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Angewandte Chemie (International Ed. in... Feb 2021Defects in the functions of RNA binding proteins (RBPs) are at the origin of many diseases; however, targeting RBPs with conventional drugs has proven difficult. PROTACs...
Defects in the functions of RNA binding proteins (RBPs) are at the origin of many diseases; however, targeting RBPs with conventional drugs has proven difficult. PROTACs are a new class of drugs that mediate selective degradation of a target protein through a cell's ubiquitination machinery. PROTACs comprise a moiety that binds the selected protein, conjugated to a ligand of an E3 ligase. Herein, we introduce RNA-PROTACs as a new concept in the targeting of RBPs. These chimeric structures employ small RNA mimics as targeting groups that dock the RNA-binding site of the RBP, whereupon a conjugated E3-recruiting peptide derived from the HIF-1α protein directs the RBP for proteasomal degradation. We performed a proof-of-concept demonstration with the degradation of two RBPs-a stem cell factor LIN28 and a splicing factor RBFOX1-and showed their use in cancer cell lines. The RNA-PROTAC approach opens the way to rapid, selective targeting of RBPs in a rational and general fashion.
Topics: Base Sequence; Binding Sites; Cell Line, Tumor; Humans; Oligonucleotides; Peptides; Proteolysis; RNA; RNA Splicing Factors; RNA-Binding Proteins; Ubiquitin-Protein Ligases
PubMed: 33108679
DOI: 10.1002/anie.202012330 -
Acta Crystallographica. Section D,... Apr 2010Coot is a molecular-graphics application for model building and validation of biological macromolecules. The program displays electron-density maps and atomic models and...
Coot is a molecular-graphics application for model building and validation of biological macromolecules. The program displays electron-density maps and atomic models and allows model manipulations such as idealization, real-space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers and Ramachandran idealization. Furthermore, tools are provided for model validation as well as interfaces to external programs for refinement, validation and graphics. The software is designed to be easy to learn for novice users, which is achieved by ensuring that tools for common tasks are 'discoverable' through familiar user-interface elements (menus and toolbars) or by intuitive behaviour (mouse controls). Recent developments have focused on providing tools for expert users, with customisable key bindings, extensions and an extensive scripting interface. The software is under rapid development, but has already achieved very widespread use within the crystallographic community. The current state of the software is presented, with a description of the facilities available and of some of the underlying methods employed.
Topics: Crystallography, X-Ray; DNA; Models, Molecular; Nucleic Acid Conformation; Protein Structure, Tertiary; Proteins; RNA; Software Design
PubMed: 20383002
DOI: 10.1107/S0907444910007493 -
Life Science Alliance Sep 2023An intronic GGGGCC repeat expansion in is a common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeats are transcribed in both sense...
An intronic GGGGCC repeat expansion in is a common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeats are transcribed in both sense and antisense directions to generate distinct dipeptide repeat proteins, of which poly(GA), poly(GR), and poly(PR) have been implicated in contributing to neurodegeneration. Poly(PR) binding to RNA may contribute to toxicity, but analysis of poly(PR)-RNA binding on a transcriptome-wide scale has not yet been carried out. We therefore performed crosslinking and immunoprecipitation (CLIP) analysis in human cells to identify the RNA binding sites of poly(PR). We found that poly(PR) binds to nearly 600 RNAs, with the sequence GAAGA enriched at the binding sites. In vitro experiments showed that poly(GAAGA) RNA binds poly(PR) with higher affinity than control RNA and induces the phase separation of poly(PR) into condensates. These data indicate that poly(PR) preferentially binds to poly(GAAGA)-containing RNAs, which may have physiological consequences.
Topics: Humans; Transcriptome; C9orf72 Protein; Gene Expression Profiling; Poly A; Dipeptides; RNA
PubMed: 37438085
DOI: 10.26508/lsa.202201824 -
Nature Communications Nov 2023PUF proteins are characterized by globular RNA-binding domains. They also interact with partner proteins that modulate their RNA-binding activities. Caenorhabditis...
PUF proteins are characterized by globular RNA-binding domains. They also interact with partner proteins that modulate their RNA-binding activities. Caenorhabditis elegans PUF protein fem-3 binding factor-2 (FBF-2) partners with intrinsically disordered Lateral Signaling Target-1 (LST-1) to regulate target mRNAs in germline stem cells. Here, we report that an intrinsically disordered region (IDR) at the C-terminus of FBF-2 autoinhibits its RNA-binding affinity by increasing the off rate for RNA binding. Moreover, the FBF-2 C-terminal region interacts with its globular RNA-binding domain at the same site where LST-1 binds. This intramolecular interaction restrains an electronegative cluster of amino acid residues near the 5' end of the bound RNA to inhibit RNA binding. LST-1 binding in place of the FBF-2 C-terminus therefore releases autoinhibition and increases RNA-binding affinity. This regulatory mechanism, driven by IDRs, provides a biochemical and biophysical explanation for the interdependence of FBF-2 and LST-1 in germline stem cell self-renewal.
Topics: Animals; RNA; Caenorhabditis elegans Proteins; Caenorhabditis elegans; Protein Binding; RNA, Messenger
PubMed: 37953271
DOI: 10.1038/s41467-023-43098-1 -
Wiley Interdisciplinary Reviews. RNA 2014RNA hairpins are the most commonly occurring secondary structural elements in RNAs and serve as nucleation sites for RNA folding, RNA-RNA, and RNA-protein interactions.... (Review)
Review
RNA hairpins are the most commonly occurring secondary structural elements in RNAs and serve as nucleation sites for RNA folding, RNA-RNA, and RNA-protein interactions. RNA hairpins are frequently capped by tetraloops, and based on sequence similarity, three broad classes of RNA tetraloops have been defined: GNRA, UNCG, and CUYG. Other classes such as the UYUN tetraloop in histone mRNAs, the UGAA in 16S rRNA, the AUUA tetraloop from the MS2 bacteriophage, and the AGNN tetraloop that binds RNase III have also been characterized. The tetraloop structure is compact and is usually characterized by a paired interaction between the first and fourth nucleotides. The two unpaired nucleotides in the loop are usually involved in base-stacking or base-phosphate hydrogen bonding interactions. Several structures of RNA tetraloops, free and complexed to other RNAs or proteins, are now available and these studies have increased our understanding of the diverse mechanisms by which this motif is recognized. RNA tetraloops can mediate RNA-RNA contacts via the tetraloop-receptor motif, kissing hairpin loops, A-minor interactions, and pseudoknots. While these RNA-RNA interactions are fairly well understood, how RNA-binding proteins recognize RNA tetraloops and tetraloop-like motifs remains unclear. In this review, we summarize the structures of RNA tetraloop-protein complexes and the general themes that have emerged on sequence- and structure-specific recognition of RNA tetraloops. We highlight how proteins achieve molecular recognition of this nucleic acid motif, the structural adaptations observed in the tetraloop to accommodate the protein-binding partner, and the role of dynamics in recognition.
Topics: Animals; Binding Sites; Humans; Models, Molecular; Nucleic Acid Conformation; Protein Structure, Secondary; RNA; RNA Folding; RNA-Binding Proteins
PubMed: 24124096
DOI: 10.1002/wrna.1196 -
Angewandte Chemie (International Ed. in... Mar 2023Small molecule targeting of RNA has emerged as a new frontier in medicinal chemistry, but compared to the protein targeting literature our understanding of chemical...
Small molecule targeting of RNA has emerged as a new frontier in medicinal chemistry, but compared to the protein targeting literature our understanding of chemical matter that binds to RNA is limited. In this study, we reported Repository Of BInders to Nucleic acids (ROBIN), a new library of nucleic acid binders identified by small molecule microarray (SMM) screening. The complete results of 36 individual nucleic acid SMM screens against a library of 24 572 small molecules were reported (including a total of 1 627 072 interactions assayed). A set of 2 003 RNA-binding small molecules was identified, representing the largest fully public, experimentally derived library of its kind to date. Machine learning was used to develop highly predictive and interpretable models to characterize RNA-binding molecules. This work demonstrates that machine learning algorithms applied to experimentally derived sets of RNA binders are a powerful method to inform RNA-targeted chemical space.
Topics: RNA; Gene Library; Machine Learning; Biological Assay; Microarray Analysis
PubMed: 36584293
DOI: 10.1002/anie.202211358 -
RNA Biology 2014Small regulatory RNAs (sRNAs) are universally distributed in all three domains of life, Archaea, Bacteria, and Eukaryotes. In bacteria, sRNAs typically function by... (Review)
Review
Small regulatory RNAs (sRNAs) are universally distributed in all three domains of life, Archaea, Bacteria, and Eukaryotes. In bacteria, sRNAs typically function by binding near the translation start site of their target mRNAs and thereby inhibit or activate translation. In eukaryotes, miRNAs and siRNAs typically bind to the 3'-untranslated region (3'-UTR) of their target mRNAs and influence translation efficiency and/or mRNA stability. In archaea, sRNAs have been identified in all species investigated using bioinformatic approaches, RNomics, and RNA-Seq. Their size can vary significantly between less than 50 to more than 500 nucleotides. Differential expression of sRNA genes has been studied using northern blot analysis, microarrays, and RNA-Seq. In addition, biological functions have been unraveled by genetic approaches, i.e., by characterization of designed mutants. As in bacteria, it was revealed that archaeal sRNAs are involved in many biological processes, including metabolic regulation, adaptation to extreme conditions, stress responses, and even in regulation of morphology and cellular behavior. Recently, the first target mRNAs were identified in archaea, including one sRNA that binds to the 5'-region of two mRNAs in Methanosarcina mazei Gö1 and a few sRNAs that bind to 3'-UTRs in Sulfolobus solfataricus, three Pyrobaculum species, and Haloferax volcanii, indicating that archaeal sRNAs appear to be able to target both the 5'-UTR or the 3'-UTRs of their respective target mRNAs. In addition, archaea contain tRNA-derived fragments (tRFs), and one tRF has been identified as a major ribosome-binding sRNA in H. volcanii, which downregulates translation in response to stress. Besides regulatory sRNAs, archaea contain further classes of sRNAs, e.g., CRISPR RNAs (crRNAs) and snoRNAs.
Topics: Archaea; Base Pairing; Clustered Regularly Interspaced Short Palindromic Repeats; Gene Expression Profiling; Gene Expression Regulation, Archaeal; Genomics; RNA, Antisense; RNA, Archaeal; RNA, Small Untranslated; RNA, Transfer
PubMed: 24755959
DOI: 10.4161/rna.28452 -
ELife Jan 2022Methyltransferase like-3 (METTL3) and METTL14 complex transfers a methyl group from -adenosyl-L-methionine to amino group of adenosine bases in RNA (mA) and DNA (mdA)....
Methyltransferase like-3 (METTL3) and METTL14 complex transfers a methyl group from -adenosyl-L-methionine to amino group of adenosine bases in RNA (mA) and DNA (mdA). Emerging evidence highlights a role of METTL3-METTL14 in the chromatin context, especially in processes where DNA and RNA are held in close proximity. However, a mechanistic framework about specificity for substrate RNA/DNA and their interrelationship remain unclear. By systematically studying methylation activity and binding affinity to a number of DNA and RNA oligos with different propensities to form inter- or intra-molecular duplexes or single-stranded molecules in vitro, we uncover an inverse relationship for substrate binding and methylation and show that METTL3-METTL14 preferentially catalyzes the formation of mdA in single-stranded DNA (ssDNA), despite weaker binding affinity to DNA. In contrast, it binds structured RNAs with high affinity, but methylates the target adenosine in RNA (mA) much less efficiently than it does in ssDNA. We also show that METTL3-METTL14-mediated methylation of DNA is largely restricted by structured RNA elements prevalent in long noncoding and other cellular RNAs.
Topics: DNA Methylation; DNA, Single-Stranded; Deoxyadenosines; Humans; Methyltransferases; RNA
PubMed: 35060905
DOI: 10.7554/eLife.67150 -
Seminars in Cell & Developmental Biology Feb 2019The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a diverse family of RNA binding proteins that function in most stages of RNA metabolism. The prototypical... (Review)
Review
The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a diverse family of RNA binding proteins that function in most stages of RNA metabolism. The prototypical member, hnRNP A1, is composed of three major domains; tandem N-terminal RNA Recognition Motifs (RRMs) and a C-terminal mostly intrinsically disordered region. HnRNP A1 is broadly implicated in basic cellular RNA processing events such as splicing, stability, nuclear export and translation. Due to its ubiquity and abundance, hnRNP A1 is also frequently usurped to control viral gene expression. Deregulation of the RNA metabolism functions of hnRNP A1 in neuronal cells contributes to several neurodegenerative disorders. Because of these roles in human pathologies, the study of hnRNP A1 provides opportunities for the development of novel therapeutics, with disruption of its RNA binding capabilities being the most promising target. The functional diversity of hnRNP A1 is reflected in the complex nature by which it interacts with various RNA targets. Indeed, hnRNP A1 binds both structured and unstructured RNAs with binding affinities that span several magnitudes. Available structures of hnRNP A1-RNA complexes also suggest a degree of plasticity in molecular recognition. Given the reinvigoration in hnRNP A1, the goal of this review is to use the available structural biochemical developments as a framework to interpret its wide-range of RNA functions.
Topics: Binding Sites; Heterogeneous Nuclear Ribonucleoprotein A1; Humans; RNA; Substrate Specificity
PubMed: 29625167
DOI: 10.1016/j.semcdb.2018.04.001 -
Wiley Interdisciplinary Reviews. RNA Jul 2018Hfq is a ubiquitous, Sm-like RNA binding protein found in most bacteria and some archaea. Hfq binds small regulatory RNAs (sRNAs), facilitates base pairing between sRNAs... (Review)
Review
Hfq is a ubiquitous, Sm-like RNA binding protein found in most bacteria and some archaea. Hfq binds small regulatory RNAs (sRNAs), facilitates base pairing between sRNAs and their mRNA targets, and directly binds and regulates translation of certain mRNAs. Because sRNAs regulate many stress response pathways in bacteria, Hfq is essential for adaptation to different environments and growth conditions. The chaperone activities of Hfq arise from multipronged RNA binding by three different surfaces of the Hfq hexamer. The manner in which the structured Sm core of Hfq binds RNA has been well studied, but recent work shows that the intrinsically disordered C-terminal domain of Hfq modulates sRNA binding, creating a kinetic hierarchy of RNA competition for Hfq and ensuring the release of double-stranded sRNA-mRNA complexes. A combination of structural, biophysical, and genetic experiments reveals how Hfq recognizes its RNA substrates and plays matchmaker for sRNAs and mRNAs in the cell. The interplay between structured and disordered domains of Hfq optimizes sRNA-mediated post-transcriptional regulation, and is a common theme in RNA chaperones. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry.
Topics: Bacteria; Host Factor 1 Protein; Molecular Chaperones; RNA Processing, Post-Transcriptional; RNA, Bacterial; RNA, Messenger; RNA, Small Untranslated
PubMed: 29633565
DOI: 10.1002/wrna.1475