scholarly journals An RNA Binding Peptide Consisting of Four Types of Amino Acid by in Vitro Selection Using cDNA Display

ACS Omega ◽  
2016 ◽  
Vol 1 (1) ◽  
pp. 52-57 ◽  
Author(s):  
Shigefumi Kumachi ◽  
Yuzuru Husimi ◽  
Naoto Nemoto
Keyword(s):  
Amino Acid ◽  
In Vitro Selection ◽  
Rna Binding ◽  
Binding Peptide

scholarly journals Integrated analysis of RNA-binding protein complexes using in vitro selection and high-throughput sequencing and sequence specificity landscapes (SEQRS)

Methods ◽  
2017 ◽  
Vol 118-119 ◽  
pp. 171-181 ◽  
Author(s):  
Tzu-Fang Lou ◽  
Chase A. Weidmann ◽  
Jordan Killingsworth ◽  
Traci M. Tanaka Hall ◽  
Aaron C. Goldstrohm ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
In Vitro Selection ◽  
Rna Binding ◽  
Protein Complexes ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Integrated Analysis ◽  
Sequence Specificity

scholarly journals The Hox transcription factor Ultrabithorax binds RNA and regulates co-transcriptional splicing through an interplay with RNA polymerase II

2021 ◽  
Author(s):  
Julie Carnesecchi ◽  
Panagiotis Boumpas ◽  
Patrick van Nierop y Sanchez ◽  
Katrin Domsch ◽  
Hugo Daniel Pinto ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Binding ◽  
Control Cell ◽  
Active Form ◽  
Pol Ii ◽  
Rna Interaction ◽  
Drosophila Cells

Transcription Factors (TFs) play a pivotal role in cell fate decision by coordinating distinct gene expression programs. Although most TFs act at the DNA regulatory layer, few TFs can bind RNA and modulate mRNA splicing. Yet, the mechanistic cues underlying TFs function in splicing remain elusive. Focusing on the Drosophila Hox TF Ultrabithorax (Ubx), our work shed light on a novel layer of Ubx function at the RNA level. Transcriptome and genome-wide binding profiles in embryonic mesoderm and Drosophila cells indicate that Ubx regulates mRNA expression and splicing to promote distinct functions in defined cellular contexts. Ubx modulates splicing via its DNA-binding domain, the Homeodomain (HD). Our results demonstrate a new RNA-binding ability of Ubx in cells and in vitro. Notably, the N51 amino acid of the HD, which mediates Ubx-DNA interaction, is non-essential for Ubx-RNA interaction in vitro but is required in vivo. We find that the N51 amino acid is necessary to mediate interaction between Ubx and the active form of the RNA Polymerase II (Pol II S2Phos) in Drosophila cells. By combining molecular and imaging approaches, our results reveal that Ubx mediates elongation-coupled splicing via a dynamic interplay with active Pol II and chromatin binding. Overall, our work uncovered a novel role of the Hox TFs at the mRNA regulatory layer. This could be an essential function for other classes of TFs to control cell diversity.

scholarly journals A crystal structure of a collaborative RNA regulatory complex reveals mechanisms to refine target specificity

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Chen Qiu ◽  
Vandita D Bhat ◽  
Sanjana Rajeev ◽  
Chi Zhang ◽  
Alexa E Lasley ◽  
...  
Keyword(s):  
Crystal Structure ◽  
In Vitro Selection ◽  
Rna Binding ◽  
Sequence Specificity ◽  
Binding Assays ◽  
Regulatory Complex ◽  
Rna Motif ◽  
Rna Complex ◽  
Motif Recognition

In the Caenorhabditis elegans germline, fem-3 Binding Factor (FBF) partners with LST-1 to maintain stem cells. A crystal structure of an FBF-2/LST-1/RNA complex revealed that FBF-2 recognizes a short RNA motif different from the characteristic 9-nt FBF binding element, and compact motif recognition coincided with curvature changes in the FBF-2 scaffold. Previously, we engineered FBF-2 to favor recognition of shorter RNA motifs without curvature change (Bhat et al., 2019). In vitro selection of RNAs bound by FBF-2 suggested sequence specificity in the central region of the compact element. This bias, reflected in the crystal structure, was validated in RNA-binding assays. FBF-2 has the intrinsic ability to bind to this shorter motif. LST-1 weakens FBF-2 binding affinity for short and long motifs, which may increase target selectivity. Our findings highlight the role of FBF scaffold flexibility in RNA recognition and suggest a new mechanism by which protein partners refine target site selection.

scholarly journals The Neural RNA-Binding Protein Musashi1 Translationally Regulates Mammalian numb Gene Expression by Interacting with Its mRNA

2001 ◽  
Vol 21 (12) ◽  
pp. 3888-3900 ◽  
Author(s):  
Takao Imai ◽  
Akinori Tokunaga ◽  
Tetsu Yoshida ◽  
Mitsuhiro Hashimoto ◽  
Katsuhiko Mikoshiba ◽  
...  
Keyword(s):  
Notch Signaling ◽  
In Vitro Selection ◽  
Neural Progenitor Cells ◽  
Rna Binding ◽  
Binding Protein ◽  
Mrna Level ◽  
Rna Binding Protein ◽  
Down Regulation

ABSTRACT Musashi1 (Msi1) is an RNA-binding protein that is highly expressed in neural progenitor cells, including neural stem cells. In this study, the RNA-binding sequences for Msi1 were determined by in vitro selection using a pool of degenerate 50-mer sequences. All of the selected RNA species contained repeats of (G/A)U n AGU (n = 1 to 3) sequences which were essential for Msi1 binding. These consensus elements were identified in some neural mRNAs. One of these, mammaliannumb (m-numb), which encodes a membrane-associated antagonist of Notch signaling, is a likely target of Msi1. Msi1 protein binds in vitro-transcribed m-numb RNA in its 3′-untranslated region (UTR) and binds endogenousm-numb mRNA in vivo, as shown by affinity precipitation followed by reverse transcription-PCR. Furthermore, adenovirus-induced Msi1 expression resulted in the down-regulation of endogenous m-Numb protein expression. Reporter assays using a chimeric mRNA that combined luciferase and the 3′-UTR of m-numb demonstrated that Msi1 decreased the reporter activity without altering the reporter mRNA level. Thus, our results suggested that Msi1 could regulate the expression of its target gene at the translational level. Furthermore, we found that Notch signaling activity was increased by Msi1 expression in connection with the posttranscriptional down-regulation of them-numb gene.

scholarly journals Identification of Amino Acid Residues of Influenza Virus Nucleoprotein Essential for RNA Binding

1999 ◽  
Vol 73 (9) ◽  
pp. 7357-7367 ◽  
Author(s):  
Debra Elton ◽  
Liz Medcalf ◽  
Konrad Bishop ◽  
Deborah Harrison ◽  
Paul Digard
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Electrostatic Interactions ◽  
Rna Binding ◽  
Mutational Analysis ◽  
High Affinity ◽  
Arginine Residues ◽  
Rna Interaction

ABSTRACT The influenza virus nucleoprotein (NP) is a single-strand-RNA-binding protein associated with genome and antigenome RNA and is one of the four virus proteins necessary for transcription and replication of viral RNA. To better characterize the mechanism by which NP binds RNA, we undertook a physical and mutational analysis of the polypeptide, with the strategy of identifying first the regions in direct contact with RNA, then the classes of amino acids involved, and finally the crucial residues by mutagenesis. Chemical fragmentation and amino acid sequencing of NP that had been UV cross linked to radiolabelled RNA showed that protein-RNA contacts occur throughout the length of the polypeptide. Chemical modification experiments implicated arginine but not lysine residues as important for RNA binding, while RNA-dependent changes in the intrinsic fluorescence spectrum of NP suggested the involvement of tryptophan residues. Supporting these observations, single-codon mutagenesis identified five tryptophan, one phenylalanine, and two arginine residues as essential for high-affinity RNA binding at physiological temperature. In addition, mutants unable to bind RNA in vitro were unable to support virus gene expression in vivo. The mutationally sensitive residues are not localized to any particular region of NP but instead are distributed throughout the protein. Overall, these data are inconsistent with previous models suggesting that the NP-RNA interaction is mediated by a discrete N-terminal domain. Instead, we propose that high-affinity binding of RNA by NP requires the concerted interaction of multiple regions of the protein with RNA and that the individual protein-RNA contacts are mediated by a combination of electrostatic interactions between positively charged residues and the phosphate backbone and planar interactions between aromatic side chains and bases.

scholarly journals In vitro selection of l-DNA aptamers that bind a structured d-RNA molecule

2020 ◽  
Vol 48 (4) ◽  
pp. 1669-1680 ◽  
Author(s):  
Sougata Dey ◽  
Jonathan T Sczepanski
Keyword(s):  
In Vitro Selection ◽  
Rna Binding ◽  
Rna Aptamers ◽  
Dna Aptamers ◽  
Binding Properties ◽  
Rna Molecules ◽  
Dna And Rna ◽  
Selection Of

Abstract The development of structure-specific RNA binding reagents remains a central challenge in RNA biochemistry and drug discovery. Previously, we showed in vitro selection techniques could be used to evolve l-RNA aptamers that bind tightly to structured d-RNAs. However, whether similar RNA-binding properties can be achieved using aptamers composed of l-DNA, which has several practical advantages compared to l-RNA, remains unknown. Here, we report the discovery and characterization of the first l-DNA aptamers against a structured RNA molecule, precursor microRNA-155, thereby establishing the capacity of DNA and RNA molecules of the opposite handedness to form tight and specific ‘cross-chiral’ interactions with each other. l-DNA aptamers bind pre-miR-155 with low nanomolar affinity and high selectivity despite the inability of l-DNA to interact with native d-RNA via Watson–Crick base pairing. Furthermore, l-DNA aptamers inhibit Dicer-mediated processing of pre-miRNA-155. The sequence and structure of l-DNA aptamers are distinct from previously reported l-RNA aptamers against pre-miR-155, indicating that l-DNA and l-RNA interact with the same RNA sequence through unique modes of recognition. Overall, this work demonstrates that l-DNA may be pursued as an alternative to l-RNA for the generation of RNA-binding aptamers, providing a robust and practical approach for targeting structured RNAs.

scholarly journals oRNAment: a database of putative RNA binding protein target sites in the transcriptomes of model species

2019 ◽  
Author(s):  
Louis Philip Benoit Bouvrette ◽  
Samantha Bovaird ◽  
Mathieu Blanchette ◽  
Eric Lécuyer
Keyword(s):  
In Vitro Selection ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Model Organisms ◽  
Rna Motifs ◽  
Non Coding Rna ◽  
Distribution Features ◽  
Available Information ◽  
User Friendly

AbstractProtein–RNA interactions are essential for controlling most aspects of RNA metabolism, including synthesis, processing, trafficking, stability and degradation. In vitro selection methods, such as RNAcompete and RNA Bind-n-Seq, have defined the consensus target motifs of hundreds of RNA-binding proteins (RBPs). However, readily available information about the distribution features of these motifs across full transcriptomes was hitherto lacking. Here, we introduce oRNAment (o RNA motifs enrichment in transcriptomes), a database that catalogues the putative motif instances of 223 RBPs, encompassing 453 motifs, in a transcriptome-wide fashion. The database covers 525 718 complete coding and non-coding RNA species across the transcriptomes of human and four prominent model organisms: Caenorhabditis elegans, Danio rerio, Drosophila melanogaster and Mus musculus. The unique features of oRNAment include: (i) hosting of the most comprehensive mapping of RBP motif instances to date, with 421 133 612 putative binding sites described across five species; (ii) options for the user to filter the data according to a specific threshold; (iii) a user-friendly interface and efficient back-end allowing the rapid querying of the data through multiple angles (i.e. transcript, RBP, or sequence attributes) and (iv) generation of several interactive data visualization charts describing the results of user queries. oRNAment is freely available at http://rnabiology.ircm.qc.ca/oRNAment/.

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