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 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.
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.
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.
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.
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.
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.
Direct RNA sequencing holds great promise for the de novo identification of RNA modifications at single-coordinate resolution; however, interpretation of raw sequencing output to discover modified ...bases remains a challenge. Using Oxford Nanopore's direct RNA sequencing technology, we developed a random forest classifier trained using experimentally detected N6-methyladenosine (m6A) sites within DRACH motifs. Our software MINES (m6A Identification using Nanopore Sequencing) assigned m6A methylation status to more than 13,000 previously unannotated DRACH sites in endogenous HEK293T transcripts and identified more than 40,000 sites with isoform-level resolution in a human mammary epithelial cell line. These sites displayed sensitivity to the m6A writer, METTL3, and eraser, ALKBH5, respectively. MINES (https://github.com/YeoLab/MINES.git) enables m6A annotation at single coordinate–level resolution from direct RNA nanopore sequencing.
RNA binding proteins (RBPs) interact with primary, precursor, and mature microRNAs (miRs) to influence mature miR levels, which in turn affect critical aspects of human development and disease. To ...understand how RBPs contribute to miR biogenesis, we analyzed human enhanced UV crosslinking followed by immunoprecipitation (eCLIP) datasets for 126 RBPs to discover miR-encoding genomic loci that are statistically enriched for RBP binding. We find that 92% of RBPs interact directly with at least one miR locus, and that some interactions are cell line specific despite expression of the miR locus in both cell lines evaluated. We validated that ILF3 and BUD13 directly interact with and stabilize miR-144 and that BUD13 suppresses mir-210 processing to the mature species. We also observed that DDX3X regulates primary miR-20a, while LARP4 stabilizes precursor mir-210. Our approach to identifying regulators of miR loci can be applied to any user-defined RNA annotation, thereby guiding the discovery of uncharacterized regulators of RNA processing.
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•Analysis of eCLIP datasets identifies 116 candidate RBPs that bind miRNA loci•∼68% of identified direct RBP:miR locus interactions are cell line specific•Knockdown of candidate RBPs affects microRNA levels•Biochemical assays reveal steps in microRNA processing that are affected
Nussbacher and Yeo perform a computational screen of eCLIP data to identify putative, endogenous regulators of microRNA biogenesis and show that this regulation can be cell type specific and both promote or inhibit processing. Their approach can be applied to any transcriptomic annotation (RNA modifications, etc.) to identify novel RBP:RNA interactions.
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