-
Nucleic Acids Research Jan 2022MicroRNAs (miRNAs) are short endogenously expressed RNAs that have the potential to regulate the expression of any RNA. This potential has led to the publication of... (Review)
Review
MicroRNAs (miRNAs) are short endogenously expressed RNAs that have the potential to regulate the expression of any RNA. This potential has led to the publication of several thousand papers each year connecting miRNAs to many different genes and human diseases. By contrast, relatively few papers appear that investigate the molecular mechanism used by miRNAs. There is a disconnect between rigorous understanding of mechanism and the extraordinary diversity of reported roles for miRNAs. Consequences of this disconnect include confusion about the assumptions underlying the basic science of human miRNAs and slow development of therapeutics that target miRNAs. Here, we present an overview of investigations into miRNAs and their impact on gene expression. Progress in our understanding of miRNAs would be aided by a greater focus on the mechanism of miRNAs and a higher burden of evidence on researchers who seek to link expression of a particular miRNA to a biological phenotype.
Topics: Animals; Antagomirs; Base Pairing; Base Sequence; Clinical Studies as Topic; Drug Development; Drug Evaluation, Preclinical; Gene Silencing; Genetic Variation; Humans; MicroRNAs; RNA Interference; Structure-Activity Relationship; Treatment Outcome
PubMed: 34967419
DOI: 10.1093/nar/gkab1256 -
Cell Oct 2022The target DNA specificity of the CRISPR-associated genome editor nuclease Cas9 is determined by complementarity to a 20-nucleotide segment in its guide RNA. However,...
The target DNA specificity of the CRISPR-associated genome editor nuclease Cas9 is determined by complementarity to a 20-nucleotide segment in its guide RNA. However, Cas9 can bind and cleave partially complementary off-target sequences, which raises safety concerns for its use in clinical applications. Here, we report crystallographic structures of Cas9 bound to bona fide off-target substrates, revealing that off-target binding is enabled by a range of noncanonical base-pairing interactions within the guide:off-target heteroduplex. Off-target substrates containing single-nucleotide deletions relative to the guide RNA are accommodated by base skipping or multiple noncanonical base pairs rather than RNA bulge formation. Finally, PAM-distal mismatches result in duplex unpairing and induce a conformational change in the Cas9 REC lobe that perturbs its conformational activation. Together, these insights provide a structural rationale for the off-target activity of Cas9 and contribute to the improved rational design of guide RNAs and off-target prediction algorithms.
Topics: RNA, Guide, CRISPR-Cas Systems; CRISPR-Cas Systems; Endonucleases; Base Pairing; Nucleotides; Gene Editing
PubMed: 36306733
DOI: 10.1016/j.cell.2022.09.026 -
Molecular Cell Apr 2020Post-transcriptional mechanisms regulate the stability and, hence, expression of coding and noncoding RNAs. Sequence-specific features within the 3' untranslated region...
Post-transcriptional mechanisms regulate the stability and, hence, expression of coding and noncoding RNAs. Sequence-specific features within the 3' untranslated region (3' UTR) often direct mRNAs for decay. Here, we characterize a genome-wide RNA decay pathway that reduces the half-lives of mRNAs based on overall 3' UTR structure formed by base pairing. The decay pathway is independent of specific single-stranded sequences, as regulation is maintained in both the original and reverse complement orientation. Regulation can be compromised by reducing the overall structure by fusing the 3' UTR with an unstructured sequence. Mutating base-paired RNA regions can also compromise this structure-mediated regulation, which can be restored by re-introducing base-paired structures of different sequences. The decay pathway requires the RNA-binding protein UPF1 and its associated protein G3BP1. Depletion of either protein increased steady-state levels of mRNAs with highly structured 3' UTRs as well as highly structured circular RNAs. This structure-dependent mechanism therefore enables cells to selectively regulate coding and noncoding RNAs.
Topics: 3' Untranslated Regions; Base Pairing; Cell Line; DNA Helicases; Gene Expression Regulation; Humans; Poly-ADP-Ribose Binding Proteins; RNA Helicases; RNA Recognition Motif Proteins; RNA Stability; RNA, Circular; RNA, Messenger; Trans-Activators
PubMed: 32017897
DOI: 10.1016/j.molcel.2020.01.021 -
Biochemistry Jun 2019Steve Benner and collaborators have recently reported an analysis of DNA containing eight nucleotide letters, the four natural letters (dG, dC, dA, and dT) and four...
Steve Benner and collaborators have recently reported an analysis of DNA containing eight nucleotide letters, the four natural letters (dG, dC, dA, and dT) and four additional letters (dP, dZ, dS, and dB). Their analysis demonstrates that the additional letters do not perturb the structure or stability of the base pairs formed between the natural letters and, remarkably, that the new base pairs, dP-dZ and dS-dB, behave virtually identically to the natural base pairs. This unprecedented result convincingly demonstrates that the thermodynamic and structural behavior previously thought to be the purview of only natural DNA is in fact not unique and can be imparted to suitably designed synthetic components. In addition, the first evidence that the eight-letter DNA can be transcribed into RNA by a mutant RNA polymerase is presented, paving the way for the transfer of more information from one biopolymer to another. Along with others working to develop unnatural DNA base pairs for both in vitro and in vivo applications, this work represents an important step toward the expansion of the genetic alphabet, a central goal of synthetic biology, and has profound implications for our understanding of the molecules and forces that can make life possible.
Topics: Base Pairing; DNA; DNA-Directed RNA Polymerases; Nucleotides; Synthetic Biology
PubMed: 31117391
DOI: 10.1021/acs.biochem.9b00274 -
Current Opinion in Chemical Biology Aug 2020The four canonical bases that make up genomic DNA are subject to a variety of chemical modifications in living systems. Recent years have witnessed the discovery of... (Review)
Review
The four canonical bases that make up genomic DNA are subject to a variety of chemical modifications in living systems. Recent years have witnessed the discovery of various new modified bases and of the enzymes responsible for their processing. Here, we review the range of DNA base modifications currently known and recent advances in chemical methodology that have driven progress in this field, in particular regarding their detection and sequencing. Elucidating the cellular functions of modifications remains an ongoing challenge; we discuss recent contributions to this area before exploring their relevance in medicine.
Topics: Animals; Base Pairing; Chromatography, Liquid; DNA; Epigenesis, Genetic; Humans; Mass Spectrometry; Sequence Analysis, DNA
PubMed: 32145439
DOI: 10.1016/j.cbpa.2020.01.014 -
Biopolymers Jan 2021Chemical modifications on RNA can regulate fundamental biological processes. Recent efforts have illuminated the chemical diversity of posttranscriptional... (Review)
Review
Chemical modifications on RNA can regulate fundamental biological processes. Recent efforts have illuminated the chemical diversity of posttranscriptional ("epitranscriptomic") modifications on eukaryotic messenger RNA and have begun to elucidate their biological roles. In this review, we discuss our current molecular understanding of epitranscriptomic RNA modifications and their effects on gene expression. In particular, we highlight the role of modifications in mediating RNA-protein interactions, RNA structure, and RNA-RNA base pairing, and how these macromolecular interactions control biological processes in the cell.
Topics: Adenosine; Base Pairing; Cytidine; Gene Expression Regulation; RNA; RNA Processing, Post-Transcriptional; RNA, Messenger
PubMed: 33001446
DOI: 10.1002/bip.23403 -
Journal of Experimental Botany May 2021The majority of the genome is transcribed to RNA in living organisms. RNA transcripts can form astonishing arrays of secondary and tertiary structures via Watson-Crick,... (Review)
Review
The majority of the genome is transcribed to RNA in living organisms. RNA transcripts can form astonishing arrays of secondary and tertiary structures via Watson-Crick, Hoogsteen, or wobble base pairing. In vivo, RNA folding is not a simple thermodynamic event of minimizing free energy. Instead, the process is constrained by transcription, RNA-binding proteins, steric factors, and the microenvironment. RNA secondary structure (RSS) plays myriad roles in numerous biological processes, such as RNA processing, stability, transportation, and translation in prokaryotes and eukaryotes. Emerging evidence has also implicated RSS in RNA trafficking, liquid-liquid phase separation, and plant responses to environmental variations such as temperature and salinity. At molecular level, RSS is correlated with splicing, polyadenylation, protein synthesis, and miRNA biogenesis and functions. In this review, we summarize newly reported methods for probing RSS in vivo and functions and mechanisms of RSS in plant physiology.
Topics: Base Pairing; Biology; Nucleic Acid Conformation; RNA; RNA Processing, Post-Transcriptional; RNA Splicing; RNA, Plant
PubMed: 33484251
DOI: 10.1093/jxb/erab030 -
Current Opinion in Microbiology Apr 2021In the past decades, Caulobacter crescentus has been extensively studied, mostly regarding its dimorphic, asymmetric life cycle. Its distinct mode of reproduction and... (Review)
Review
In the past decades, Caulobacter crescentus has been extensively studied, mostly regarding its dimorphic, asymmetric life cycle. Its distinct mode of reproduction and the need to optimally adapt to ever-changing environmental conditions require tight coordination of gene regulation. Post-transcriptional regulation through non-coding RNAs and RNA-binding proteins constitutes an important layer of expression control in bacteria, but its principles and mechanisms in Caulobacter have only recently been explored. RNA-binding proteins including the RNA chaperone Hfq and ribonuclease RNase E contribute to the activity of regulatory RNAs. Riboswitches and RNA thermometers govern expression of downstream open reading frames, while the small regulatory RNAs CrfA, ChvR and GsrN instead control targets encoded in trans by direct base-pairing interactions.
Topics: Bacterial Proteins; Base Pairing; Caulobacter crescentus; Gene Expression Regulation, Bacterial; RNA, Bacterial; RNA, Messenger; RNA, Small Untranslated
PubMed: 33529919
DOI: 10.1016/j.mib.2021.01.002 -
Cellular and Molecular Life Sciences :... Dec 2021Bacteriophage genomes are the richest source of modified nucleobases of any life form. Of these, 2,6 diaminopurine, which pairs with thymine by forming three hydrogen... (Review)
Review
Bacteriophage genomes are the richest source of modified nucleobases of any life form. Of these, 2,6 diaminopurine, which pairs with thymine by forming three hydrogen bonds violates Watson and Crick's base pairing. 2,6 diaminopurine initially found in the cyanophage S-2L is more widespread than expected and has also been detected in phage infecting Gram-negative and Gram-positive bacteria. The biosynthetic pathway for aminoadenine containing DNA as well as the exclusion of adenine are now elucidated. This example of a natural deviation from the genetic code represents only one of the possibilities explored by nature and provides a proof of concept for the synthetic biology of non-canonical nucleic acids.
Topics: Adenine; Adenine Nucleotides; Bacteriophages; Base Pairing; DNA, Viral; Genome, Viral; Hydrogen Bonding
PubMed: 34910247
DOI: 10.1007/s00018-021-04055-7 -
ChemistryOpen Apr 2020Dynamic and reversible non-covalent interactions endow synthetic systems and materials with smart adaptive functions that allow them to response to diverse stimuli,... (Review)
Review
Dynamic and reversible non-covalent interactions endow synthetic systems and materials with smart adaptive functions that allow them to response to diverse stimuli, interact with external agents, or repair structural defects. Inspired by the outstanding performance and selectivity of DNA in living systems, scientists are increasingly employing Watson-Crick nucleobase pairing to control the structure and properties of self-assembled materials. Two sets of complementary purine-pyrimidine pairs (guanine:cytosine and adenine:thymine(uracil)) are available that provide selective and directional H-bonding interactions, present multiple metal-coordination sites, and exhibit rich redox chemistry. In this review, we highlight several recent examples that profit from these features and employ nucleobase interactions in functional systems and materials, covering the fields of energy/electron transfer, charge transport, adaptive nanoparticles, porous materials, macromolecule self-assembly, or polymeric materials with adhesive or self-healing ability.
Topics: Adenine; Base Pairing; Coordination Complexes; Cytosine; DNA; Electron Transport; Energy Transfer; Guanine; Molecular Conformation; Oxidation-Reduction; Surface Properties; Thymine; Uracil
PubMed: 32257750
DOI: 10.1002/open.201900363