Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells

Post-transcriptional modification of RNA nucleosides occurs in all living organisms. Pseudouridine, the most abundant modified nucleoside in non-coding RNAs, enhances the function of transfer RNA and ribosomal RNA by stabilizing the RNA structure. Messenger RNAs were not known to contain pseudouridine, but artificial pseudouridylation dramatically affects mRNA function—it changes the genetic code by facilitating non-canonical base pairing in the ribosome decoding centre. However, without evidence of naturally occurring mRNA pseudouridylation, its physiological relevance was unclear. Here we present a comprehensive analysis of pseudouridylation in Saccharomyces cerevisiae and human RNAs using Pseudo-seq, a genome-wide, single-nucleotide-resolution method for pseudouridine identification. Pseudo-seq accurately identifies known modification sites as well as many novel sites in non-coding RNAs, and reveals hundreds of pseudouridylated sites in mRNAs. Genetic analysis allowed us to assign most of the new modification sites to one of seven conserved pseudouridine synthases, Pus1–4, 6, 7 and 9. Notably, the majority of pseudouridines in mRNA are regulated in response to environmental signals, such as nutrient deprivation in yeast and serum starvation in human cells. These results suggest a mechanism for the rapid and regulated rewiring of the genetic code through inducible mRNA modifications. Our findings reveal unanticipated roles for pseudouridylation and provide a resource for identifying the targets of pseudouridine synthases implicated in human disease.American Cancer Society (Robbie Sue Mudd Kidney Cancer Research Scholar Grant RSG-13-396-01-RMC)National Institutes of Health (U.S.) (GM094303)National Institutes of Health (U.S.) (GM081399)American Cancer Society. New England Division (Ellison Foundation Postdoctoral Fellowship)American Cancer Society (Postdoctoral Fellowship PF-13-319-01-RMC)National Institutes of Health (U.S.) (Pre-doctoral Training Grant T32GM007287

Application of FTIR Spectroscopy to Analyze RNA Structure

Fourier transform infrared (FTIR) spectroscopy has been widely used for the analysis of both protein and nucleic acid secondary structure. This is one of the vibration spectroscopy methods that are extremely sensitive to any change in molecular structure. While numerous reports describe how to proceed to analyze protein and deoxyribonucleic acid (DNA) structures using FTIR, reports related to the analyses of ribonucleic acids (RNAs) are few. Nevertheless, RNAs are versatile molecules involved in a multitude of roles in the cell. In this chapter, we present applications of FTIR for the structural analysis of RNA, including the analysis of helical parameters and noncanonical base pairing, often found in RNA. The effect of temperature pretreatment, which has a great impact on RNA folding, will also be discussed. © 2020, Springer Science+Business Media, LLC, part of Springer Nature

Pseudouridine synthetase Pus1 of Saccharomyces cerevisiae: kinetic characterisation, tRNA structural requirement and real-time analysis of its complex with tRNA.

International audiencePseudouridine synthetase Pus1 from Saccharomyces cerevisiae is a multisite-specific enzyme that catalyses the formation of pseudouridine residues at different positions in several tRNA transcripts. Recombinant Pus1, tagged with six histidine residues at its N terminus was expressed in Escherichia coli and purified. Transcripts of yeast tRNAValand intronless yeast tRNAIlewere used as substrates to measure pseudouridine formation at position 27. The catalytic parameters Kmand kcatfor tRNAValand tRNAIlewere 420(+/-100) nM and 0.4(+/-0.1) min-1, 740(+/-100) nM and 0.5(+/-0.1) min-1, respectively. Pus1 possesses a general affinity for tRNA, irrespective of whether they are substrates. Its equilibrium dissociation constant ranges from 15 nM for the substrate yeast tRNAValand non-substrate yeast intronless tRNAPhe, to 150 nM for the substrate yeast intronless tRNAIle. The difference in the affinity for the different tRNA species is not reflected in the specific activity of the enzyme, indicating that the binding of Pus1 to tRNA is not the kinetically limiting step. The importance of tertiary base-pairs was investigated with several variants of yeast tRNAs. Although dispensable for activity, both the presence of a D-stem-loop and the presence of a G26.A44 base-pair, near the target uridine U27, are important elements for Pus1 tRNA high affinity recognition. The presence of a G26.A44 base-pair in tRNA increases its association constant rate with Pus1 (ka) by a factor of approximately 100, resulting in a decrease of the overall equilibrium dissociation constant (Kd). The dissociation rate (kd) is the same, independent of the presence of a G26.A44 base-pair in the tRNA. A model describing the interaction of Pus1 with tRNA is proposed

Structure of the H-NS-DNA nucleoprotein complex.

Nucleoid associated proteins (NAPs) play a key role in the compaction and expression of the prokaryotic genome. Here we report the organisation of a major NAP, the protein H-NS on a double stranded DNA fragment. For this purpose we have carried out a small angle neutron scattering study in conjunction with contrast variation to obtain the contributions to the scattering (structure factors) from DNA and H-NS. The H-NS structure factor agrees with a heterogeneous, two-state binding model with sections of the DNA duplex surrounded by protein and other sections having protein bound to the major groove. In the presence of magnesium chloride, we observed a structural rearrangement through a decrease in cross-sectional diameter of the nucleoprotein complex and an increase in fraction of major groove bound H-NS. The two observed binding modes and their modulation by magnesium ions provide a structural basis for H-NS-mediated genome organisation and expression regulation

Sm-like protein Hfq: Location of the ATP-binding site and the effect of ATP on Hfq–RNA complexes

Sm-like proteins are ubiquitous ring-shaped oligomers that exhibit a variety of nucleic acid-binding activities. They have been linked functionally to various cellular events involving RNA, and it is generally believed that their activity is exerted via the passive binding of nucleic acids. Our earlier studies of the Sm-like Escherichia coli protein Hfq provided the first evidence indicating that Hfq is an ATP-binding protein. Using a combination of biochemical and genetic techniques, we have now determined a plausible ATP-binding site in Hfq and tested Hfq's ATP-binding affinity and stoichiometry. The results of RNA footprinting and binding analyses suggest that ATP binding by the Hfq–RNA complex results in its significant destabilization. RNA footprinting indicates deprotection of Hfq-bound RNA tracts in the presence of ATP, suggestive of their release by the protein. The results reported herein broaden the scope of potential in vivo roles for Hfq and other Sm-like proteins