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Nature Reviews. Molecular Cell Biology Jul 2024Many steps of RNA processing occur during transcription by RNA polymerases. Co-transcriptional activities are deemed commonplace in prokaryotes, in which the lack of... (Review)
Review
Many steps of RNA processing occur during transcription by RNA polymerases. Co-transcriptional activities are deemed commonplace in prokaryotes, in which the lack of membrane barriers allows mixing of all gene expression steps, from transcription to translation. In the past decade, an extraordinary level of coordination between transcription and RNA processing has emerged in eukaryotes. In this Review, we discuss recent developments in our understanding of co-transcriptional gene regulation in both eukaryotes and prokaryotes, comparing methodologies and mechanisms, and highlight striking parallels in how RNA polymerases interact with the machineries that act on nascent RNA. The development of RNA sequencing and imaging techniques that detect transient transcription and RNA processing intermediates has facilitated discoveries of transcription coordination with splicing, 3'-end cleavage and dynamic RNA folding and revealed physical contacts between processing machineries and RNA polymerases. Such studies indicate that intron retention in a given nascent transcript can prevent 3'-end cleavage and cause transcriptional readthrough, which is a hallmark of eukaryotic cellular stress responses. We also discuss how coordination between nascent RNA biogenesis and transcription drives fundamental aspects of gene expression in both prokaryotes and eukaryotes.
Topics: Transcription, Genetic; Prokaryotic Cells; DNA-Directed RNA Polymerases; Eukaryotic Cells; Humans; Gene Expression Regulation; Animals; Eukaryota; RNA Splicing; RNA Processing, Post-Transcriptional; RNA
PubMed: 38509203
DOI: 10.1038/s41580-024-00706-2 -
RNA (New York, N.Y.) Sep 2023k-Junctions are elaborated forms of kink turns with an additional helix on the nonbulged strand, thus forming a three-way helical junction. Two were originally...
k-Junctions are elaborated forms of kink turns with an additional helix on the nonbulged strand, thus forming a three-way helical junction. Two were originally identified in the structures of and thiamine pyrophosphate (TPP) riboswitches, and another called DUF-3268 was tentatively identified from sequence information. In this work we show that the and riboswitch k-junctions fold in response to the addition of magnesium or sodium ions, and that atomic mutations that should disrupt key hydrogen bonding interactions greatly impair folding. Using X-ray crystallography, we have determined the structure of the DUF-3268 RNA and thus confirmed that it is a k-junction. It also folds upon the addition of metal ions, though requiring a 40-fold lower concentration of either divalent or monovalent ions. The key difference between the DUF-3268 and riboswitch k-junctions is the lack of nucleotides inserted between G1b and A2b in the former. We show that this insertion is primarily responsible for the difference in folding properties. Finally, we show that the DUF-3268 can functionally substitute for the k-junction in the TPP riboswitch such that the chimera can bind the TPP ligand, although less avidly.
Topics: Riboswitch; Escherichia coli; Arabidopsis; RNA Folding; Thiamine Pyrophosphate; Ions; Nucleic Acid Conformation
PubMed: 37311599
DOI: 10.1261/rna.079678.123 -
Life Science Alliance Jan 2024Protein-RNA complexes exist in many forms within the cell, from stable machines such as the ribosome to transient assemblies like the spliceosome. All protein-RNA... (Review)
Review
Protein-RNA complexes exist in many forms within the cell, from stable machines such as the ribosome to transient assemblies like the spliceosome. All protein-RNA assemblies rely on spatially and temporally coordinated interactions between specific proteins and RNAs to achieve a functional form. RNA folding and structure are often critical for successful protein binding and protein-RNA complex formation. RNA modifications change the chemical nature of a given RNA and often alter its folding kinetics. Both these alterations can affect how and if proteins or other RNAs can interact with the modified RNA and assemble into complexes. N-methyladenosine (mA) is the most common base modification on mRNAs and regulatory noncoding RNAs and has been shown to impact RNA structure and directly modulate protein-RNA interactions. In this review, focusing on the mechanisms and available quantitative information, we discuss first how the METTL3/14 mA writer complex is specifically targeted to RNA assisted by protein-RNA and other interactions to enable site-specific and co-transcriptional RNA modification and, once introduced, how the mA modification affects RNA folding and protein-RNA interactions.
Topics: RNA; RNA, Messenger; RNA, Untranslated; Protein Binding; Ribosomes
PubMed: 37935465
DOI: 10.26508/lsa.202302240 -
Angewandte Chemie (International Ed. in... May 2024Incorporating stimuli-responsive components into RNA constructs provides precise spatiotemporal control over RNA structures and functions. Despite considerable...
Incorporating stimuli-responsive components into RNA constructs provides precise spatiotemporal control over RNA structures and functions. Despite considerable advancements, the utilization of redox-responsive stimuli for the activation of caged RNAs remains scarce. In this context, we present a novel strategy that leverages post-synthetic acylation coupled with redox-responsive chemistry to exert control over RNA. To achieve this, we design and synthesize a series of acylating reagents specifically tailored for introducing disulfide-containing acyl adducts into the 2'-OH groups of RNA ("cloaking"). Our data reveal that these acyl moieties can be readily appended, effectively blocking RNA catalytic activity and folding. We also demonstrate the traceless release and reactivation of caged RNAs ("uncloaking") through reducing stimuli. By employing this strategy, RNA exhibits rapid cellular uptake, effective distribution and activation in the cytosol without lysosomal entrapment. We anticipate that our methodology will be accessible to laboratories engaged in RNA biology and holds promise as a versatile platform for RNA-based applications.
Topics: Acylation; Oxidation-Reduction; RNA; Humans; Disulfides
PubMed: 38480851
DOI: 10.1002/anie.202402178 -
Nature Communications Oct 2023YTHDF2 has been extensively studied and typified as an RNA-binding protein that specifically recognizes and destabilizes RNAs harboring N-methyladenosine (mA), the most...
YTHDF2 has been extensively studied and typified as an RNA-binding protein that specifically recognizes and destabilizes RNAs harboring N-methyladenosine (mA), the most prevalent internal modification found in eukaryotic RNAs. In this study, we unravel the mA-independent role of YTHDF2 in the formation of an aggresome, where cytoplasmic protein aggregates are selectively sequestered upon failure of protein homeostasis mediated by the ubiquitin-proteasome system. Downregulation of YTHDF2 in HeLa cells reduces the circularity of aggresomes and the rate of movement of misfolded polypeptides, inhibits aggresome formation, and thereby promotes cellular apoptosis. Mechanistically, YTHDF2 is recruited to a misfolded polypeptide-associated complex composed of UPF1, CTIF, eEF1A1, and DCTN1 through its interaction with UPF1. Subsequently, YTHDF2 increases the interaction between the dynein motor protein and the misfolded polypeptide-associated complex, facilitating the diffusion dynamics of the movement of misfolded polypeptides toward aggresomes. Therefore, our data reveal that YTHDF2 is a cellular factor involved in protein quality control.
Topics: Humans; Cytoplasm; Dyneins; HeLa Cells; Peptides; Protein Folding; RNA Helicases; RNA-Binding Proteins; Trans-Activators; Transcription Factors; Proteolysis; Organelles
PubMed: 37803021
DOI: 10.1038/s41467-023-42015-w -
Current Opinion in Structural Biology Jun 2024In the last two decades, our existing notion that most foldable proteins have a unique native state has been challenged by the discovery of metamorphic proteins, which... (Review)
Review
In the last two decades, our existing notion that most foldable proteins have a unique native state has been challenged by the discovery of metamorphic proteins, which reversibly interconvert between multiple, sometimes highly dissimilar, native states. As the number of known metamorphic proteins increases, several computational and experimental strategies have emerged for gaining insights about their refolding processes and identifying unknown metamorphic proteins amongst the known proteome. In this review, we describe the current advances in biophysically and functionally ascertaining the structural interconversions of metamorphic proteins and how coevolution can be harnessed to identify novel metamorphic proteins from sequence information. We also discuss the challenges and ongoing efforts in using artificial intelligence-based protein structure prediction methods to discover metamorphic proteins and predict their corresponding three-dimensional structures.
Topics: Proteins; Protein Folding; Protein Conformation; Models, Molecular; Humans; Artificial Intelligence
PubMed: 38537533
DOI: 10.1016/j.sbi.2024.102807 -
The Journal of Chemical Physics May 2024The folding of RNA and DNA strands plays crucial roles in biological systems and bionanotechnology. However, studying these processes with high-resolution numerical...
The folding of RNA and DNA strands plays crucial roles in biological systems and bionanotechnology. However, studying these processes with high-resolution numerical models is beyond current computational capabilities due to the timescales and system sizes involved. In this article, we present a new coarse-grained model for investigating the folding dynamics of nucleic acids. Our model represents three nucleotides with a patchy particle and is parameterized using well-established nearest-neighbor models. Thanks to the reduction of degrees of freedom and to a bond-swapping mechanism, our model allows for simulations at timescales and length scales that are currently inaccessible to more detailed models. To validate the performance of our model, we conducted extensive simulations of various systems: We examined the thermodynamics of DNA hairpins, capturing their stability and structural transitions, the folding of an MMTV pseudoknot, which is a complex RNA structure involved in viral replication, and also explored the folding of an RNA tile containing a k-type pseudoknot. Finally, we evaluated the performance of the new model in reproducing the melting temperatures of oligomers and the dependence on the toehold length of the displacement rate in toehold-mediated displacement processes, a key reaction used in molecular computing. All in all, the successful reproduction of experimental data and favorable comparisons with existing coarse-grained models validate the effectiveness of the new model.
Topics: RNA; DNA; Nucleic Acid Conformation; Thermodynamics; Molecular Dynamics Simulation; Models, Molecular; RNA Folding
PubMed: 38814009
DOI: 10.1063/5.0202829 -
Observation of coordinated RNA folding events by systematic cotranscriptional RNA structure probing.Nature Communications Nov 2023RNA begins to fold as it is transcribed by an RNA polymerase. Consequently, RNA folding is constrained by the direction and rate of transcription. Understanding how RNA...
RNA begins to fold as it is transcribed by an RNA polymerase. Consequently, RNA folding is constrained by the direction and rate of transcription. Understanding how RNA folds into secondary and tertiary structures therefore requires methods for determining the structure of cotranscriptional folding intermediates. Cotranscriptional RNA chemical probing methods accomplish this by systematically probing the structure of nascent RNA that is displayed from an RNA polymerase. Here, we describe a concise, high-resolution cotranscriptional RNA chemical probing procedure called variable length Transcription Elongation Complex RNA structure probing (TECprobe-VL). We demonstrate the accuracy and resolution of TECprobe-VL by replicating and extending previous analyses of ZTP and fluoride riboswitch folding and mapping the folding pathway of a ppGpp-sensing riboswitch. In each system, we show that TECprobe-VL identifies coordinated cotranscriptional folding events that mediate transcription antitermination. Our findings establish TECprobe-VL as an accessible method for mapping cotranscriptional RNA folding pathways.
Topics: RNA Folding; RNA; Nucleic Acid Conformation; Riboswitch; Transcription, Genetic; DNA-Directed RNA Polymerases
PubMed: 38030633
DOI: 10.1038/s41467-023-43395-9 -
International Journal of Molecular... Jan 2024Baculoviruses are viral pathogens that infect different species of Lepidoptera, Diptera, and Hymenoptera, with a global distribution. Due to their biological... (Review)
Review
Baculoviruses are viral pathogens that infect different species of Lepidoptera, Diptera, and Hymenoptera, with a global distribution. Due to their biological characteristics and the biotechnological applications derived from these entities, the family is an important subject of study and manipulation in the natural sciences. With the advent of RNA interference mechanisms, the presence of baculoviral genes that do not code for proteins but instead generate transcripts similar to microRNAs (miRNAs) has been described. These miRNAs are functionally associated with the regulation of gene expression, both in viral and host sequences. This article provides a comprehensive review of miRNA biogenesis, function, and characterization in general, with a specific focus on those identified in baculoviruses. Furthermore, it delves into the specific roles of baculoviral miRNAs in regulating viral and host genes and presents structural and thermodynamic stability studies that are useful for detecting shared characteristics with predictive utility. This review aims to expand our understanding of the baculoviral miRNAome, contributing to improvements in the production of baculovirus-based biopesticides, management of resistance phenomena in pests, enhancement of recombinant protein production systems, and development of diverse and improved BacMam vectors to meet biomedical demands.
Topics: MicroRNAs; Baculoviridae; RNA Interference; Biological Control Agents; Biotechnology
PubMed: 38203774
DOI: 10.3390/ijms25010603 -
Wiley Interdisciplinary Reviews. RNA 2023Cell functionality relies on the existing pool of proteins and their folding into functional conformations. This is achieved through the regulation of protein synthesis,... (Review)
Review
Cell functionality relies on the existing pool of proteins and their folding into functional conformations. This is achieved through the regulation of protein synthesis, which requires error-free mRNAs and ribosomes. Ribosomes are quality control hubs for mRNAs and proteins. Problems during translation elongation slow down the decoding rate, leading to ribosome halting and the eventual collision with the next ribosome. Collided ribosomes form a specific disome structure recognized and solved by ribosome quality control (RQC) mechanisms. RQC pathways orchestrate the degradation of the problematic mRNA by no-go decay and the truncated nascent peptide, the repression of translation initiation, and the recycling of the stalled ribosomes. All these events maintain protein homeostasis and return valuable ribosomes to translation. As such, cell homeostasis and function are maintained at the mRNA level by preventing the production of aberrant or unnecessary proteins. It is becoming evident that the crosstalk between RQC and the protein homeostasis network is vital for cell function, as the absence of RQC components leads to the activation of stress response and neurodegenerative diseases. Here, we review the molecular events of RQC discovered through well-designed stalling reporters. Given the impact of RQC in proteostasis, we discuss the relevance of identifying endogenous mRNA regulated by RQC and their preservation in stress conditions. This article is categorized under: RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms Translation > Regulation.
Topics: Proteostasis; Saccharomyces cerevisiae Proteins; Saccharomyces cerevisiae; Ribosomes; Protein Biosynthesis; RNA, Messenger
PubMed: 37488089
DOI: 10.1002/wrna.1809