RNA contains over 100 post-transcriptional chemical modifications. Of these, the most abundant is pseudouridine (Ψ), the 5’ ribosyl isomer of uridine. The formation of Ψ occurs at the polynucleotide level, and is catalyzed by Ψ synthase enzymes. To date, five families of Ψ synthases have been identified. Our work deals with the fifth and last family to be discovered, the Ψ synthase TruD family. TruD homologs are present in organisms across the three kingdoms of life. A sequence alignment of TruD homologs reveals distinct sequence insertions at specific and conserved locations in homologs from higher organisms. We have carried out extensive bioinformatics searches in an effort to characterize these insertion sequences, and have found that one of these insertions has a high degree of similarity to the R3H single-strand nucleic acid binding domain. To further understand the nature of this insertion, we examined its role in the enzymatic activity of the yeast TruD homolog, Pus7p, and found that mutating this insert decreased the enzyme’s activity by almost half. The human genome codes for two TruD homologs, PUS7 and PUS7L. These putative pseudouridine synthases were named based on their similarity to the yeast TruD homolog Pus7p, but their function has only been inferred based on sequence, and neither their activity, nor their structure have been examined. In an effort to further our understanding about the TruD synthase family, we have taken a closer look at PUS7 and PUS7L. We have determined the optimal conditions for over-expression of the enzymes in a bacterial expression system, and shown that the proteins are soluble, and can be purified using serial chromatography. In addition, we have queried these enzymes’ ability to recognize canonical TruD substrates and found that they are unable to complement an E. coli truD deletion or an S. cerevisiae pus7 deletion. Instead, the human PUS7 enzyme proved to be highly specific to the human tRNAGlu sequence, displaying activity targeted to U13. This activity appears to be independent of accessory factors. Taken together, this work strives to further our knowledge of Ψ synthases by examining TruD homologs present in higher organisms. TruD has the least sequence identity to the other four synthase families and does not possess any of the known RNA binding motifs. This work expands our knowledge base of the TruD family, the most divergent of the five Ψ synthase families.

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.