Specificity and nonspecificity in RNA–protein interactions

Jankowsky, Eckhard; Harris, Michael E.

doi:10.1038/nrm4032

Cited by 234 publications

(260 citation statements)

References 135 publications

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“…For example, the RNA substrates for key enzymes such as the ribosome, spliceosome, tRNA and mRNA processing enzymes can vary greatly in sequence and/or structure[1–5]. Given the broad range of alternative substrates that are recognized by these enzymes, their specificity cannot be entirely captured by sequence motif analysis, homology modeling, or similar approaches that consider only genomically encoded or optimal substrates[6]. Moreover, it is well established that a biologically relevant investigation of enzyme specificity involves understanding how substrates compete for enzyme association [7][8][9].…”

Section: Introductionmentioning

confidence: 99%

Optimization of high-throughput sequencing kinetics for determining enzymatic rate constants of thousands of RNA substrates

Niland

Jankowsky

Harris

2016

Analytical Biochemistry

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Quantification of the specificity of RNA binding proteins and RNA processing enzymes is essential to understanding their fundamental roles in biological processes. High Throughput Sequencing Kinetics (HTS-Kin) uses high throughput sequencing and internal competition kinetics to simultaneously monitor the processing rate constants of thousands of substrates by RNA processing enzymes. This technique has provided unprecedented insight into the substrate specificity of the tRNA processing endonuclease ribonuclease P. Here, we investigate the accuracy and robustness of measurements associated with each step of the HTS-Kin procedure. We examine the effect of substrate concentration on the observed rate constant, determine the optimal kinetic parameters, and provide guidelines for reducing error in amplification of the substrate population. Importantly, we find that high-throughput sequencing, and experimental reproducibility contribute their own sources of error, and these are the main sources of imprecision in the quantified results when otherwise optimized guidelines are followed.

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Section: Introductionmentioning

confidence: 99%

Optimization of high-throughput sequencing kinetics for determining enzymatic rate constants of thousands of RNA substrates

Niland

Jankowsky

Harris

2016

Analytical Biochemistry

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“…RBPs regulate every step of RNA life through their association with RNA sequences and/or secondary structures (Glisovic et al 2008;Jankowsky and Harris 2015). Several RBPs that regulate maternal mRNA degradation, a hallmark of MZT in all metazoans , have been identified.…”

Section: [Supplemental Materials Is Available For This Article]mentioning

confidence: 99%

Dynamic RNA–protein interactions underlie the zebrafish maternal-to-zygotic transition

Despic

Dejung

et al. 2017

Genome Res.

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During the maternal-to-zygotic transition (MZT), transcriptionally silent embryos rely on post-transcriptional regulation of maternal mRNAs until zygotic genome activation (ZGA). RNA-binding proteins (RBPs) are important regulators of posttranscriptional RNA processing events, yet their identities and functions during developmental transitions in vertebrates remain largely unexplored. Using mRNA interactome capture, we identified 227 RBPs in zebrafish embryos before and during ZGA, hereby named the zebrafish MZT mRNA-bound proteome. This protein constellation consists of many conserved RBPs, some of which are potential stage-specific mRNA interactors that likely reflect the dynamics of RNA-protein interactions during MZT. The enrichment of numerous splicing factors like hnRNP proteins before ZGA was surprising, because maternal mRNAs were found to be fully spliced. To address potentially unique roles of these RBPs in embryogenesis, we focused on Hnrnpa1. iCLIP and subsequent mRNA reporter assays revealed a function for Hnrnpa1 in the regulation of poly(A) tail length and translation of maternal mRNAs through sequence-specific association with 3 ′ UTRs before ZGA. Comparison of iCLIP data from two developmental stages revealed that Hnrnpa1 dissociates from maternal mRNAs at ZGA and instead regulates the nuclear processing of pri-mir-430 transcripts, which we validated experimentally. The shift from cytoplasmic to nuclear RNA targets was accompanied by a dramatic translocation of Hnrnpa1 and other pre-mRNA splicing factors to the nucleus in a transcription-dependent manner. Thus, our study identifies global changes in RNAprotein interactions during vertebrate MZT and shows that Hnrnpa1 RNA-binding activities are spatially and temporally coordinated to regulate RNA metabolism during early development.

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“…1 In mammalian cells, more than 1,000 diverse proteins interact with RNA. 2 The interactions of proteins with RNA have been generally explained using different types of motifs such as RNA recognition motif, double stranded RNA binding motif, Arginine rich motif, GXXG motif, tetra loops in RNA and so on. 3 The RNA-recognition motif (RRM) is the most abundant RNA-binding domain in higher vertebrates, which is present in about 0.5%-1.0% of human genes.…”

Section: Introductionmentioning

confidence: 99%

Exploring the molecular basis of RNA recognition by the dimeric RNA-binding protein via molecular simulation methods

Chang

Zhang

et al. 2016

RNA Biology

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RNA-binding protein with multiple splicing (RBPMS) is critical for axon guidance, smooth muscle plasticity, and regulation of cancer cell proliferation and migration. Recently, different states of the RNA-recognition motif (RRM) of RBPMS, one in its free form and another in complex with CAC-containing RNA, were determined by Xray crystallography. In this article, the free RRM domain, its wild type complex and 2 mutant complex systems are studied by molecular dynamics (MD) simulations. Through comparison of free RRM domain and complex systems, it's found that the RNA binding facilitates stabilizing the RNA-binding interface of RRM domain, especially the C-terminal loop. Although both R38Q and T103A/K104A mutations reduce the binding affinity of RRM domain and RNA, the underlining mechanisms are different. Principal component analysis (PCA) and Molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) methods were used to explore the dynamical and recognition mechanisms of RRM domain and RNA. R38Q mutation is positioned on the homodimerization interface and mainly induces the large fluctuations of RRM domains. This mutation does not directly act on the RNA-binding interface, but some interfacial hydrogen bonds are weakened. In contrast, T103A/K104A mutations are located on the RNA-binding interface of RRM domain. These mutations obviously break most of high occupancy hydrogen bonds in the RNA-binding interface. Meanwhile, the key interfacial residues lose their favorable energy contributions upon RNA binding. The ranking of calculated binding energies in 3 complex systems is well consistent with that of experimental binding affinities. These results will be helpful in understanding the RNA recognition mechanisms of RRM domain.

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Specificity and nonspecificity in RNA–protein interactions

Cited by 234 publications

References 135 publications

Optimization of high-throughput sequencing kinetics for determining enzymatic rate constants of thousands of RNA substrates

Optimization of high-throughput sequencing kinetics for determining enzymatic rate constants of thousands of RNA substrates

Dynamic RNA–protein interactions underlie the zebrafish maternal-to-zygotic transition

Exploring the molecular basis of RNA recognition by the dimeric RNA-binding protein via molecular simulation methods

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