is required cell autonomously in cardiac cells for regulating heart rate. Interestingly, these functional defects are not accompanied by any obvious structural abnormalities, suggesting that dFmr1 may regulate a different repertoire of targets in Drosophila than in vertebrates. Taken together, our findings support the hypothesis that dFmr1 protein is essential for proper cardiac function and establish the fly as a new model for studying the role(s) of FraX proteins in the heart