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Molecular Cancer Dec 2020Circular RNAs (CircRNAs) are single-stranded, covalently closed RNA molecules that are ubiquitous across species ranging from viruses to mammals. Important advances have... (Review)
Review
Circular RNAs (CircRNAs) are single-stranded, covalently closed RNA molecules that are ubiquitous across species ranging from viruses to mammals. Important advances have been made in the biogenesis, regulation, localization, degradation and modification of circRNAs. CircRNAs exert biological functions by acting as transcriptional regulators, microRNA (miR) sponges and protein templates. Moreover, emerging evidence has revealed that a group of circRNAs can serve as protein decoys, scaffolds and recruiters. However, the existing research on circRNA-protein interactions is quite limited. Hence, in this review, we briefly summarize recent progress in the metabolism and functions of circRNAs and elaborately discuss the patterns of circRNA-protein interactions, including altering interactions between proteins, tethering or sequestering proteins, recruiting proteins to chromatin, forming circRNA-protein-mRNA ternary complexes and translocating or redistributing proteins. Many discoveries have revealed that circRNAs have unique expression signatures and play crucial roles in a variety of diseases, enabling them to potentially act as diagnostic biomarkers and therapeutic targets. This review systematically evaluates the roles and mechanisms of circRNAs, with the hope of advancing translational medicine involving circRNAs.
Topics: Animals; Chromatin; Humans; Models, Biological; Proteins; RNA Stability; RNA, Circular; Transcription, Genetic
PubMed: 33317550
DOI: 10.1186/s12943-020-01286-3 -
Cell Feb 2009From the earliest comparisons of RNA production with steady-state levels, it has been clear that cells transcribe more RNA than they accumulate, implying the existence... (Review)
Review
From the earliest comparisons of RNA production with steady-state levels, it has been clear that cells transcribe more RNA than they accumulate, implying the existence of active RNA degradation systems. In general, RNA is degraded at the end of its useful life, which is long for a ribosomal RNA but very short for excised introns or spacer fragments, and is closely regulated for most mRNA species. RNA molecules with defects in processing, folding, or assembly with proteins are identified and rapidly degraded by the surveillance machinery. Because RNA degradation is ubiquitous in all cells, it is clear that it must be carefully controlled to accurately recognize target RNAs. How this is achieved is perhaps the most pressing question in the field.
Topics: Animals; Humans; Metabolic Networks and Pathways; RNA Stability; Ribonucleoproteins; Transcription, Genetic
PubMed: 19239894
DOI: 10.1016/j.cell.2009.01.019 -
Methods in Molecular Biology (Clifton,... 2018RNA interference (RNAi) is a biological process by which double-stranded RNA (dsRNA) induces sequence-specific gene silencing by targeting mRNA for degradation. As a... (Review)
Review
RNA interference (RNAi) is a biological process by which double-stranded RNA (dsRNA) induces sequence-specific gene silencing by targeting mRNA for degradation. As a tool for knocking down the expression of individual genes posttranscriptionally, RNAi has been widely used to study the cellular function of genes. In this chapter, I describe procedures for using gene-specific, synthetic, short interfering RNA (siRNA) to induce gene silencing in mammalian cells. Protocols for using lipid-based transfection reagents and electroporation techniques are provided. Potential challenges and problems associated with the siRNA technology are also discussed.
Topics: Animals; Electroporation; Gene Knockdown Techniques; Humans; RNA Interference; RNA Stability; Transfection
PubMed: 29423805
DOI: 10.1007/978-1-4939-7471-9_16 -
Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics.Nature Communications Mar 2022Therapeutic mRNAs and vaccines are being developed for a broad range of human diseases, including COVID-19. However, their optimization is hindered by mRNA instability...
Therapeutic mRNAs and vaccines are being developed for a broad range of human diseases, including COVID-19. However, their optimization is hindered by mRNA instability and inefficient protein expression. Here, we describe design principles that overcome these barriers. We develop an RNA sequencing-based platform called PERSIST-seq to systematically delineate in-cell mRNA stability, ribosome load, as well as in-solution stability of a library of diverse mRNAs. We find that, surprisingly, in-cell stability is a greater driver of protein output than high ribosome load. We further introduce a method called In-line-seq, applied to thousands of diverse RNAs, that reveals sequence and structure-based rules for mitigating hydrolytic degradation. Our findings show that highly structured "superfolder" mRNAs can be designed to improve both stability and expression with further enhancement through pseudouridine nucleoside modification. Together, our study demonstrates simultaneous improvement of mRNA stability and protein expression and provides a computational-experimental platform for the enhancement of mRNA medicines.
Topics: COVID-19; Humans; Pseudouridine; RNA; RNA Stability; RNA, Messenger
PubMed: 35318324
DOI: 10.1038/s41467-022-28776-w -
Cell Proliferation Jan 2022YTHDF1 is known as a m A reader protein, and many researches of YTHDF1 focused on the regulation of mRNA translation efficiency. However, YTHDF1 is also related to RNA...
OBJECTIVES
YTHDF1 is known as a m A reader protein, and many researches of YTHDF1 focused on the regulation of mRNA translation efficiency. However, YTHDF1 is also related to RNA degradation, but how YTHDF1 regulates mRNA degradation is indefinite. Liquid-liquid phase separation (LLPS) underlies the formation of membraneless compartments in mammal cells, and there are few reports focused on the correlation of RNA degradation with LLPS. In this research, we focused on the mechanism of YTHDF1 degraded mRNA through LLPS.
MATERIALS AND METHODS
The CRISPR/Cas9 knock out system was used to establish the YTHDF1 knock out (YTHDF1-KO) cell lines (HEK293 and HeLa) and METTL14 knock out (METTL14-KO) cell line (HEK293). 4SU-TT-seq was used to check the half-life changes of mRNAs. Actinomycin D and qPCR were used to test the half-life changes of individual mRNA. RNA was stained with SYTO RNA-select dye in wild type (WT) and YTHDF1-KO HeLa cell lines. Co-localization of YTHDF1 and AGO2 was identified by immunofluorescence. The interaction domain of YTHDF1 and AGO2 was identified by western blot. Phase separation of YTHDF1 was performed in vitro and in vivo. Fluorescence recovery after photobleaching (FRAP) was performed on droplets as an assessment of their liquidity.
RESULTS
In this research, we found that deletion of YTHDF1 led to massive RNA patches deposited in cytoplasm. The results of 4SU-TT-seq showed that deletion of YTHDF1 would prolong the half-life of mRNAs. Immunofluorescence data showed that YTHDF1 and AGO2 could co-localize in P-body, and Co-IP results showed that YTHDF1 could interact with AGO2 through YT521-B homology (YTH) domain. We confirmed that YTHDF1 could undergo phase separation in vitro and in vivo, and compared with AGO2, YTHDF1 was more important in P-body formation. The FRAP results showed that liquid AGO2 droplets would convert to gel/solid when YTHDF1 was deleted. As AGO2 plays important roles in miRISCs, we also found that miRNA-mediate mRNA degradation is related to YTHDF1.
CONCLUSIONS
YTHDF1 recruits AGO2 through the YTH domain. YTHDF1 degrades targeting mRNAs by promoting P-body formation through LLPS. The deletion of YTHDF1 causes the P-body to change from liquid droplets to gel/solid droplets, and form AGO2/RNA patches, resulting in a degradation delay of mRNAs. These findings reveal a previously unrecognized crosstalk between YTHDF1 and AGO2, raising a new sight of mRNA post-transcriptional regulation by YTHDF1.
Topics: Argonaute Proteins; Base Sequence; Cytoplasm; HEK293 Cells; HeLa Cells; Humans; MicroRNAs; Protein Binding; Protein Domains; RNA Stability; RNA-Binding Proteins
PubMed: 34821414
DOI: 10.1111/cpr.13157 -
Molecular Cell Apr 2022Messenger RNA (mRNA) translation by the ribosome represents the final step of a complicated molecular dance from DNA to protein. Although classically considered a... (Review)
Review
Messenger RNA (mRNA) translation by the ribosome represents the final step of a complicated molecular dance from DNA to protein. Although classically considered a decipherer that translates a 64-word genetic code into a proteome of astonishing complexity, the ribosome can also shape the transcriptome by controlling mRNA stability. Recent work has discovered that the ribosome is an arbiter of the general mRNA degradation pathway, wherein the ribosome transit rate serves as a major determinant of transcript half-lives. Specifically, members of the degradation complex sense ribosome translocation rates as a function of ribosome elongation rates. Central to this notion is the concept of codon optimality: although all codons impact translation rates, some are deciphered quickly, whereas others cause ribosome hesitation as a consequence of relative cognate tRNA concentration. These transient pauses induce a unique ribosome conformational state that is probed by the deadenylase complex, thereby inducing an orchestrated set of events that enhance both poly(A) shortening and cap removal. Together, these data imply that the coding region of an mRNA not only encodes for protein content but also impacts protein levels through determining the transcript's fate.
Topics: Codon; Protein Biosynthesis; Proteins; RNA Stability; RNA, Messenger; Ribosomes
PubMed: 35452615
DOI: 10.1016/j.molcel.2022.03.032 -
Molecular Cell Feb 2023The relationship between mRNA translation and decay is incompletely understood, with conflicting reports suggesting that translation can either promote decay or...
The relationship between mRNA translation and decay is incompletely understood, with conflicting reports suggesting that translation can either promote decay or stabilize mRNAs. The effect of translation on mRNA decay has mainly been studied using ensemble measurements and global transcription and translation inhibitors, which can have pleiotropic effects. We developed a single-molecule imaging approach to control the translation of a specific transcript that enabled simultaneous measurement of translation and mRNA decay. Our results demonstrate that mRNA translation reduces mRNA stability, and mathematical modeling suggests that this process is dependent on ribosome flux. Furthermore, our results indicate that miRNAs mediate efficient degradation of both translating and non-translating target mRNAs and reveal a predominant role for mRNA degradation in miRNA-mediated regulation. Simultaneous observation of translation and decay of single mRNAs provides a framework to directly study how these processes are interconnected in cells.
Topics: RNA, Messenger; Nonsense Mediated mRNA Decay; Single Molecule Imaging; RNA Stability; Ribosomes; MicroRNAs; Protein Biosynthesis
PubMed: 36731471
DOI: 10.1016/j.molcel.2023.01.013 -
Deadenylase-dependent mRNA decay of GDF15 and FGF21 orchestrates food intake and energy expenditure.Cell Metabolism Apr 2022Hepatokines, secretory proteins from the liver, mediate inter-organ communication to maintain a metabolic balance between food intake and energy expenditure. However,...
Hepatokines, secretory proteins from the liver, mediate inter-organ communication to maintain a metabolic balance between food intake and energy expenditure. However, molecular mechanisms by which hepatokine levels are rapidly adjusted following stimuli are largely unknown. Here, we unravel how CNOT6L deadenylase switches off hepatokine expression after responding to stimuli (e.g., exercise and food) to orchestrate energy intake and expenditure. Mechanistically, CNOT6L inhibition stabilizes hepatic Gdf15 and Fgf21 mRNAs, increasing corresponding serum protein levels. The resulting upregulation of GDF15 stimulates the hindbrain to suppress appetite, while increased FGF21 affects the liver and adipose tissues to induce energy expenditure and lipid consumption. Despite the potential of hepatokines to treat metabolic disorders, their administration therapies have been challenging. Using small-molecule screening, we identified a CNOT6L inhibitor enhancing GDF15 and FGF21 hepatokine levels, which dramatically improves diet-induced metabolic syndrome. Our discovery, therefore, lays the foundation for an unprecedented strategy to treat metabolic syndrome.
Topics: Animals; Eating; Energy Metabolism; Fibroblast Growth Factors; Growth Differentiation Factor 15; Humans; Liver; Metabolic Syndrome; Mice; RNA Stability; Ribonucleases
PubMed: 35385705
DOI: 10.1016/j.cmet.2022.03.005 -
Communications Biology Dec 2022Circular RNAs (CircRNAs) are a class of noncoding RNAs formed by backsplicing during cotranscriptional and posttranscriptional processes, and they widely exist in... (Review)
Review
Circular RNAs (CircRNAs) are a class of noncoding RNAs formed by backsplicing during cotranscriptional and posttranscriptional processes, and they widely exist in various organisms. CircRNAs have multiple biological functions and are associated with the occurrence and development of many diseases. While the biogenesis and biological function of circRNAs have been extensively studied, there are few studies on circRNA degradation and only a few pathways for specific circRNA degradation have been identified. Here we outline basic information about circRNAs, summarize the research on the circRNA degradation mechanisms and discusses where this field might head, hoping to provide some inspiration and guidance for scholars who aim to study the degradation of circRNAs.
Topics: RNA, Circular; RNA; RNA Stability
PubMed: 36494488
DOI: 10.1038/s42003-022-04262-3 -
Advances in Neurobiology 2018Ribonucleic acid (RNA) homeostasis is dynamically modulated in response to changing physiological conditions. Tight regulation of RNA abundance through both... (Review)
Review
Ribonucleic acid (RNA) homeostasis is dynamically modulated in response to changing physiological conditions. Tight regulation of RNA abundance through both transcription and degradation determines the amount, timing, and location of protein translation. This balance is of particular importance in neurons, which are among the most metabolically active and morphologically complex cells in the body. As a result, any disruptions in RNA degradation can have dramatic consequences for neuronal health. In this chapter, we will first discuss mechanisms of RNA stabilization and decay. We will then explore how the disruption of these pathways can lead to neurodegenerative disease.
Topics: Humans; Neurodegenerative Diseases; RNA Stability; RNA, Messenger
PubMed: 29916018
DOI: 10.1007/978-3-319-89689-2_5