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Annual Review of Biochemistry 2015Precursor messenger RNA (pre-mRNA) splicing is a critical step in the posttranscriptional regulation of gene expression, providing significant expansion of the... (Review)
Review
Precursor messenger RNA (pre-mRNA) splicing is a critical step in the posttranscriptional regulation of gene expression, providing significant expansion of the functional proteome of eukaryotic organisms with limited gene numbers. Split eukaryotic genes contain intervening sequences or introns disrupting protein-coding exons, and intron removal occurs by repeated assembly of a large and highly dynamic ribonucleoprotein complex termed the spliceosome, which is composed of five small nuclear ribonucleoprotein particles, U1, U2, U4/U6, and U5. Biochemical studies over the past 10 years have allowed the isolation as well as compositional, functional, and structural analysis of splicing complexes at distinct stages along the spliceosome cycle. The average human gene contains eight exons and seven introns, producing an average of three or more alternatively spliced mRNA isoforms. Recent high-throughput sequencing studies indicate that 100% of human genes produce at least two alternative mRNA isoforms. Mechanisms of alternative splicing include RNA-protein interactions of splicing factors with regulatory sites termed silencers or enhancers, RNA-RNA base-pairing interactions, or chromatin-based effects that can change or determine splicing patterns. Disease-causing mutations can often occur in splice sites near intron borders or in exonic or intronic RNA regulatory silencer or enhancer elements, as well as in genes that encode splicing factors. Together, these studies provide mechanistic insights into how spliceosome assembly, dynamics, and catalysis occur; how alternative splicing is regulated and evolves; and how splicing can be disrupted by cis- and trans-acting mutations leading to disease states. These findings make the spliceosome an attractive new target for small-molecule, antisense, and genome-editing therapeutic interventions.
Topics: Alternative Splicing; Animals; Disease; Gene Expression Regulation; Humans; Mutation; RNA Precursors; RNA Splicing; RNA, Catalytic; Ribonucleoproteins, Small Nuclear; Spliceosomes
PubMed: 25784052
DOI: 10.1146/annurev-biochem-060614-034316 -
Cell Feb 2023Viroids and viroid-like covalently closed circular (ccc) RNAs are minimal replicators that typically encode no proteins and hijack cellular enzymes for replication. The...
Viroids and viroid-like covalently closed circular (ccc) RNAs are minimal replicators that typically encode no proteins and hijack cellular enzymes for replication. The extent and diversity of viroid-like agents are poorly understood. We developed a computational pipeline to identify viroid-like cccRNAs and applied it to 5,131 metatranscriptomes and 1,344 plant transcriptomes. The search yielded 11,378 viroid-like cccRNAs spanning 4,409 species-level clusters, a 5-fold increase compared to the previously identified viroid-like elements. Within this diverse collection, we discovered numerous putative viroids, satellite RNAs, retrozymes, and ribozy-like viruses. Diverse ribozyme combinations and unusual ribozymes within the cccRNAs were identified. Self-cleaving ribozymes were identified in ambiviruses, some mito-like viruses and capsid-encoding satellite virus-like cccRNAs. The broad presence of viroid-like cccRNAs in diverse transcriptomes and ecosystems implies that their host range is far broader than currently known, and matches to CRISPR spacers suggest that some cccRNAs replicate in prokaryotes.
Topics: RNA, Circular; Viroids; RNA, Catalytic; RNA, Viral; Ecosystem; Plant Diseases
PubMed: 36696902
DOI: 10.1016/j.cell.2022.12.039 -
Nature Biotechnology Jun 2019RNA aptamers and RNA aptamer-based devices can be genetically encoded and expressed in cells to probe and manipulate cellular function. However, their usefulness in the...
RNA aptamers and RNA aptamer-based devices can be genetically encoded and expressed in cells to probe and manipulate cellular function. However, their usefulness in the mammalian cell is limited by low expression and rapid degradation. Here we describe the Tornado (Twister-optimized RNA for durable overexpression) expression system for achieving rapid RNA circularization, resulting in RNA aptamers with high stability and expression levels. Tornado-expressed transcripts contain an RNA of interest flanked by Twister ribozymes. The ribozymes rapidly undergo autocatalytic cleavage, leaving termini that are ligated by the ubiquitous endogenous RNA ligase RtcB. Using this approach, protein-binding aptamers that otherwise have minimal effects in cells become potent inhibitors of cellular signaling. Additionally, an RNA-based fluorescent metabolite biosensor for S-adenosyl methionine (SAM) that is expressed at low levels when expressed as a linear RNA achieves levels sufficient for detection of intracellular SAM dynamics when expressed as a circular RNA. The Tornado expression system thus markedly enhances the utility of RNA-based approaches in the mammalian cell.
Topics: Aptamers, Nucleotide; Catalysis; Gene Expression Regulation; Ligases; Nucleic Acid Conformation; RNA; RNA, Catalytic; RNA, Circular; S-Adenosylmethionine
PubMed: 30962542
DOI: 10.1038/s41587-019-0090-6 -
ACS Chemical Biology Aug 2020Biocatalysis is dominated by protein enzymes, and only a few classes of ribozymes are known to contribute to the task of promoting biochemical transformations. The RNA...
Biocatalysis is dominated by protein enzymes, and only a few classes of ribozymes are known to contribute to the task of promoting biochemical transformations. The RNA World theory encompasses the notion that earlier forms of life made use of a much greater diversity of ribozymes and other functional RNAs to guide complex metabolic states long before proteins had emerged in evolution. In recent years, the discoveries of various classes of ribozymes, riboswitches, and other noncoding RNAs in bacteria have provided additional support for the hypothesis that RNA molecules indeed have the catalytic competence to promote diverse chemical reactions without the aid of protein enzymes. Herein, some of the most striking observations made from examinations of natural riboswitches that bind small ligands are highlighted and used as a basis to imagine the characteristics and functions of long-extinct ribozymes from the RNA World.
Topics: Biocatalysis; Ligands; Nucleic Acid Conformation; RNA; RNA, Catalytic; Riboswitch
PubMed: 32687319
DOI: 10.1021/acschembio.0c00214 -
Nature Aug 2021Single-particle cryogenic electron microscopy (cryo-EM) has become a standard technique for determining protein structures at atomic resolution. However, cryo-EM studies...
Single-particle cryogenic electron microscopy (cryo-EM) has become a standard technique for determining protein structures at atomic resolution. However, cryo-EM studies of protein-free RNA are in their early days. The Tetrahymena thermophila group I self-splicing intron was the first ribozyme to be discovered and has been a prominent model system for the study of RNA catalysis and structure-function relationships, but its full structure remains unknown. Here we report cryo-EM structures of the full-length Tetrahymena ribozyme in substrate-free and bound states at a resolution of 3.1 Å. Newly resolved peripheral regions form two coaxially stacked helices; these are interconnected by two kissing loop pseudoknots that wrap around the catalytic core and include two previously unforeseen (to our knowledge) tertiary interactions. The global architecture is nearly identical in both states; only the internal guide sequence and guanosine binding site undergo a large conformational change and a localized shift, respectively, upon binding of RNA substrates. These results provide a long-sought structural view of a paradigmatic RNA enzyme and signal a new era for the cryo-EM-based study of structure-function relationships in ribozymes.
Topics: Apoenzymes; Cryoelectron Microscopy; Holoenzymes; Models, Molecular; Nucleic Acid Conformation; RNA, Catalytic; Tetrahymena thermophila
PubMed: 34381213
DOI: 10.1038/s41586-021-03803-w -
The Journal of Biological Chemistry Feb 2020The ribosome and RNase P are cellular ribonucleoprotein complexes that perform peptide bond synthesis and phosphodiester bond cleavage, respectively. Both are ancient... (Review)
Review
The ribosome and RNase P are cellular ribonucleoprotein complexes that perform peptide bond synthesis and phosphodiester bond cleavage, respectively. Both are ancient biological assemblies that were already present in the last universal common ancestor of all life. The large subunit rRNA in the ribosome and the RNA subunit of RNase P are the ribozyme components required for catalysis. Here, we explore the idea that these two large ribozymes may have begun their evolutionary odyssey as an assemblage of RNA "fragments" smaller than the contemporary full-length versions and that they transitioned through distinct stages along a pathway that may also be relevant for the evolution of other non-coding RNAs.
Topics: Evolution, Molecular; Models, Molecular; RNA, Catalytic; RNA, Ribosomal; Ribonuclease P
PubMed: 31953324
DOI: 10.1074/jbc.REV119.009929 -
Acta Biochimica Polonica 2016Despite the wealth of data on RNA secondary structure, conformational dynamics and tertiary structure in vitro and in vivo, predicting RNA biological activity in...
Despite the wealth of data on RNA secondary structure, conformational dynamics and tertiary structure in vitro and in vivo, predicting RNA biological activity in cellular environments remains difficult. Here, we present a comparison between in silico RNA fingerprinting and published experimental data that sheds light on efficient design of the hammerhead ribozyme molecules with a high intracellular efficiency. Our method, which we call RNA dactyloscopy, is a reliable tool for assessing the catalytic properties, modeling and design of RNA.
Topics: Base Sequence; Biocatalysis; Models, Molecular; Nucleic Acid Conformation; RNA, Catalytic; Thermodynamics
PubMed: 27904888
DOI: 10.18388/abp.2016_1341 -
International Journal of Molecular... Mar 2021Viroids are non-coding circular RNA molecules with rod-like or branched structures. They are often ribozymes, characterized by catalytic RNA. They can perform many basic... (Review)
Review
Viroids are non-coding circular RNA molecules with rod-like or branched structures. They are often ribozymes, characterized by catalytic RNA. They can perform many basic functions of life and may have played a role in evolution since the beginning of life on Earth. They can cleave, join, replicate, and undergo Darwinian evolution. Furthermore, ribozymes are the essential elements for protein synthesis of cellular organisms as parts of ribosomes. Thus, they must have preceded DNA and proteins during evolution. Here, we discuss the current evidence for viroids or viroid-like RNAs as a likely origin of life on Earth. As such, they may also be considered as models for life on other planets or moons in the solar system as well as on exoplanets.
Topics: Animals; Gene Silencing; Genetic Complementation Test; Humans; Meteoroids; Nucleic Acid Conformation; Origin of Life; Plant Diseases; RNA Interference; RNA, Catalytic; RNA, Viral; Ribosomes; Symbiosis; Viroids; Virus Diseases; Virus Replication
PubMed: 33800543
DOI: 10.3390/ijms22073476 -
Nature Chemical Biology Apr 2021The RNA World theory encompasses the hypothesis that sophisticated ribozymes and riboswitches were the primary drivers of metabolic processes in ancient organisms.... (Review)
Review
The RNA World theory encompasses the hypothesis that sophisticated ribozymes and riboswitches were the primary drivers of metabolic processes in ancient organisms. Several types of catalytic RNAs and many classes of ligand-sensing RNA switches still exist in modern cells. Curiously, allosteric ribozymes formed by the merger of RNA enzyme and RNA switch components are largely absent in today's biological systems. This is true despite the striking abundances of various classes of both self-cleaving ribozymes and riboswitch aptamers. Here we present the known types of ligand-controlled ribozymes and riboswitches and discuss the possible reasons why fused ribozyme-aptamer constructs have been disfavored through evolution.
Topics: Allosteric Regulation; Animals; Aptamers, Nucleotide; Evolution, Molecular; Genetic Engineering; Humans; Nucleic Acid Conformation; RNA; RNA, Catalytic; Riboswitch
PubMed: 33495645
DOI: 10.1038/s41589-020-00713-2 -
Journal of the American Chemical Society Aug 2015Ribozymes are highly structured RNA sequences that can be tailored to recognize and cleave specific stretches of mRNA. Their current therapeutic efficacy remains low due...
Ribozymes are highly structured RNA sequences that can be tailored to recognize and cleave specific stretches of mRNA. Their current therapeutic efficacy remains low due to their large size and structural instability compared to shorter therapeutically relevant RNA such as small interfering RNA (siRNA) and microRNA (miRNA). Herein, a synthetic strategy that makes use of the spherical nucleic acid (SNA) architecture to stabilize ribozymes and transfect them into live cells is reported. The properties of this novel ribozyme-SNA are characterized in the context of the targeted knockdown of O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein involved in chemotherapeutic resistance of solid tumors, foremost glioblastoma multiforme (GBM). Data showing the direct cleavage of full-length MGMT mRNA, knockdown of MGMT protein, and increased sensitization of GBM cells to therapy-mediated apoptosis, independent of transfection agents, provide compelling evidence for the promising properties of this new chemical architecture.
Topics: Biological Transport; Caspases; Cell Line, Tumor; DNA Modification Methylases; Enzyme Activation; Gene Silencing; Humans; RNA, Catalytic; Transfection
PubMed: 26271335
DOI: 10.1021/jacs.5b07104