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Methods in Molecular Biology (Clifton,... 2024The structure of RNA molecules is absolutely critical to their functions in a biological system. RNA structure is dynamic and changes in response to cellular needs....
The structure of RNA molecules is absolutely critical to their functions in a biological system. RNA structure is dynamic and changes in response to cellular needs. Within the last few decades, there has been an increased interest in studying the structure of RNA molecules and how they change to support the needs of the cell in different conditions. Selective 2'-hydroxyl acylation-based mutational profiling using high-throughput sequencing is a powerful method to predict the secondary structure of RNA molecules both in vivo and in immunopurified samples. Selective 2'-hydroxyl acylation-based mutational profiling using high-throughput sequencing works by adding bulky groups onto accessible "flexible" bases in an RNA molecule that are not involved in any base-pairing or RNA-protein interactions. When the RNA is reverse transcribed into cDNA, the bulky groups are incorporated as base mutations, which can be compared to an unmodified control to identify the locations of flexible bases. The comparison of sequence data between modified and unmodified samples allows the computer software program (developed to generate reactivity profiles) to generate RNA secondary structure models. These models can be compared in a variety of conditions to determine how specific stimuli influence RNA secondary structures.
Topics: RNA Folding; RNA; Mutation; High-Throughput Nucleotide Sequencing; Nucleic Acid Conformation; Software; Acylation
PubMed: 38907926
DOI: 10.1007/978-1-0716-3918-4_20 -
Bioinformatics (Oxford, England) Sep 2023Understanding RNA folding at the level of secondary structures can give important insights concerning the function of a molecule. We are interested to learn how...
MOTIVATION
Understanding RNA folding at the level of secondary structures can give important insights concerning the function of a molecule. We are interested to learn how secondary structures change dynamically during transcription, as well as whether particular secondary structures form already during or only after transcription. While different approaches exist to simulate cotranscriptional folding, the current strategies for visualization are lagging behind. New, more suitable approaches are necessary to help with exploring the generated data from cotranscriptional folding simulations.
RESULTS
We present DrForna, an interactive visualization app for viewing the time course of a cotranscriptional RNA folding simulation. Specifically, users can scroll along the time axis and see the population of structures that are present at any particular time point.
AVAILABILITY AND IMPLEMENTATION
DrForna is a JavaScript project available on Github at https://github.com/ViennaRNA/drforna and deployed at https://viennarna.github.io/drforna.
Topics: Computer Simulation; RNA Folding
PubMed: 37682108
DOI: 10.1093/bioinformatics/btad555 -
Accounts of Chemical Research Oct 2023The function of cellular RNA is modulated by a host of post-transcriptional chemical modifications installed by dedicated RNA-modifying enzymes. RNA modifications are...
The function of cellular RNA is modulated by a host of post-transcriptional chemical modifications installed by dedicated RNA-modifying enzymes. RNA modifications are widespread in biology, occurring in all kingdoms of life and in all classes of RNA molecules. They regulate RNA structure, folding, and protein-RNA interactions, and have important roles in fundamental gene expression processes involving mRNA, tRNA, rRNA, and other types of RNA species. Our understanding of RNA modifications has advanced considerably; however, there are still many outstanding questions regarding the distribution of modifications across all RNA transcripts and their biological function. One of the major challenges in the study of RNA modifications is the lack of sequencing methods for the transcriptome-wide mapping of different RNA-modification structures. Furthermore, we lack general strategies to characterize RNA-modifying enzymes and RNA-modification reader proteins. Therefore, there is a need for new approaches to enable integrated studies of RNA-modification chemistry and biology.In this Account, we describe our development and application of chemoproteomic strategies for the study of RNA-modification-associated proteins. We present two orthogonal methods based on nucleoside and oligonucleotide chemical probes: 1) RNA-mediated activity-based protein profiling (RNABPP), a metabolic labeling strategy based on reactive modified nucleoside probes to profile RNA-modifying enzymes in cells and 2) photo-cross-linkable diazirine-containing synthetic oligonucleotide probes for identifying RNA-modification reader proteins.We use RNABPP with C5-modified cytidine and uridine nucleosides to capture diverse RNA-pyrimidine-modifying enzymes including methyltransferases, dihydrouridine synthases, and RNA dioxygenase enzymes. Metabolic labeling facilitates the mechanism-based cross-linking of RNA-modifying enzymes with their native RNA substrates in cells. Covalent RNA-protein complexes are then isolated by denaturing oligo(dT) pulldown, and cross-linked proteins are identified by quantitative proteomics. Once suitable modified nucleosides have been identified as mechanism-based proteomic probes, they can be further deployed in transcriptome-wide sequencing experiments to profile the substrates of RNA-modifying enzymes at nucleotide resolution. Using 5-fluorouridine-mediated RNA-protein cross-linking and sequencing, we analyzed the substrates of human dihydrouridine synthase DUS3L. 5-Ethynylcytidine-mediated cross-linking enabled the investigation of ALKBH1 substrates. We also characterized the functions of these RNA-modifying enzymes in human cells by using genetic knockouts and protein translation reporters.We profiled RNA readers for -methyladenosine (mA) and -methyladenosine (mA) using a comparative proteomic workflow based on diazirine-containing modified oligonucleotide probes. Our approach enables quantitative proteome-wide analysis of the preference of RNA-binding proteins for modified nucleotides across a range of affinities. Interestingly, we found that YTH-domain proteins YTHDF1/2 can bind to both mA and mA to mediate transcript destabilization. Furthermore, mA also inhibits stress granule proteins from binding to RNA.Taken together, we demonstrate the application of chemical probing strategies, together with proteomic and transcriptomic workflows, to reveal new insights into the biological roles of RNA modifications and their associated proteins.
Topics: Humans; Nucleosides; Adenosine; Proteomics; Diazomethane; Oligonucleotide Probes; RNA; AlkB Homolog 1, Histone H2a Dioxygenase
PubMed: 37733063
DOI: 10.1021/acs.accounts.3c00450 -
Briefings in Bioinformatics Nov 2023RNA biology has risen to prominence after a remarkable discovery of diverse functions of noncoding RNA (ncRNA). Most untranslated transcripts often exert their... (Review)
Review
RNA biology has risen to prominence after a remarkable discovery of diverse functions of noncoding RNA (ncRNA). Most untranslated transcripts often exert their regulatory functions into RNA-RNA complexes via base pairing with complementary sequences in other RNAs. An interplay between RNAs is essential, as it possesses various functional roles in human cells, including genetic translation, RNA splicing, editing, ribosomal RNA maturation, RNA degradation and the regulation of metabolic pathways/riboswitches. Moreover, the pervasive transcription of the human genome allows for the discovery of novel genomic functions via RNA interactome investigation. The advancement of experimental procedures has resulted in an explosion of documented data, necessitating the development of efficient and precise computational tools and algorithms. This review provides an extensive update on RNA-RNA interaction (RRI) analysis via thermodynamic- and comparative-based RNA secondary structure prediction (RSP) and RNA-RNA interaction prediction (RIP) tools and their general functions. We also highlighted the current knowledge of RRIs and the limitations of RNA interactome mapping via experimental data. Then, the gap between RSP and RIP, the importance of RNA homologues, the relationship between pseudoknots, and RNA folding thermodynamics are discussed. It is hoped that these emerging prediction tools will deepen the understanding of RNA-associated interactions in human diseases and hasten treatment processes.
Topics: Humans; RNA; Computational Biology; RNA, Untranslated; Genomics; RNA Folding; Nucleic Acid Conformation; Algorithms
PubMed: 38040490
DOI: 10.1093/bib/bbad421 -
Nature Communications Nov 2023T-box riboswitches are unique riboregulators where gene regulation is mediated through interactions between two highly structured RNAs. Despite extensive structural...
T-box riboswitches are unique riboregulators where gene regulation is mediated through interactions between two highly structured RNAs. Despite extensive structural insights, how RNA-RNA interactions drive the folding and structural transitions of T-box to achieve functional conformations remains unclear. Here, by combining SAXS, single-molecule FRET and computational modeling, we elaborate the folding energy landscape of a translational T-box aptamer consisting of stems I, II and IIA/B, which Mg-induced global folding and tRNA binding are cooperatively coupled. smFRET measurements reveal that high Mg stabilizes IIA/B and its stacking on II, which drives the pre-docking of I and II into a competent conformation, subsequent tRNA binding promotes docking of I and II to form a high-affinity tRNA binding groove, of which the essentiality of IIA/B and S-turn in II is substantiated with mutational analysis. We highlight a delicate balance among Mg, the intra- and intermolecular RNA-RNA interactions in modulating RNA folding and function.
Topics: Riboswitch; Nucleic Acid Conformation; Scattering, Small Angle; X-Ray Diffraction; RNA, Transfer; RNA Folding; RNA
PubMed: 37968328
DOI: 10.1038/s41467-023-43232-z -
Nature Communications Sep 2023The transcriptional intermediates of RNAs fold into secondary structures with multiple regulatory roles, yet the details of such cotranscriptional RNA folding are...
The transcriptional intermediates of RNAs fold into secondary structures with multiple regulatory roles, yet the details of such cotranscriptional RNA folding are largely unresolved in eukaryotes. Here, we present eSPET-seq (Structural Probing of Elongating Transcripts in eukaryotes), a method to assess the cotranscriptional RNA folding in Saccharomyces cerevisiae. Our study reveals pervasive structural transitions during cotranscriptional folding and overall structural similarities between nascent and mature RNAs. Furthermore, a combined analysis with genome-wide R-loop and mutation rate approximations provides quantitative evidence for the antimutator effect of nascent RNA folding through competitive inhibition of the R-loops, known to facilitate transcription-associated mutagenesis. Taken together, we present an experimental evaluation of cotranscriptional folding in eukaryotes and demonstrate the antimutator effect of nascent RNA folding. These results suggest genome-wide coupling between the processing and transmission of genetic information through RNA folding.
Topics: Antimutagenic Agents; Eukaryotic Cells; Mutagenesis; RNA; Saccharomyces cerevisiae
PubMed: 37730811
DOI: 10.1038/s41467-023-41550-w -
Journal of Fungi (Basel, Switzerland) Nov 2023Moonlighting proteins combine multiple functions in one polypeptide chain. An increasing number of moonlighting proteins are being found in diverse fungal taxa that vary... (Review)
Review
Moonlighting proteins combine multiple functions in one polypeptide chain. An increasing number of moonlighting proteins are being found in diverse fungal taxa that vary in morphology, life cycle, and ecological niche. In this mini-review we discuss examples of moonlighting proteins in fungi that illustrate their roles in transcription and DNA metabolism, translation and RNA metabolism, protein folding, and regulation of protein function, and their interaction with other cell types and host proteins.
PubMed: 37998912
DOI: 10.3390/jof9111107 -
Current Opinion in Structural Biology Feb 2024During protein synthesis, the growing nascent peptide chain moves inside the polypeptide exit tunnel of the ribosome from the peptidyl transferase center towards the... (Review)
Review
During protein synthesis, the growing nascent peptide chain moves inside the polypeptide exit tunnel of the ribosome from the peptidyl transferase center towards the exit port where it emerges into the cytoplasm. The ribosome defines the unique energy landscape of the pioneering round of protein folding. The spatial confinement and the interactions of the nascent peptide with the tunnel walls facilitate formation of secondary structures, such as α-helices. The vectorial nature of protein folding inside the tunnel favors local intra- and inter-molecular interactions, thereby inducing cotranslational folding intermediates that do not form upon protein refolding in solution. Tertiary structures start to fold in the lower part of the tunnel, where interactions with the ribosome destabilize native protein folds. The present review summarizes the recent progress in understanding the driving forces of nascent protein folding inside the tunnel and at the surface of the ribosome.
Topics: Protein Folding; Ribosomes; Protein Biosynthesis; Proteins; Peptides
PubMed: 38071940
DOI: 10.1016/j.sbi.2023.102740 -
Accounts of Chemical Research Oct 2023Protein biosynthesis is a central process in all living cells that is catalyzed by a complex molecular machine─the ribosome. This process is termed translation because... (Review)
Review
Protein biosynthesis is a central process in all living cells that is catalyzed by a complex molecular machine─the ribosome. This process is termed translation because the language of nucleotides in mRNAs is translated into the language of amino acids in proteins. Transfer RNA (tRNA) molecules charged with amino acids serve as adaptors and recognize codons of mRNA in the decoding center while simultaneously the individual amino acids are assembled into a peptide chain in the peptidyl transferase center (PTC). As the nascent peptide emerges from the ribosome, it is threaded through a long tunnel referred to as a nascent peptide exit tunnel (NPET). The PTC and NPET are the sites targeted by many antibiotics and are thus of tremendous importance from a biomedical perspective and for drug development in the pharmaceutical industry.Researchers have achieved much progress in characterizing ribosomal translation at the molecular level; an impressive number of high-resolution structures of different functional and inhibited states of the ribosome are now available. These structures have significantly contributed to our understanding of how the ribosome interacts with its key substrates, namely, mRNA, tRNAs, and translation factors. In contrast, much less is known about the mechanisms of how small molecules, especially antibiotics, affect ribosomal protein synthesis. This mainly concerns the structural basis of small molecule-NPET interference with cotranslational protein folding and the regulation of protein synthesis. Growing biochemical evidence suggests that NPET plays an active role in the regulation of protein synthesis.Much-needed progress in this field is hampered by the fact that during the preparation of ribosome complexes for structural studies (i.e., X-ray crystallography, cryoelectron microscopy, and NMR spectroscopy) the aminoacyl- or peptidyl-tRNAs are unstable and become hydrolyzed. A solution to this problem is the application of hydrolysis-resistant mimics of aminoacyl- or peptidyl-tRNAs.In this Account, we present an overview of synthetic methods for the generation of peptidyl-tRNA analogs. Modular approaches have been developed that combine () RNA and peptide solid-phase synthesis on 3'-aminoacylamino-adenosine resins, () native chemical ligations and Staudinger ligations, () tailoring of tRNAs by the selective cleavage of natural native tRNAs with DNAzymes followed by reassembly with enzymatic ligation to synthetic peptidyl-RNA fragments, and () enzymatic tailing and cysteine charging of the tRNA to obtain modified CCA termini of a tRNA that are chemically ligated to the peptide moiety of interest. With this arsenal of tools, in principle, any desired sequence of a stably linked peptidyl-tRNA mimic is accessible. To underline the significance of the synthetic conjugates, we briefly point to the most critical applications that have shed new light on the molecular mechanisms underlying the context-specific activity of ribosome-targeting antibiotics, ribosome-dependent incorporation of multiple consecutive proline residues, the incorporation of d-amino acids, and tRNA mischarging.Furthermore, we discuss new types of stably charged tRNA analogs, relying on triazole- and squarate (instead of amide)-linked conjugates. Those have pushed forward our mechanistic understanding of nonribosomal peptide synthesis, where aminoacyl-tRNA-dependent enzymes are critically involved in various cellular processes in primary and secondary metabolism and in bacterial cell wall synthesis.
Topics: Cryoelectron Microscopy; RNA, Transfer; RNA; Amino Acids; Protein Biosynthesis; Peptides; Anti-Bacterial Agents; RNA, Messenger; Biology
PubMed: 37728742
DOI: 10.1021/acs.accounts.3c00412 -
Trends in Biochemical Sciences Jul 2023The mitochondrial ribosome (mitoribosome) is a multicomponent machine that has unique structural features. Biogenesis of the human mitoribosome includes correct... (Review)
Review
The mitochondrial ribosome (mitoribosome) is a multicomponent machine that has unique structural features. Biogenesis of the human mitoribosome includes correct maturation and folding of the mitochondria-encoded RNA components (12S and 16S mt-rRNAs, and mt-tRNAVal) and their assembly together with 82 nucleus-encoded mitoribosomal proteins. This complex process requires the coordinated action of multiple assembly factors. Recent advances in single-particle cryo-electron microscopy (cryo-EM) have provided detailed insights into the specific functions of several mitoribosome assembly factors and have defined their timing. In this review we summarize mitoribosomal small (mtSSU) and large subunit (mtLSU) biogenesis based on structural findings, and we discuss potential crosstalk between mtSSU and mtLSU assembly pathways as well as coordination between mitoribosome biogenesis and other processes involved in mitochondrial gene expression.
Topics: Humans; Cryoelectron Microscopy; Mitochondrial Ribosomes; RNA, Ribosomal, 16S; Mitochondrial Proteins; Ribosomal Proteins
PubMed: 37169615
DOI: 10.1016/j.tibs.2023.04.002