Review

Authors: Stefanie Gerstberger, Markus Hafner, Thomas Tuschl...

Post-transcriptional gene regulation (PTGR) concerns processes involved in the maturation, transport, stability and translation of coding and non-coding RNAs. RNA-binding proteins (RBPs) and ribonucleoproteins coordinate RNA processing and PTGR. The introduction of large-scale quantitative methods, such as next-generation sequencing and modern protein mass spectrometry, has renewed interest in the investigation of PTGR and the protein factors involved at a systems-biology level. Here, we present a census of 1,542 manually curated RBPs that we have analysed for their interactions with different classes of RNA, their evolutionary conservation, their abundance and their tissue-specific expression. Our analysis is a critical step towards the comprehensive characterization of proteins involved in human RNA metabolism.

Topics: Animals; Gene Expression Regulation; Humans; RNA; RNA-Binding Proteins

PubMed: 25365966
DOI: 10.1038/nrg3813

Authors: Ozgur Oksuz, Jonathan E Henninger, Robert Warneford-Thomson...

Transcription factors (TFs) orchestrate the gene expression programs that define each cell's identity. The canonical TF accomplishes this with two domains, one that binds specific DNA sequences and the other that binds protein coactivators or corepressors. We find that at least half of TFs also bind RNA, doing so through a previously unrecognized domain with sequence and functional features analogous to the arginine-rich motif of the HIV transcriptional activator Tat. RNA binding contributes to TF function by promoting the dynamic association between DNA, RNA, and TF on chromatin. TF-RNA interactions are a conserved feature important for vertebrate development and disrupted in disease. We propose that the ability to bind DNA, RNA, and protein is a general property of many TFs and is fundamental to their gene regulatory function.

Topics: Transcription Factors; RNA; Binding Sites; Protein Binding; DNA

PubMed: 37402367
DOI: 10.1016/j.molcel.2023.06.012

Authors: Alice Ghidini, Antoine Cléry, François Halloy...

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

Review

Authors: Xian Zhao, Youxiu Zhong, Xudong Wang...

Circular RNA (circRNA) is a novel endogenous non-coding RNA (ncRNA) that, like microRNA (miRNA), is a rapidly emerging RNA research topic. CircRNA, unlike traditional linear RNAs (which have 5' and 3' ends), has a closed-loop structure that is unaffected by RNA exonucleases. Thus, circRNA has sustained expression and is less sensitive to degradation. Since circRNAs have many miRNAs binding sites, eliminating their repressive effects on their target genes can strongly enhance their expression. CircRNAs serve an important regulatory role in disease onset and progression via specific circRNA-miRNA interactions. We summarized the current progress in elucidating mechanisms and biogenesis of circRNAs in this review. In particular, circRNAs can function mainly as miRNA sponges, regulating host gene expression and protein transportation. Finally, we discussed the application prospects and significant challenges for the development of circRNA-based therapeutics.

Topics: Binding Sites; MicroRNAs; RNA; RNA, Circular

PubMed: 35813288
DOI: 10.7150/ijms.71840

Authors: Sergio Leone, Avinash Srivastava, Andrés Herrero-Ruiz...

Molecular chaperones are critical for protein homeostasis and are implicated in several human pathologies such as neurodegeneration and cancer. While the binding of chaperones to nascent and misfolded proteins has been studied in great detail, the direct interaction between chaperones and RNA has not been systematically investigated. Here, we provide the evidence for widespread interaction between chaperones and RNA in human cells. We show that the major chaperone heat shock protein 70 (HSP70) binds to non-coding RNA transcribed by RNA polymerase III (RNA Pol III) such as tRNA and 5S rRNA. Global chromatin profiling revealed that HSP70 binds genomic sites of transcription by RNA Pol III. Detailed biochemical analyses showed that HSP70 alleviates the inhibitory effect of cognate tRNA transcript on tRNA gene transcription. Thus, our study uncovers an unexpected role of HSP70-RNA interaction in the biogenesis of a specific class of non-coding RNA with wider implications in cancer therapeutics.

Topics: Humans; HSP70 Heat-Shock Proteins; Molecular Chaperones; Neoplasms; RNA; RNA Polymerase III; RNA, Transfer; RNA, Untranslated

PubMed: 38266641
DOI: 10.1016/j.molcel.2024.01.001

Authors: Wen Y Wu, Prarthana Mohanraju, Chunyu Liao...

CRISPR-Cas are prokaryotic adaptive immune systems. Cas nucleases generally use CRISPR-derived RNA guides to specifically bind and cleave DNA or RNA targets. Here, we describe the experimental characterization of a bacterial CRISPR effector protein Cas12m representing subtype V-M. Despite being less than half the size of Cas12a, Cas12m catalyzes auto-processing of a crRNA guide, recognizes a 5'-TTN' protospacer-adjacent motif (PAM), and stably binds a guide-complementary double-stranded DNA (dsDNA). Cas12m has a RuvC domain with a non-canonical catalytic site and accordingly is incapable of guide-dependent cleavage of target nucleic acids. Despite lacking target cleavage activity, the high binding affinity of Cas12m to dsDNA targets allows for interference as demonstrated by its ability to protect bacteria against invading plasmids through silencing invader transcription and/or replication. Based on these molecular features, we repurposed Cas12m by fusing it to a cytidine deaminase that resulted in base editing within a distinct window.

Topics: CRISPR-Associated Proteins; CRISPR-Cas Systems; DNA; Plasmids; RNA; RNA, Guide, CRISPR-Cas Systems

PubMed: 36427491
DOI: 10.1016/j.molcel.2022.11.003

Authors: Yuxiang Sun, Hui Dai, Xiaoxia Dai...

Microsatellite repeat expansions within genes contribute to a number of neurological diseases. The accumulation of toxic proteins and RNA molecules with repetitive sequences, and/or sequestration of RNA-binding proteins by RNA molecules containing expanded repeats are thought to be important contributors to disease aetiology. Here we reveal that the adenosine in CAG repeat RNA can be methylated to N-methyladenosine (mA) by TRMT61A, and that mA can be demethylated by ALKBH3. We also observed that the mA/adenosine ratio in CAG repeat RNA increases with repeat length, which is attributed to diminished expression of ALKBH3 elicited by the repeat RNA. Additionally, TDP-43 binds directly and strongly with mA in RNA, which stimulates the cytoplasmic mis-localization and formation of gel-like aggregates of TDP-43, resembling the observations made for the protein in neurological diseases. Moreover, mA in CAG repeat RNA contributes to CAG repeat expansion-induced neurodegeneration in Caenorhabditis elegans and Drosophila. In sum, our study offers a new paradigm of the mechanism through which nucleotide repeat expansion contributes to neurological diseases and reveals a novel pathological function of mA in RNA. These findings may provide an important mechanistic basis for therapeutic intervention in neurodegenerative diseases emanating from CAG repeat expansion.

Topics: Animals; Humans; Adenosine; Caenorhabditis elegans; DNA-Binding Proteins; Drosophila melanogaster; Neurodegenerative Diseases; RNA; Trinucleotide Repeat Expansion; Cytoplasm; Disease Models, Animal

PubMed: 37938769
DOI: 10.1038/s41586-023-06701-5

Authors: P Emsley, B Lohkamp, W G Scott...

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

Authors: Jan-Niklas Tants, Lasse Oberstrass, Julia E Weigand...

Zinc finger (ZnF) domains appear in a pool of structural contexts and despite their small size achieve varying target specificities, covering single-stranded and double-stranded DNA and RNA as well as proteins. Combined with other RNA-binding domains, ZnFs enhance affinity and specificity of RNA-binding proteins (RBPs). The ZnF-containing immunoregulatory RBP Roquin initiates mRNA decay, thereby controlling the adaptive immune system. Its unique ROQ domain shape-specifically recognizes stem-looped cis-elements in mRNA 3'-untranslated regions (UTR). The N-terminus of Roquin contains a RING domain for protein-protein interactions and a ZnF, which was suggested to play an essential role in RNA decay by Roquin. The ZnF domain boundaries, its RNA motif preference and its interplay with the ROQ domain have remained elusive, also driven by the lack of high-resolution data of the challenging protein. We provide the solution structure of the Roquin-1 ZnF and use an RBNS-NMR pipeline to show that the ZnF recognizes AU-rich RNAs. We systematically refine the contributions of adenines in a poly(U)-background to specific complex formation. With the simultaneous binding of ROQ and ZnF to a natural target transcript of Roquin, our study for the first time suggests how Roquin integrates RNA shape and sequence features through the ROQ-ZnF tandem.

Topics: Zinc Fingers; RNA-Binding Proteins; Protein Binding; Humans; Models, Molecular; RNA; Binding Sites; 3' Untranslated Regions; RNA, Messenger; RNA Stability; Ubiquitin-Protein Ligases

PubMed: 38953172
DOI: 10.1093/nar/gkae555

Authors: Yajun Wang, Wei Zhu, Yumi Jang...

Telomeric repeat-containing RNA (TERRA) and its formation of RNA:DNA hybrids (or TERRA R-loops), influence telomere maintenance, particularly in human cancer cells that use homologous recombination-mediated alternative lengthening of telomeres. Here, we report that the RNA-binding motif protein 14 (RBM14) is associated with telomeres in human cancer cells. RBM14 negatively regulates TERRA expression. It also binds to TERRA and inhibits it from forming TERRA R-loops at telomeres. RBM14 depletion has several effects, including elevated TERRA levels, telomeric R-loops, telomere dysfunction-induced DNA damage foci formation, particularly in the presence of DNA replication stress, pRPA32 accumulation at telomeres and telomere signal-free ends. Thus, RBM14 protects telomere integrity via modulating TERRA levels and its R-loop formation at telomeres.

Topics: Humans; DNA; R-Loop Structures; RNA; RNA, Long Noncoding; RNA-Binding Motifs; Telomere; Telomere Homeostasis; Neoplasms

PubMed: 37930826
DOI: 10.1093/nar/gkad967