RNA-binding proteins (RBPs) comprise a large class of over 2,000 proteins that interact with transcripts in all manner of RNA-driven processes. The structures and mechanisms that RBPs use to bind and ...regulate RNA are incredibly diverse. In this review, we take a look at the components of protein-RNA interaction, from the molecular level to multi-component interaction. We first summarize what is known about protein-RNA molecular interactions based on analyses of solved structures. We additionally describe software currently available for predicting protein-RNA interaction and other resources useful for the study of RBPs. We then review the structure and function of seventeen known RNA-binding domains and analyze the hydrogen bonds adopted by protein-RNA structures on a domain-by-domain basis. We conclude with a summary of the higher-level mechanisms that regulate protein-RNA interactions.
RNA-binding proteins (RBPs) hold the key to RNA regulation in all domains of life. Corley, Burns, and Yeo review molecular aspects of how RNA-binding domains within RBPs engage with RNA and major themes of their modes of action.
To identify endogenous miRNA-target sites, we isolated AGO-bound RNAs from Caenorhabditis elegans by individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP), which fortuitously also ...produced miRNA-target chimeric reads. Through the analysis of thousands of reproducible chimeras, pairing to the miRNA seed emerged as the predominant motif associated with functional interactions. Unexpectedly, we discovered that additional pairing to 3′ sequences is prevalent in the majority of target sites and leads to specific targeting by members of miRNA families. By editing an endogenous target site, we demonstrate that 3′ pairing determines targeting by specific miRNA family members and that seed pairing is not always sufficient for functional target interactions. Finally, we present a simplified method, chimera PCR (ChimP), for the detection of specific miRNA-target interactions. Overall, our analysis revealed that sequences in the 5′ as well as the 3′ regions of a miRNA provide the information necessary for stable and specific miRNA-target interactions in vivo.
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•AGO iCLIP miRNA-target chimeras reveal miRNA targeting landscape in C. elegans•miRNA families target non-overlapping sets of target sites•miRNA 3′ end interactions contribute to target site specificity in vivo•Chimera PCR (ChimP) identifies specific miRNA-target sites of interest
Argonaute iCLIP produces miRNA-target chimeras that identify miRNA targets. Broughton et al. analyze these chimeras, reporting that 3′ regions of miRNAs confer specificity to miRNA-target interactions in vivo. They develop a simple, versatile, and economic method called chimera PCR (ChimP) for testing miRNA-target interactions.
RNA binding proteins are critical to the maintenance of the transcriptome via controlled regulation of RNA processing and transport. Alterations of these proteins impact multiple steps of the RNA ...life cycle resulting in various molecular phenotypes such as aberrant RNA splicing, transport, and stability. Disruption of RNA binding proteins and widespread RNA processing defects are increasingly recognized as critical determinants of neurological diseases. Here, we describe distinct mechanisms by which the homeostasis of RNA binding proteins is compromised in neurological disorders through their reduced expression level, increased propensity to aggregate or sequestration by abnormal RNAs. These mechanisms all converge toward altered neuronal function highlighting the susceptibility of neurons to deleterious changes in RNA expression and the central role of RNA binding proteins in preserving neuronal integrity. Emerging therapeutic approaches to mitigate or reverse alterations of RNA binding proteins in neurological diseases are discussed.
Disruption of RNA binding proteins with widespread perturbations of the transcriptome is at the heart of several neurological diseases. Nussbacher et al. review the diverse mechanisms by which this occurs (reduced expression, increased aggregation, and sequestration by repeat-containing RNAs and proteins), and emerging therapeutic approaches to ameliorate these pathologies.
RNA processing plays a central role in accurately transmitting genetic information into functional RNA and protein regulators. To fully appreciate the RNA life-cycle, tools to observe RNA with high ...spatial and temporal resolution are critical. Here we review recent advances in RNA imaging and highlight how they will propel the field of RNA biology. We discuss current trends in RNA imaging and their potential to elucidate unanswered questions in RNA biology.
Throughout their lifetimes, messenger RNAs (mRNAs) associate with proteins to form ribonucleoproteins (mRNPs). Since the discovery of the first mRNP component more than 40 years ago, what is known as ...the mRNA interactome now comprises >1,000 proteins. These proteins bind mRNAs in myriad ways with varying affinities and stoichiometries, with many assembling onto nascent RNAs in a highly ordered process during transcription and precursor mRNA (pre-mRNA) processing. The nonrandom distribution of major mRNP proteins observed in transcriptome-wide studies leads us to propose that mRNPs are organized into three major domains loosely corresponding to 5′ untranslated regions (UTRs), open reading frames, and 3′ UTRs. Moving from the nucleus to the cytoplasm, mRNPs undergo extensive remodeling as they are first acted upon by the nuclear pore complex and then by the ribosome. When not being actively translated, cytoplasmic mRNPs can assemble into large multi-mRNP assemblies or be permanently disassembled and degraded. In this review, we aim to give the reader a thorough understanding of past and current eukaryotic mRNP research.
Stress granules (SGs) are transient ribonucleoprotein (RNP) aggregates that form during cellular stress and are increasingly implicated in human neurodegeneration. To study the proteome and ...compositional diversity of SGs in different cell types and in the context of neurodegeneration-linked mutations, we used ascorbate peroxidase (APEX) proximity labeling, mass spectrometry, and immunofluorescence to identify ∼150 previously unknown human SG components. A highly integrated, pre-existing SG protein interaction network in unstressed cells facilitates rapid coalescence into larger SGs. Approximately 20% of SG diversity is stress or cell-type dependent, with neuronal SGs displaying a particularly complex repertoire of proteins enriched in chaperones and autophagy factors. Strengthening the link between SGs and neurodegeneration, we demonstrate aberrant dynamics, composition, and subcellular distribution of SGs in cells from amyotrophic lateral sclerosis (ALS) patients. Using three Drosophila ALS/FTD models, we identify SG-associated modifiers of neurotoxicity in vivo. Altogether, our results highlight SG proteins as central to understanding and ultimately targeting neurodegeneration.
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•APEX proximity labeling reveals ∼150 unknown SG proteins in a dense protein network•SG composition varies by stress and cell type, especially in neuronal cells•ALS motor neurons contain SGs with distinct content and subcellular distribution•SG proteins modify ALS-mutant-mediated toxicity in fly models of neurodegeneration
Interactions between stress granule proteins exist ahead of a stress response and candidate SG proteins modify disease phenotypes in vivo.
RNA-programmed genome editing using CRISPR/Cas9 from Streptococcus pyogenes has enabled rapid and accessible alteration of specific genomic loci in many organisms. A flexible means to target RNA ...would allow alteration and imaging of endogenous RNA transcripts analogous to CRISPR/Cas-based genomic tools, but most RNA targeting methods rely on incorporation of exogenous tags. Here, we demonstrate that nuclease-inactive S. pyogenes CRISPR/Cas9 can bind RNA in a nucleic-acid-programmed manner and allow endogenous RNA tracking in living cells. We show that nuclear-localized RNA-targeting Cas9 (RCas9) is exported to the cytoplasm only in the presence of sgRNAs targeting mRNA and observe accumulation of ACTB, CCNA2, and TFRC mRNAs in RNA granules that correlate with fluorescence in situ hybridization. We also demonstrate time-resolved measurements of ACTB mRNA trafficking to stress granules. Our results establish RCas9 as a means to track RNA in living cells in a programmable manner without genetically encoded tags.
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•RNA-targeting Cas9 (RCas9) enabled recognition of endogenous, unmodified mRNAs•RCas9 did not influence mRNA abundance or amount of translated protein•Subcellular distribution of RCas9 was highly correlated with RNA-FISH•RCas9 revealed trafficking of mRNAs to stress granules in live cells
RNA-targeting Cas9 enables tracking of endogenous, untagged mRNA, establishing CRISPR/Cas9 as a programmable system to recognize RNA in live cells.
Structural and transcriptional changes during early brain maturation follow fixed developmental programs defined by genetics. However, whether this is true for functional network activity remains ...unknown, primarily due to experimental inaccessibility of the initial stages of the living human brain. Here, we developed human cortical organoids that dynamically change cellular populations during maturation and exhibited consistent increases in electrical activity over the span of several months. The spontaneous network formation displayed periodic and regular oscillatory events that were dependent on glutamatergic and GABAergic signaling. The oscillatory activity transitioned to more spatiotemporally irregular patterns, and synchronous network events resembled features similar to those observed in preterm human electroencephalography. These results show that the development of structured network activity in a human neocortex model may follow stable genetic programming. Our approach provides opportunities for investigating and manipulating the role of network activity in the developing human cortex.
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•Long-term, single-cell transcriptomics reveals cortical organoid developmental dynamics•Cortical organoids exhibit phase-amplitude coupling during network-synchronous events•Differential role of glutamate and GABA in initiating and maintaining oscillations•Network-level events are similar to the human preterm neonatal EEG features
Oscillatory activity is a candidate mechanism for how neural populations are temporally organized. Cortical organoids exhibit periodic and highly regular nested oscillatory network events that are dependent on glutamatergic and GABAergic signaling. The emerging development of network activity transitions to more spatiotemporally complex activity, capturing features of preterm infant electroencephalography.
Base editing: advances and therapeutic opportunities Porto, Elizabeth M; Komor, Alexis C; Slaymaker, Ian M ...
Nature reviews. Drug discover/Nature reviews. Drug discovery,
12/2020, Letnik:
19, Številka:
12
Journal Article
Recenzirano
Odprti dostop
Base editing - the introduction of single-nucleotide variants (SNVs) into DNA or RNA in living cells - is one of the most recent advances in the field of genome editing. As around half of known ...pathogenic genetic variants are due to SNVs, base editing holds great potential for the treatment of numerous genetic diseases, through either temporary RNA or permanent DNA base alterations. Recent advances in the specificity, efficiency, precision and delivery of DNA and RNA base editors are revealing exciting therapeutic opportunities for these technologies. We expect the correction of single point mutations will be a major focus of future precision medicine.
Mutations in genes that encode RNA-binding proteins (RBPs) have emerged as critical determinants of neurological diseases, especially motor neuron disorders such as amyotrophic lateral sclerosis ...(ALS). RBPs are involved in all aspects of RNA processing, controlling the life cycle of RNAs from synthesis to degradation. Hallmark features of RBPs in neuron dysfunction include misregulation of RNA processing, mislocalization of RBPs to the cytoplasm, and abnormal aggregation of RBPs. Much progress has been made in understanding how ALS-associated mutations in RBPs drive pathogenesis. Here, we focus on several key RBPs involved in ALS—TDP-43, HNRNP A2/B1, HNRNP A1, FUS, EWSR1, and TAF15—and review our current understanding of how mutations in these proteins cause disease.
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