Over the past few years, intercalated motifs (i‐motifs) have attracted attention due to the direct visualization of their existence in the nuclei of human cells. Traditionally, i‐motifs have been ...studied using expensive and complicated NMR, and/or relatively inexpensive but less common circular dichroism spectrometry. The aim of this study was to investigate the feasibility of using less expensive, less complicated, and more widely available CE as an alternative for i‐motif related research. The mobilities of two DNA and RNA i‐motifs in CE were determined under different pH conditions. Our results demonstrate that CE is able to identify and differentiate mostly folded, partially folded, and mostly unfolded DNA and RNA i‐motifs through changes in peak shape and migration time, thus providing a new method to study both i‐motif conformation and the interactions between i‐motifs and their ligands.
Summary
The pentatricopeptide repeat (PPR) proteins form one of the largest protein families in land plants. They are characterised by tandem 30–40 amino acid motifs that form an extended binding ...surface capable of sequence‐specific recognition of RNA strands. Almost all of them are post‐translationally targeted to plastids and mitochondria, where they play important roles in post‐transcriptional processes including splicing, RNA editing and the initiation of translation. A code describing how PPR proteins recognise their RNA targets promises to accelerate research on these proteins, but making use of this code requires accurate definition and annotation of all of the various nucleotide‐binding motifs in each protein. We have used a structural modelling approach to define 10 different variants of the PPR motif found in plant proteins, in addition to the putative deaminase motif that is found at the C‐terminus of many RNA‐editing factors. We show that the super‐helical RNA‐binding surface of RNA‐editing factors is potentially longer than previously recognised. We used the redefined motifs to develop accurate and consistent annotations of PPR sequences from 109 genomes. We report a high error rate in PPR gene models in many public plant proteomes, due to gene fusions and insertions of spurious introns. These consistently annotated datasets across a wide range of species are valuable resources for future comparative genomics studies, and an essential pre‐requisite for accurate large‐scale computational predictions of PPR targets. We have created a web portal (http://www.plantppr.com) that provides open access to these resources for the community.
Significance Statement
Accurate prediction of the RNA targets of pentatricopeptide repeat (PPR) proteins requires accurate annotation of their RNA‐binding motifs. Here we used structural modelling to define 10 different variants of PPR motifs in plant proteins and used these redefined motifs to develop improved annotations of PPRs from 109 genomes. These consistently annotated datasets are valuable resources for comparative genomics studies and for large‐scale prediction of PPR function.
Review of structural and signaling roles of evolutionary conserved motifs and residues in vertebrate chemokine receptors.
Chemokine receptors regulate cell migration and homing. They belong to the ...rhodopsin‐like family of GPCRs. Their ancestor genes emerged in the early stages of vertebrate evolution. Since then, the family has been greatly expanded through whole and segmental genome duplication events. During evolution, many amino acid changes have been introduced in individual chemokine receptors, but certain motifs and residues are highly conserved. Previously, we proposed a nomenclature system of the vertebrate chemokine receptors based on their evolutionary history and phylogenetic analyses. With the use of this classification system, we are now able to confidently assign the species orthologs of vertebrate chemokine receptors. Here, we systematically analyze conserved motifs and residues of each group of orthologous chemokine receptors that may play important roles in their signaling and biologic functions. Our present analysis may provide useful information on how individual chemokine receptors are activated upon ligand binding.
RNA helicases and E3 ubiquitin ligases mediate many critical functions in cells, but their actions have largely been studied in distinct biological contexts. Here, we uncover evolutionarily conserved ...rules of engagement between RNA helicases and tripartite motif (TRIM) E3 ligases that lead to their functional coordination in vertebrate innate immunity. Using cryoelectron microscopy and biochemistry, we show that RIG-I-like receptors (RLRs), viral RNA receptors with helicase domains, interact with their cognate TRIM/TRIM-like E3 ligases through similar epitopes in the helicase domains. Their interactions are avidity driven, restricting the actions of TRIM/TRIM-like proteins and consequent immune activation to RLR multimers. Mass spectrometry and phylogeny-guided biochemical analyses further reveal that similar rules of engagement may apply to diverse RNA helicases and TRIM/TRIM-like proteins. Our analyses suggest not only conserved substrates for TRIM proteins but also, unexpectedly, deep evolutionary connections between TRIM proteins and RNA helicases, linking ubiquitin and RNA biology throughout animal evolution.
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•TRIM65 selectively recognizes MDA5 filaments using PSpry bivalency•TRIM65 PSpry binds α1/α3 helices in the Hel2 domain of MDA5•RIPLET recognizes RIG-I using a similar epitope in the helicase domain•Distinct TRIM proteins recognize common epitopes in diverse helicases using bivalency
Proper immune function requires multiple layers of checks and balances. Kato et al. show a conserved mechanism by which the antiviral proteins RIG-I-like receptors collaborate with a family of E3 ligases, TRIM-like proteins, to ensure high fidelity and robustness of the antiviral immune response.
Mitogen‐activated protein kinases (MAPKs; ERK1/2, p38, JNK, and ERK5) have evolved to transduce environmental and developmental signals (growth factors, stress) into adaptive and programmed responses ...(differentiation, inflammation, apoptosis). Almost 20 years ago, it was discovered that MAPKs contain a docking site in the C‐terminal lobe that binds a conserved 13‐16 amino acid sequence known as the D‐ or KIM‐motif (kinase interaction motif). Recent crystal structures of MAPK:KIM‐peptide complexes are leading to a precise understanding of how KIM sequences contribute to MAPK selectivity. In addition, new crystal and especially NMR studies are revealing how residues outside the canonical KIM motif interact with specific MAPKs and contribute further to MAPK selectivity and signaling pathway fidelity. In this review, we focus on these recent studies, with an emphasis on the use of NMR spectroscopy, isothermal titration calorimetry and small angle X‐ray scattering to investigate these processes.
Previous studies demonstrate the usefulness of using multiple tools and methods for improving the accuracy of motif detection. Over the past years, numerous motif discovery pipelines have been ...developed. However, they typically report only the top ranked results either from individual motif finders or from a combination of multiple tools and algorithms.
Here we present MODSIDE, a motif discovery pipeline and similarity detector. The pipeline integrated four de novo motif finders: ChIPMunk, MEME, Weeder, and XXmotif. It also incorporated a motif similarity detection tool MOTIFSIM. MODSIDE was designed for delivering not only the predictive results from individual motif finders but also the comparison results for multiple tools. The results include the common significant motifs from multiple tools, the motifs detected by some tools but not by others, and the best matches for each motif in the motif collection of multiple tools. MODSIDE also possesses other useful features for merging similar motifs and clustering motifs into motif trees.
We evaluated MODSIDE and its adopted motif finders on 16 benchmark datasets. The statistical results demonstrate MODSIDE achieves better accuracy than individual motif finders. We also compared MODSIDE with two popular motif discovery pipelines: MEME-ChIP and RSAT peak-motifs. The comparison results reveal MODSIDE attains similar performance as RSAT peak-motifs but better accuracy than MEME-ChIP. In addition, MODSIDE is able to deliver various comparison results that are not offered by MEME-ChIP, RSAT peak-motifs, and other existing motif discovery pipelines.
Long noncoding RNAs (lncRNA) have been associated with various types of cancer; however, the precise role of many lncRNAs in tumorigenesis remains elusive. Here we demonstrate that the cytosolic ...lncRNA P53RRA is downregulated in cancers and functions as a tumor suppressor by inhibiting cancer progression. Chromatin remodeling proteins LSH and Cfp1 silenced or increased P53RRA expression, respectively. P53RRA bound Ras GTPase-activating protein-binding protein 1 (G3BP1) using nucleotides 1 and 871 of P53RRA and the RRM interaction domain of G3BP1 (aa 177-466). The cytosolic P53RRA-G3BP1 interaction displaced p53 from a G3BP1 complex, resulting in greater p53 retention in the nucleus, which led to cell-cycle arrest, apoptosis, and ferroptosis. P53RRA promoted ferroptosis and apoptosis by affecting transcription of several metabolic genes. Low P53RRA expression significantly correlated with poor survival in patients with breast and lung cancers harboring wild-type p53. These data show that lncRNAs can directly interact with the functional domain of signaling proteins in the cytoplasm, thus regulating p53 modulators to suppress cancer progression.
A cytosolic lncRNA functions as a tumor suppressor by activating the p53 pathway.
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Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed ...unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins—structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.
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•Global calcineurin signaling in humans revealed through systematic substrate mapping•Discovery of calcineurin-binding sequences enables robust in silico SLiM predictions•BioID uncovers SLiM-dependent calcineurin proximity to nuclear pores and centrosomes•Calcineurin dephosphorylates nuclear pore proteins and regulates transport in vivo
Wigington, Roy, et al. elucidate a signaling network for the Ca2+-activated phosphatase calcineurin by systematically uncovering calcineurin-binding SLiMs throughout the human proteome. Using in vitro, in silico, and in vivo methods, they discover multiple calcineurin substrates, including Notch1 and several nucleoporins, leading to the unexpected finding that calcineurin regulates nuclear transport.
Transposable elements represent nearly half of mammalian genomes and are generally described as parasites, or “junk DNA.” The LINE1 retrotransposon is the most abundant class and is thought to be ...deleterious for cells, yet it is paradoxically highly expressed during early development. Here, we report that LINE1 plays essential roles in mouse embryonic stem cells (ESCs) and pre-implantation embryos. In ESCs, LINE1 acts as a nuclear RNA scaffold that recruits Nucleolin and Kap1/Trim28 to repress Dux, the master activator of a transcriptional program specific to the 2-cell embryo. In parallel, LINE1 RNA mediates binding of Nucleolin and Kap1 to rDNA, promoting rRNA synthesis and ESC self-renewal. In embryos, LINE1 RNA is required for Dux silencing, synthesis of rRNA, and exit from the 2-cell stage. The results reveal an essential partnership between LINE1 RNA, Nucleolin, Kap1, and peri-nucleolar chromatin in the regulation of transcription, developmental potency, and ESC self-renewal.
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•LINE1 RNA is abundant and nuclear in mouse ESCs and pre-implantation embryos•LINE1 knockdown inhibits ESC self-renewal and induces transition to a 2C state•LINE1 RNA recruits Nucleolin/Kap1 to repress Dux and activate rRNA synthesis•In embryos, LINE1 inhibition causes persistence of the 2C program and impairs ZGA
RNA transcribed from LINE1 elements acts as a nuclear scaffold to direct gene expression programs essential for ESC self-renewal and pre-implantation embryo development.
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