RNA-binding Proteins Research Articles

Inhibition of RNA splicing triggers CHMP7 nuclear entry, impacting TDP-43 function and leading to the onset of ALS cellular phenotypes.

Amyotrophic lateral sclerosis (ALS) is linked to the reduction of certain nucleoporins in neurons. Increased nuclear localization of charged multivesicular body protein 7 (CHMP7), a protein involved in nuclear pore surveillance, has been identified as a key factor damaging nuclear pores and disrupting transport. Using CRISPR-based microRaft, followed by gRNA identification (CRaft-ID), we discovered 55 RNA-binding proteins (RBPs) that influence CHMP7 localization, including SmD1, a survival of motor neuron (SMN) complex component. Immunoprecipitation-mass spectrometry (IP-MS) and enhanced crosslinking and immunoprecipitation (CLIP) analyses revealed CHMP7's interactions with SmD1, small nuclear RNAs, and splicing factor mRNAs in motor neurons (MNs). ALS induced pluripotent stem cell (iPSC)-MNs show reduced SmD1 expression, and inhibiting SmD1/SMN complex increased CHMP7 nuclear localization. Crucially, overexpressing SmD1 in ALS iPSC-MNs restored CHMP7's cytoplasmic localization and corrected STMN2 splicing. Our findings suggest that early ALS pathogenesis is driven by SMN complex dysregulation.

Impact of RNA Enzymes and RNA Protein Complexes in Unleashing RNAInterference for Manyfold Crop Improvements: A Review

RNA interference, which enables more accurate downregulation of gene expression without interfering with the expression of various genes, is a beneficial instrument for gene silencing and a potential gene regulatory strategy in functional genomics that has a big impact on crop progress. Small interfering RNA molecules swift up the RNAi gene silencing machinery, effectually suppressing a target gene. Subsequently transgene proteins not present from genetically modified RNAi plants, it has a sum of rewards over orthodox transgenic methods. Dicer, Drosha which are RNA enzymes, along with protein complexes, RISC and Argonaute, institute at the epicenter of this technology. These explicit RNA binding proteins and ribonucleases regulate the synthesis and production of tiny regulating RNAs. Several latent usages for this ground-breaking process in agriculture have been identified for improving crops, such as the construction of disease-resistant, biotic or abiotic, high-yielding, stress-tolerant and exclusive assortments; enhancement of nutritional quality; removal of allergens and toxins; and postponing the ripening of fruits and vegetables for extended storage periods. This ground-breaking procedure has the latent to be further utilized to pave the way for the creation of environmentally friendly biotech methods for crop protection and enhancement. Improving research and development activities in this auspicious field is imperative to ensure the efficient appropriate and safe application of these instruments as enduring remedies for agricultural enhancement.

HnRNP A1, hnRNP A2B1, and hnRNP K are dysregulated in tauopathies, but do not colocalize with tau pathology.

Tau interacts with multiple heterogeneous nuclear ribonucleoproteins (hnRNPs)-a family of RNA binding proteins that regulate multiple known cellular functions, including mRNA splicing, mRNA transport, and translation regulation. We have previously demonstrated particularly significant interactions between phosphorylated tau and three hnRNPs (hnRNP A1, hnRNP A2B1, and hnRNP K). Although multiple hnRNPs have been previously implicated in tauopathies, knowledge of whether these hnRNPs colocalize with tau aggregates or show cellular mislocalization in disease is limited. Here, we performed a neuropathological study examining the colocalization between hnRNP A1, hnRNP A2B1, hnRNP K, and phosphorylated tau in two brain regions (hippocampus and frontal cortex) in six disease groups (Alzheimer's disease, mild cognitive impairment, progressive supranuclear palsy, corticobasal degeneration, Pick's disease, and controls). Contrary to expectations, hnRNP A1, hnRNP A2B1, and hnRNP K did not colocalize with AT8-immunoreactive phosphorylated tau pathology in any of the tauopathies examined. However, we did observe significant cellular mislocalization of hnRNP A1, hnRNP A2B1 and hnRNP K in tauopathies, with unique patterns of mislocalization observed for each hnRNP. These data point to broad dysregulation of hnRNP A1, A2B1 and K across tauopathies with implications for disease processes and RNA regulation.

Regulation of synapse density by Pumilio RNA-binding proteins

Regulation of synapse density by Pumilio RNA-binding proteins

RNA-Binding Protein-Mediated Alternative Splicing Regulates Abiotic Stress Responses in Plants.

The alternative splicing of pre-mRNA generates distinct mRNA variants from a pre-mRNA, thereby modulating a gene's function. The splicing of pre-mRNA depends on splice sites and regulatory elements in pre-mRNA, as well as the snRNA and proteins that recognize these sequences. Among these, RNA-binding proteins (RBPs) are the primary regulators of pre-mRNA splicing and play a critical role in the regulation of alternative splicing by recognizing the elements in pre-mRNA. However, little is known about the function of RBPs in stress response in plants. Here, we summarized the RBPs involved in the alternative splicing of pre-mRNA and their recognizing elements in pre-mRNA, and the recent advance in the role of RBP-mediated alternative splicing in response to abiotic stresses in plants. This review proposes that the regulation of pre-mRNA alternative splicing by RBPs is an important way for plants to adapt to abiotic stresses, and the regulation of alternative splicing by RBPs is a promising direction for crop breeding.

HuR (ELAVL1) regulates the CCHFV minigenome and HAZV replication by associating with viral genomic RNA.

Crimean-Congo Hemorrhagic Fever virus (CCHFV) is a tick-borne pathogen that causes severe acute fever disease in humans and requires a biosafety level 4 laboratory for handling. Hazara virus (HAZV), belonging to the same virus genus as CCHFV, does not exhibit pathogenesis in humans. To investigate host RNA-binding proteins (RBPs) that regulate CCHFV replication, we generated a series of mutant RAW264.7 cells by CRISPR/Cas9 system and these cells were infected with HAZV. The viral titers in the supernatant of these cells was investigated, and HuR (ELAVL1) was identified. HuR KO RAW264.7 cells reduced HAZV replication. HuR is an RBP that enhances mRNA stability by binding to adenyl-uridine (AU)-rich regions in their 3' non-coding region (NCR). HuR regulates innate immune response by binding to host mRNAs of signaling molecules. The expression of cytokine genes such as Ifnb, Il6, and Tnf was reduced in HuR KO cells after HAZV infection. Although HuR supports the innate immune response during HAZV infection, we found that innate immune activation by HAZV infection did not affect its replication. We then investigated whether HuR regulates HAZV genome RNA stability. HAZV RNA genome was precipitated with an anti-HuR antibody, and HAZV genome RNA stability was lowered in HuR KO cells. We found that HuR associated with HAZV RNA and stabilized it to enhance HAZV replication. Furthermore, HuR-deficiency reduced CCHFV minigenome replication. CCHFV is a negative-strand RNA virus and positive-strand RNA is produced during replication. HuR was associated with positive-strand RNA rather than negative-strand RNA, and AU-rich region in 3'-NCR of S segment was responsible for immunoprecipitation with anti-HuR antibody and minigenome replication. Additionally, HuR inhibitor treatment reduced CCHFV minigenome replication. Our results indicate that HuR aids replication of the CCHFV minigenome by associating with the AU-rich region in the 3'-NCR.

Comprehensive Profiling of Roquin Binding Preferences for RNA Stem-Loops.

The cellular levels of mRNAs are controlled post-transcriptionally by cis-regulatory elements located in the 3'-untranslated region. These linear or structured elements are recognized by RNA-binding proteins (RBPs) to modulate mRNA stability. The Roquin-1 and -2 proteins specifically recognize RNA stem-loop motifs, the trinucleotide loop-containing constitutive decay elements (CDEs) and the hexanucleotide loop-containing alternative decay elements (ADEs), with their unique ROQ domain to initiate mRNA degradation. However, the RNA-binding capacity of Roquin towards different classes of stem-loops has not been rigorously characterized, leaving its exact binding preferences unclear. Here, we map the RNA-binding preference of the ROQ domain at nucleotide resolution introducing sRBNS (structured RNA Bind-n-Seq), a customized RBNS workflow with pre-structured RNA libraries. We found a clear preference of Roquin towards specific loop sizes and extended the consensus motifs for CDEs and ADEs. The newly identified motifs are recognized with nanomolar affinity through the canonical RNA-ROQ interface. Using these new stem-loop variants as blueprints, we predicted novel Roquin target mRNAs and verified the expanded target space in cells. The study demonstrates the power of high-throughput assays including RNA structure formation for the systematic investigation of (structural) RNA-binding preferences to comprehensively identify mRNA targets and elucidate the biological function of RBPs.

Activity-Based Acylome Profiling with N-(Cyanomethyl)-N-(phenylsulfonyl)amides for Targeted Lysine Acylation and Post-Translational Control of Protein Function in Cells.

Lysine acylations are ubiquitous and structurally diverse post-translational modifications that vastly expand the functional heterogeneity of the human proteome. Hence, the targeted acylation of lysine residues has emerged as a strategic approach to exert biomimetic control over the protein function. However, existing strategies for targeted lysine acylation in cells often rely on genetic intervention, recruitment of endogenous acylation machinery, or nonspecific acylating agents and lack methods to quantify the magnitude of specific acylations on a global level. In this study, we develop activity-based acylome profiling (ABAP), a chemoproteomic strategy that exploits elaborate N-(cyanomethyl)-N-(phenylsulfonyl)amides and lysine-centric probes for site-specific introduction and proteome-wide mapping of posttranslational lysine acylations in human cells. Harnessing this framework, we quantify various artificial acylations and rediscover numerous endogenous lysine acylations. We validate site-specific acetylation of target lysines and establish a structure-activity relationship for N-(cyanomethyl)-N-(phenylsulfonyl)amides in proteins from diverse structural and functional classes. We identify paralog-selective chemical probes that acetylate conserved lysines within interferon-stimulated antiviral RNA-binding proteins, generating de novo proteoforms with obstructed RNA interactions. We further demonstrate that targeted acetylation of a key enzyme in retinoid metabolism engenders a proteoform with a conformational change in the protein structure, leading to a gain-of-function phenotype and reduced drug potency. These findings underscore the versatility of our strategy in biomimetic control over protein function through targeted delivery and global profiling of endogenous and artificial lysine acylations, potentially advancing therapeutic modalities and our understanding of biological processes orchestrated by these post-translational modifications.

RNA-binding protein Trx regulates alternative splicing and promotes metastasis of HCC via interacting with LINC00152.

Epithelial-mesenchymal transition (EMT) is central to HCC metastasis, in which RNA-binding proteins (RBPs) play a key role. To explore the role of RBPs in metastasis of hepatocellular carcinoma (HCC), whole transcriptome sequencing was conducted to identify differential RBPs between HCC with metastasis and HCC without metastasis. The influence of RBPs on metastasis of HCC was verified by in vitro and in vivo experiments. The interaction of RBPs with non-coding RNAs was evaluated by RNA immunoprecipitation and pull-down assays. RNA sequencing, whole-genome sequencing, and alternative splicing analysis were further performed to clarify post-transcriptional regulation mechanisms. Whole transcriptome sequencing results showed that expression of thioredoxin (Trx) was significantly upregulated in HCC patients with metastasis. Trx was also found to be associated with poor prognosis in HCC patients. Overexpression of Trx could promote migration and invasion of HCC cells in vitro and increase the rate of lung metastasis of HCC cells in vivo. Moreover, binding assays showed that Trx could bind to LINC00152. As a result, LINC00152 was verified to determine the pro-metastasis function of Trx by knockdown assay. Furthermore, we revealed that Trx could regulate metastasis-associated alternative splicing program. Specifically, angiopoietin 1 (ANGPT1) was the splicing target; the splicing isoform switching of ANGPT1 could activate the PI3K-Akt pathway, upregulate EMT-associated proteins, and promote migration and invasion of HCC cells. We found that Trx could interact with LINC00152 and promote HCC metastasis via regulating alternative splicing, indicating that Trx may serve as a novel therapeutic target for HCC treatment.

IrCLIP-RNP and Re-CLIP reveal patterns of dynamic protein associations on RNA.

RNA binding proteins ( RBPs ) control varied processes, including RNA splicing, stability, transport, and translation 1-3 . Dysfunctional RNA-RBP interactions contribute to the pathogenesis of human disease 1,4,5 , however, characterizing the nature and dynamics of multiprotein assemblies on RNA has been challenging. To address this, non-isotopic ligation-based ultraviolet crosslinking immunoprecipitation 6 was combined with mass spectrometry ( irCLIP-RNP ) to identify RNA-dependent associated proteins ( RDAPs ) co-bound to RNA with any RBP of interest. irCLIP-RNP defined landscapes of multimeric protein assemblies on RNA, uncovering previously unknown patterns of RBP-RNA associations, including cell-type-selective combinatorial relationships between RDAPs and primary RBPs. irCLIP-RNP also defined dynamic RDAP remodeling in response to epidermal growth factor ( EGF ), uncovering EGF-induced recruitment of UPF1 adjacent to HNRNPC to effect splicing surveillance of cell proliferation mRNAs. To identify the RNAs simultaneously co-bound by multiple studied RBPs, a sequential immunoprecipitation irCLIP ( Re-CLIP ) method was also developed. Re-CLIP confirmed binding relationships seen in irCLIP-RNP and detected simultaneous HNRNPC and UPF1 co-binding on RND3 and DDX3X mRNAs. irCLIP-RNP and Re-CLIP provide a framework to identify and characterize dynamic RNA-protein assemblies in living cells.

Capture of RNA-binding proteins across mouse tissues using HARD-AP

RNA-binding proteins (RBPs) modulate all aspects of RNA metabolism, but a comprehensive picture of RBP expression across tissues is lacking. Here, we describe our development of the method we call HARD-AP that robustly retrieves RBPs and tightly associated RNA regulatory complexes from cultured cells and fresh tissues. We successfully use HARD-AP to establish a comprehensive atlas of RBPs across mouse primary organs. We then systematically map RNA-binding sites of these RBPs using machine learning-based modeling. Notably, the modeling reveals that the LIM domain as an RNA-binding domain in many RBPs. We validate the LIM-domain-only protein Csrp1 as a tissue-dependent RNA binding protein. Taken together, HARD-AP is a powerful approach that can be used to identify RBPomes from any type of sample, allowing comprehensive and physiologically relevant networks of RNA-protein interactions.

Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) in hematological diseases.

The oncofetal mRNA-binding protein IGF2BP1 belongs to a conserved family of RNA-binding proteins. It primarily promotes RNA stability, regulates translation and RNA localization, and mediates gene expression through its downstream effectors. Numerous studies have demonstrated that IGF2BP1 plays crucial roles in embryogenesis and carcinogenesis. IGF2BP1-modulated cell proliferation, invasion, and chemo-resistance in solid tumors have attracted researchers' attention. Additionally, several studies have highlighted the importance of IGF2BP1 in hematologic malignancies and hematological genetic diseases, positioning it as a promising therapeutic target for hematological disorders. However, there is a lack of systematic summaries regarding the IGF2BP1 gene within the hematological field. In this review, we provide a comprehensive overview of the discovery and molecular structure of IGF2BP1, along with recent studies on its role in regulating embryogenesis. We also focus on the mechanisms by which IGF2BP1 regulates hematological malignancies through its interactions with its targeted mRNAs. Furthermore, we systematically elucidate the function and mechanism of IGF2BP1 in promoting fetal hemoglobin expression in adult hematopoietic stem/progenitor cells. Finally, we discuss the limitations and challenges of IGF2BP1 as a therapeutic target, offering insights into its prospects.

RNA-Binding Proteins as Novel Effectors in Osteoblasts and Osteoclasts: A Systems Biology Approach to Dissect the Transcriptional Landscape.

Bone health is ensured by the coordinated action of two types of cells-the osteoblasts that build up bone structure and the osteoclasts that resorb the bone. The loss of balance in their action results in pathological conditions such as osteoporosis. Central to this study is a class of RNA-binding proteins (RBPs) that regulates the biogenesis of miRNAs. In turn, miRNAs represent a critical level of regulation of gene expression and thus control multiple cellular and biological processes. The impact of miRNAs on the pathobiology of various multifactorial diseases, including osteoporosis, has been demonstrated. However, the role of RBPs in bone remodeling is yet to be elucidated. The aim of this study is to dissect the transcriptional landscape of genes encoding the compendium of 180 RBPs in bone cells. We developed and applied a multi-modular integrative analysis algorithm. The core methodology is gene expression analysis using the GENEVESTIGATOR platform, which is a database and analysis tool for manually curated and publicly available transcriptomic data sets, and gene network reconstruction using the Ingenuity Pathway Analysis platform. In this work, comparative insights into gene expression patterns of RBPs in osteoblasts and osteoclasts were obtained, resulting in the identification of 24 differentially expressed genes. Furthermore, the regulation patterns upon different treatment conditions revealed 20 genes as being significantly up- or down-regulated. Next, novel gene-gene associations were dissected and gene networks were reconstructed. Additively, a set of osteoblast- and osteoclast-specific gene signatures were identified. The consolidation of data and information gained from each individual analytical module allowed nominating novel promising candidate genes encoding RBPs in osteoblasts and osteoclasts and will significantly enhance the understanding of potential regulatory mechanisms directing intracellular processes in the course of (patho)physiological bone turnover.

Altered mRNA transport and local translation in iNeurons with RNA binding protein knockdown.

Neurons rely on mRNA transport and local translation to facilitate rapid protein synthesis in processes far from the cell body. These processes allow precise spatial and temporal control of translation and are mediated by RNA binding proteins (RBPs), including those known to be associated with neurodegenerative diseases. Here, we use proteomics, transcriptomics, and microscopy to investigate the impact of RBP knockdown on mRNA transport and local translation in iPSC-derived neurons. We find thousands of transcripts enriched in neurites and that many of these transcripts are locally translated, possibly due to the shorter length of transcripts in neurites. Loss of frontotemporal dementia/amyotrophic lateral sclerosis (FTD/ALS)-associated RBPs TDP- 43 and hnRNPA1 lead to distinct alterations in the neuritic proteome and transcriptome. TDP-43 knockdown (KD) leads to increased neuritic mRNA and translation. In contrast, hnRNPA1 leads to increased neuritic mRNA, but not translation, and more moderate effects on local mRNA profiles, possibly due to compensation by hnRNPA3. These results highlight the crucial role of FTD/ALS-associated RBPs in mRNA transport and local translation in neurons and the importance of these processes in neuron health and disease.

RNA structures within Venezuelan equine encephalitis virus E1 alter macrophage replication fitness and contribute to viral emergence.

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne +ssRNA virus belonging to the Togaviridae. VEEV is found throughout Central and South America and is responsible for periodic epidemic/epizootic outbreaks of febrile and encephalitic disease in equines and humans. Endemic/enzootic VEEV is transmitted between Culex mosquitoes and sylvatic rodents, whereas epidemic/epizootic VEEV is transmitted between mosquitoes and equids, which serve as amplification hosts during outbreaks. Epizootic VEEV emergence has been shown to arise from mutation of enzootic VEEV strains. Specifically, epizootic VEEV has been shown to acquire amino acid mutations in the E2 viral glycoprotein that facilitate viral entry and equine amplification. However, the abundance of synonymous mutations which accumulate across the epizootic VEEV genome suggests that other viral determinants such as RNA secondary structure may also play a role in VEEV emergence. In this study we identify novel RNA structures in the E1 gene which specifically alter replication fitness of epizootic VEEV in macrophages but not other cell types. We show that SNPs are conserved within epizootic lineages and that RNA structures are conserved across different lineages. We also identified several novel RNA-binding proteins that are necessary for altered macrophage replication. These results suggest that emergence of VEEV in nature requires multiple mutations across the viral genome, some of which alter cell-type specific replication fitness in an RNA structure-dependent manner.

Human cells contain myriad excised linear intron RNAs with links to gene regulation and potential utility as biomarkers.

A previous study using Thermostable Group II Intron Reverse Transcriptase sequencing (TGIRT-seq) found human plasma contains short (≤300 nt) structured full-length excised linear intron (FLEXI) RNAs with potential to serve as blood-based biomarkers. Here, TGIRT-seq identified >9,000 different FLEXI RNAs in human cell lines, including relatively abundant FLEXIs with cell-type-specific expression patterns. Analysis of public CLIP-seq datasets identified 126 RNA-binding proteins (RBPs) that have binding sites within the region corresponding to the FLEXI or overlapping FLEXI splice sites in pre-mRNAs, including 53 RBPs with binding sites for ≥30 different FLEXIs. These included splicing factors, transcription factors, a chromatin remodeling protein, cellular growth regulators, and proteins with cytoplasmic functions. Analysis of ENCODE datasets identified subsets of these RBPs whose knockdown impacted FLEXI host gene mRNA levels or proximate alternative splicing, indicating functional interactions. Hierarchical clustering identified six subsets of RBPs whose FLEXI binding sites were co-enriched in six subsets of functionally related host genes: AGO1-4 and DICER, including but not limited to agotrons or mirtron pre-miRNAs; DKC1, NOLC1, SMNDC1, and AATF (Apoptosis Antagonizing Transcription Factor), including but not limited to snoRNA-encoding FLEXIs; two subsets of alternative splicing factors; and two subsets that included RBPs with cytoplasmic functions (e.g., LARP4, PABPC4, METAP2, and ZNF622) together with regulatory proteins. Cell fractionation experiments showed cytoplasmic enrichment of FLEXI RNAs with binding sites for RBPs with cytoplasmic functions. The subsets of host genes encoding FLEXIs with binding sites for different subsets of RBPs were co-enriched with non-FLEXI other short and long introns with binding sites for the same RBPs, suggesting overarching mechanisms for coordinately regulating expression of functionally related genes. Our findings identify FLEXIs as a previously unrecognized large class of cellular RNAs and provide a comprehensive roadmap for further analyzing their biological functions and the relationship of their RBPs to cellular regulatory mechanisms.

Conserved role for spliceosomal component PRPF40A in microexon splicing.

Microexons (exons ≤30 nts) are important features of neuronal transcriptomes, but pose mechanistic challenges to the splicing machinery. We previously showed that PRP-40, a component of the U1 spliceosome, is globally required for microexon splicing in C. elegans. Here we show that the homologous PRPF40A is also globally required for microexon splicing in mouse neuroblastoma cells. We find that PRPF40A co-regulates microexons along with SRRM4, a neuron-specific regulator of microexon splicing. The relationship between exon size and dependence on PRPF40A/SRRM4 is distinct, with SRRM4-dependence exhibiting a size threshold (~30 nts) and PRPF40A-dependence exhibiting a graded decrease as exon size increases. Finally, we show that PRPF40A knockdown causes an increase in productive splicing of its spliceosomal binding partner Luc7l by skipping of a small "poison exon." Similar homeostatic cross-regulation is often observed across paralogous RNA binding proteins. Here we find this concept likewise applies across evolutionarily unrelated but functionally and physically coupled spliceosomal components.

Structural features within the NORAD long noncoding RNA underlie efficient repression of Pumilio activity.

Long noncoding RNAs (lncRNAs) are increasingly appreciated for their important functions in mammalian cells. However, how their functional capacities are encoded in their sequences and manifested in their structures remains largely unknown. Some lncRNAs bind to and modulate the availability of RNA-binding proteins, but the structural principles that underlie this mode of regulation are unknown. The NORAD lncRNA is a known decoy for Pumilio proteins, which modulate the translation and stability of hundreds of messenger RNAs and, consequently, a regulator of genomic stability and aging. Here we probed the RNA structure and long-range RNA-RNA interactions formed by human NORAD inside cells under different stressful conditions. We discovered a highly modular structure consisting of well-defined domains that contribute independently to NORAD function. Following arsenite stress, most structural domains undergo relaxation and form interactions with other RNAs that are targeted to stress granules. We further revealed a unique structural organization that spatially clusters the multiple Pumilio binding sites along NORAD and consequently contributes to the derepression of Pumilio targets. We then applied these structural principles to design an effective artificial decoy for the let-7 microRNA. Our work demonstrates how the sequence of a lncRNA spatially clusters its function into separated domains and how structural principles can be employed for the rational design of lncRNAs with desired activities.

Knockout of rbm24a and rbm24b genes in zebrafish impairs skeletal and cardiac muscle integrity and function during development.

Skeletal and cardiac muscles are contractile tissues whose development and function are dependent on genetic programs that must be precisely orchestrated in time and space. In addition to transcription factors, RNA-binding proteins tightly regulate gene expression by controlling the fate of RNA transcripts, thus specific proteins levels within the cell. Rbm24 has been identified as a key player of myogenesis and cardiomyogenesis in several vertebrates, by controlling various aspects of post-transcriptional regulation, including pre-mRNA alternative splicing and mRNA stabilization. In zebrafish, knockdown of rbm24a or rbm24b also causes skeletal and cardiac muscle phenotypes, but how their combined loss affects muscle integrity and function remains elusive. By genome editing, we have generated rbm24a and rbm24b single mutants as well as double mutants. Structural analyses indicate that homozygous rbm24a and rbm24b double mutants exhibit severe somitic muscle and cardiac phenotypes, although rbm24b single mutants are obviously normal. We further show that the loss of rbm24a and rbm24b disrupts sarcomere organization, impairing functional contractility and motility of skeletal and cardiac muscles. The rbm24 mutant zebrafish represents a new genetic tool for in-depth studies of Rbm24-mediated post-transcriptional regulation of skeletal and cardiac muscle development, disease and regeneration.

An atlas of RNA-dependent proteins in cell division reveals the riboregulation of mitotic protein-protein interactions.

Ribonucleoprotein complexes are dynamic assemblies of RNA with RNA-binding proteins (RBPs), which can modulate the fate of the RNA molecules from transcription to degradation. Vice versa, RNA can regulate the interactions and functions of the associated proteins. Dysregulation of RBPs is linked to diseases such as cancer and neurological disorders. RNA and RBPs are present in mitotic structures like the centrosomes and spindle microtubules, but their influence on mitotic spindle integrity remains unknown. Thus, we applied the R-DeeP strategy for the proteome-wide identification of RNA-dependent proteins and complexes to cells synchronized in mitosis versus interphase. The resulting atlas of RNA-dependent proteins in cell division can be accessed through the R-DeeP 3.0 database (R-DeeP3.dkfz.de). It revealed key mitotic factors as RNA-dependent such as AURKA, KIFC1 and TPX2 that were linked to RNA despite their lack of canonical RNA-binding domains. KIFC1 was identified as a new interaction partner and phosphorylation substrate of AURKA at S349 and T359. In addition, KIFC1 interacted with both, AURKA and TPX2, in an RNA-dependent manner. Our data suggest a riboregulation of mitotic protein-protein interactions during spindle assembly, offering new perspectives on the control of cell division processes by RNA-protein complexes.