Archael proteins Nop10 and Gar1 increase the catalytic activity of Cbf5 in pseudouridylating tRNA

Sherpa Romeo green journal. Open access article. Creative Commons Attribution 4.0 International License (CC BY 4.0) appliesCbf5 is a pseudouridine synthase that usually acts in a guide RNA-dependent manner as part of H/ACA small ribonucleoproteins; however archaeal Cbf5 can also act independently of guide RNA in modifying uridine 55 in tRNA. This guide-independent activity of Cbf5 is enhanced by proteins Nop10 and Gar1 which are also found in H/ACA small ribonucleoproteins. Here, we analyzed the specific contribution of Nop10 and Gar1 for Cbf5-catalyzed pseudouridylation of tRNA. Interestingly, both Nop10 and Gar1 not only increase Cbf5’s affinity for tRNA, but they also directly enhance Cbf5’s catalytic activity by increasing the kcat of the reaction. In contrast to the guide RNA-dependent reaction, Gar1 is not involved in product release after tRNA modification. These results in conjunction with structural information suggest that Nop10 and Gar1 stabilize Cbf5 in its active conformation; we hypothesize that this might also be true for guide-RNA dependent pseudouridine formation by Cbf5.Ye

Pre-steady-state kinetic analysis of the three Escherichia coli pseudouridine synthases TruB, TruA, and RluA reveals uniformly slow catalysis

Sherpa Romeo green journal. Open access article. Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0) appliesPseudouridine synthases catalyze formation of the most abundant modification of functional RNAs by site-specifically isomerizing uridines to pseudouridines. While the structure and substrate specificity of these enzymes have been studied in detail, the kinetic and the catalytic mechanism of pseudouridine synthases remain unknown. Here, the first pre-steady-state kinetic analysis of three Escherichia coli pseudouridine synthases is presented. A novel stopped-flow absorbance assay revealed that substrate tRNA binding by TruB takes place in two steps with an overall rate of 6 sec 1. In order to observe catalysis of pseudouridine formation directly, the traditional tritium release assay was adapted for the quench-flow technique, allowing, for the first time, observation of a single round of pseudouridine formation. Thereby, the single-round rate constant of pseudouridylation (kC) by TruB was determined to be 0.5 sec 1. This rate constant is similar to the kcat obtained under multiple-turnover conditions in steady-state experiments, indicating that catalysis is the rate-limiting step for TruB. In order to investigate if pseudouridine synthases are characterized by slow catalysis in general, the rapid kinetic quench-flow analysis was also performed with two other E. coli enzymes, RluA and TruA, which displayed rate constants of pseudouridine formation of 0.7 and 0.35 sec 1, respectively. Hence, uniformly slow catalysis might be a general feature of pseudouridine synthases that share a conserved catalytic domain and supposedly use the same catalytic mechanism.Ye

An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation

Sherpa Romeo green journal. Permission to archive final published versionPseudouridine synthases introduce the most common RNA modification and likely use the same catalytic mechanism. Besides a catalytic aspartate residue, the contributions of other residues for catalysis of pseudouridine formation are poorly understood. Here, we have tested the role of a conserved basic residue in the active site for catalysis using the bacterial pseudouridine synthase TruB targeting U55 in tRNAs. Substitution of arginine 181 with lysine results in a 2500-fold reduction of TruB’s catalytic rate without affecting tRNA binding. Furthermore, we analyzed the function of a second-shell aspartate residue (D90) that is conserved in all TruB enzymes and interacts with C56 of tRNA. Site-directed mutagenesis, biochemical and kinetic studies reveal that this residue is not critical for substrate binding but influences catalysis significantly as replacement of D90 with glutamate or asparagine reduces the catalytic rate 30- and 50-fold, respectively. In agreement with molecular dynamics simulations of TruB wild type and TruB D90N, we propose an electrostatic network composed of the catalytic aspartate (D48), R181 and D90 that is important for catalysis by finetuning the D48-R181 interaction. Conserved, negatively charged residues similar to D90 are found in a number of pseudouridine synthases, suggesting that this might be a general mechanism.Ye

Investigating Crosstalk Between DNA Repair and Ribosome Biogenesis

Although typically perceived as separate pathways, DNA repair and ribosome biogenesis have surprisingly been associated with one another by the reported interaction between the human DNA repair enzyme SMUG1 and the pseudouridine synthase dyskerin (DCK1, Cbf5 in yeast). Specifically, this interaction has been shown to excise 5-hydeoxylmethyl deoxyuridine, but not uridine or pseudouridine from RNA molecules. Uracil glycosylases, such as SMUG1, remove damaged bases from DNA, leaving an abasic site open for repair. In particular, uracil glycosylases serve a role in protecting the genome from mutation in the event of cytosine deamination to uracil. Unrepaired U:G mismatches can give rise to transition mutations in replication, resulting in altered gene expression. Dyskerin, a ribosome biogenesis factor, is part of a ribonucleoprotein complex that modifies ribosomal RNA by catalyzing the isomerization of uridine to pseudouridine. The function of dyskerin offers an interesting link to cancer, in which large ribosome quantities are generated within an enlarged nucleolus for rapid protein synthesis. Additionally, RNA pseudouridine synthases have been found to be overexpressed in many cancers and linked to a poor prognosis. Following the overexpression and purification of SMUG1 and Cbf5 (dyskerin homologue), the study looks to establish an electrophoresis and fluorescence based uracil-DNA glycosylase assay utilizing fluorescently labelled DNA molecules and DNA endonucleases. In light of the unclear SMUG1-dyskerin interaction, this fluorescence based assay, will be used to characterize the effect of Cbf5 on SMUG1 activity and vice versa. The affinity of SMUG1 to RNA in the presence and absence of Cbf5 will also be investigated via binding studies, with the aim of testing the hypothesis that dyskerin acts as an RNA tether for SMUG1. Studying these protein-protein and RNA-protein interactions will provide insight into the crosstalk between the two important, and previously thought to be unrelated, cellular pathways of DNA repair and ribosome biogenesis with potential implications for new cancer treatment strategies. *Indicates presente

Survival of the Fittest: Elucidating Fitness of Escherichia coli strains Lacking Pseudouridine Synthases

The ribosome is a fundamental component of gene expression, producing proteins from corresponding mRNA sequences. The ribosome is comprised largely of ribosomal RNA (rRNA), which is known to contain various nucleotide modifications. The most abundant of these modifications is pseudouridine, the 5-ribosyl isomer of uridine, whose isomerization is catalyzed by pseudouridine synthases. The peptidyltransferase center (PTC) of the E. coli ribosome contains 6 pseudouridines generated by four pseudouridine synthases. The objective of this study is to examine the contributions of ribosomal pseudouridine synthases to bacterial fitness by characterizing growth phenotypes of knockout strains. Each of the four pseudouridine synthases acting on the ribosomal PTC were sequentially deleted from wild type E. coli, and the effects on cellular fitness were assessed using competition assays and growth curves in both minimal medium and in the presence of antibiotic. The competition assays revealed that each knockout strain (single, double, triple, and quadruple) exhibited a severely reduced fitness, as they were quickly outcompeted by the wildtype strain. The growth curves showed that certain pseudouridine synthases became more important under different stress conditions. In minimal media, it was found that RluB and RluF are more important than the alternative pseudouridine synthases in counteracting the effects of nutrient limitation, both with and without antibiotic present. Additionally, deleting RluE seemed to rescue the growth phenotype in the multiple knockouts. Together, these findings suggest that pseudouridines play different roles in ribosomal activity within the cell depending on the growth conditions, and provide selective advantages for E. coli when competing for limited resources

Investigating the function of snR30 during ribosome biogenesis

snR30 is an essential and atypical H/ACA box guide RNA conserved across all eukaryotes. In general, the H/ACA ribonucleic protein (RNP) complex is formed by Cbf5, Gar1, Nop10, and Nhp2 associating with an H/ACA guide RNA. This complex is typically involved in the conversion of uridine to pseudouridine. The snR30 RNP, however, is essential for processing of precursor ribosomal RNA (rRNA) at three sites. These cleavages are required for production of mature 18S rRNA which is necessary for ribosome biogenesis. snR30 differs from other H/ACA guide RNAs in that the binding of the target RNA occurs in the lower half of the unpaired pocket within the terminal hairpin compared to traditional H/ACA RNAs. Additional proteins including Utp23, Utp24, Rrp7, Rok1, and others interact either with snR30 or snR30-interacting proteins. This project aims at reconstituting and characterizing a purified snR30 RNP complex to study the most critical function of H/ACA RNPs during ribosome biogenesis. The H/ACA core protein complex Cbf5-Nop10-Gar1 was purified using glutathione- and Nickel-sepharose chromatography. Synthesis and purification of RNA was achieved through a series of different techniques. First, purified yeast genomic DNA was used for the amplification of snR30 and 35S rRNA. Sequences were cloned into a plasmid. Next, PCR products of these sequences were used as templates in in vitro transcription to generate RNA. Finally, short target RNA comprising a fragment of rRNA was purified using anion exchange chromatography while snR30 was purified by size exclusion chromatography. The target RNA was fluorescently labeled with a fluorescein dye at the oxidized 3’ end. With these components, it now becomes possible to study how snR30 interacts with the H/ACA proteins and how the snR30 RNP complex binds to and dissociates from target RNA, e.g by determining the affinity for rRNA. *Indicates presente

Regulating gene expression through targeted RNA modification

Pseudouridylation refers to the isomerization of uridine to pseudouridine in RNA and is catalyzed by enzymes known as pseudouridine synthases. In eukaryotes, pseudouridylation of rRNA is primarily directed by a complex of proteins and a box H/ACA guide RNA. The guide RNA specifies a target by forming transient base pair interactions on either side of the target uridine, although other standalone pseudouridine synthases may employ different targeting mechanisms. With this project, we aim to modify naturally occurring guide RNAs to allow for specific targeting in a location different from the original. Pseudouridylation of a premature stop codon in mRNA results in translational readthrough and production of a functional protein. Here, we utilize the CUP1 gene from baker’s yeast as a model gene to be targeted by pseudouridylation that enables survival in the presence of high copper concentrations. First, we have generated and purified two guide RNAs never previously worked with in our lab to test their effectiveness at targeting a specific uridine. Also, the proteins associating with these H/ACA guide RNAs have been expressed and purified for biochemical investigations. Second, a premature stop codon was introduced at the ninth codon of the CUP1 gene (Q9UAA). As a control to mimic the presence of a pseudouridylated stop codon, we have generated the mutants cup1p(Q9S) and cup1p(Q9T) mimicking readthrough of the premature stop codon and tested their ability to chelate copper in vivo. Interestingly, these experiments revealed that not all premature stop codons can be rescued by pseudouridylation. Current work focuses on the insertion of test sequences harboring premature termination codons upstream of CUP1. These test sequences are predicted to be targeted by known pseudouridine synthases. Ultimately, this experimental system will identify which sequences harboring premature stop codons can be targeted by pseudouridylation to re-activate gene expression. This new system to regulate gene expression has applications in bioengineering and possibly the treatment of inherited diseases resulting from mutations that cause premature stop codons. *Indicates presente

Detecting chemical modifications of Ribonucleic Acid

Transfer Ribonucleic acid (tRNA) is one of the highly chemically modified RNA in the cell, and the isomerization of uridine to pseudouridine (ᴪ) is the most abundant RNA modification found in all domains of life. Although the function of RNA modification is not yet fully understood, the chemical changes in RNA have been implicated in cellular fitness and development of genetic diseases. For example, the bacterial proteins TruA and RluA are both pseudouridine synthases which catalyze the isomerization of uridine to pseudouridine. TruA is capable of modifying several nearby sites in tRNAs while RluA modifies different RNAs at positions with a shared sequence and structure. These modifications are conserved in all forms of life, but little is known about their function. The objective of this research project was to modify tRNA using modification enzymes and to establish a method to detect multiple RNA modifications at once using High Performance Liquid Chromatography (HPLC). Using a reverse phase chromatography column will allow to separate samples based on hydrophobicity. On one side, tRNA was successfully in vitro transcribed, purified and digested into nucleosides, which were then separated using the HPLC column. On the other side, TruA and RluA wild-type proteins were successfully overexpressed and purified, in order to prepare modified tRNA. In the future, this system will enable us to detect and quantify the presence of pseudouridine as well as other chemical modifications in any type of RNA. This tool will thereby greatly advance research in the Kothe lab to understand the mechanisms and functions of RNA modifications. *Indicates presente