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Muscleblind-like proteins use modular domains to localize RNAs by riding kinesins and docking to membranes

ABSTRACT: RNA transport and local translation provide spatial control of gene expression, and RNA binding proteins (RBPs) act as critical adapters in this multi-step process. Muscleblind-like (MBNL) RNA binding proteins, implicated in Myotonic Dystrophy and cancer, localize RNAs to myoblast membranes and distal neurites, through unknown mechanisms. We found that MBNLs form motile and anchored granules in neurons and myoblasts, and selectively associate with kinesins Kif1bα and Kif1c through its zinc finger (ZnF) domains. Other RBPs with similar ZnFs also associate with these kinesins, implicating a motor-RBP specificity code. Live cell imaging and fractionation revealed that membrane anchoring is mediated through the unstructured carboxy-terminal tail of MBNL1. Both kinesin- and membrane-recruitment functions were reconstituted using MBNL-MS2 coat protein fusions; this approach, termed RBP Module Recruitment and Imaging (RBP-MRI), decouples RNA binding, kinesin recruitment, and membrane anchoring functions, while also establishing general strategies for studying multi-functional, modular domains of RBPs.

ORGANISM(S): Mus musculus

PROVIDER: GSE207597 | GEO | 2022/07/09

REPOSITORIES: GEO

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rec-Y2H matrix in S. cerevisiae screening reveals a vast potential for direct protein-protein interactions among RNA binding proteins

Project description:RNA-binding proteins (RBPs) are crucial factors of post-transcriptional gene regulation and their modes of action are intensely investigated. At the center of attention are RNA motifs that guide where RBPs bind. However, sequence motifs recognized by RBPs are typically a poor predictor of RBP-RNA interactions in vivo. It is hence believed that many RBPs recognize RNAs as complexes, to increase specificity and regulatory potential. To probe the potential for RBP–RBP complex formation, we assembled a library of 978 mammalian RBPs and used rec-Y2H screening to detect direct interactions between RBPs, sampling >1M possible interactions. We discovered 1994 new interactions and demonstrate that our interaction screening discovers RBP pairs that bind RNAs adjacently. We further find that the mRNA binding region preferences of an RBP can deviate, depending on its adjacently binding interaction partner. Finally, we reveal novel RBP–RBP interaction networks among major RNA processing steps and show that RBP mutations observed in cancer rewire spliceosomal interaction networks.

2021-09-01 | E-MTAB-9612 | biostudies-arrayexpress

Specific kinesin expression profiles associated with taxane resistance in breast cancer

Project description:Purpose: Breast cancer is a genetically heterogenous disease with subtypes differing in prognosis and chemosensitivity. The basal-like breast cancer (BLBC) molecular subtype is associated with poorer outcomes, but is more responsive to taxane-based chemotherapy. We evaluated the role of kinesins, motor proteins interacting with microtubules, in influencing taxane resistance. Experimental Design: Kinesin (KIF) expression was studied in one local dataset comprising all taxane resistant breast cancers in relation to taxane resistance. Data in the NCI-60 cell line dataset (GSE5846) nd the MDACC dataset (GSE20194) is separately detailed. Results: In the local dataset, the kinesin KIF26B is overexpressed in taxane-resistant residual breast cancers post-chemotherapy. Conclusions: We show that kinesin overexpression correlates with taxane resistance in BLBC cell lines and tissue. Our results suggest a potential approach to overcoming taxane resistance through concurrent or sequential use of kinesin inhibitors, highlighting the ATP-binding domain as a drug development target. Kinesin (KIF) expression was studied in one local dataset comprising all taxane resistant breast cancers in relation to taxane resistance. Data in the NCI-60 cell line dataset (GSE5846) and the MDACC dataset (GSE20194) is separately detailed.

2012-12-01 | E-GEOD-22796 | biostudies-arrayexpress

The RBPome of influenza A virus mRNA reveals a role for TDP-43 in viral replication

Project description:Recent technical advances have resulted in a significant increase in our understanding of the RNA-binding protein (RBP) repertoire present within eukaryotic cells. A particular focus has been on the RBPs that interact with cellular polyadenylated mRNAs. However, recent studies utilising the same technologies have begun to tease apart the RBP interactome of viral mRNAs, most notably SARS-CoV-2, demonstrating clear similarities but also differences between the RBP profiles of viral and cellular mRNAs. Herein, for the first time we comprehensively mapped the RBPs that associate with the NP mRNA of an influenza A virus. Moreover, we provide evidence that the viral polymerase is essential for the recruitment of RPBs to viral mRNAs through direct polymerase-RBP interactions during transcription. We show that loss of TDP-43, that associates with the viral mRNA, results in a decrease in viral mRNA availability within infected cells, with an impact on the accumulation of viral genomic RNA and the yield of infectious viral particles. Overall, we uncover an important role for TDP-43 in the influenza A virus replication cycle via a direct interaction with viral mRNAs and we demonstrate an essential role of the viral polymerase in orchestrating the assembly of viral mRNPs.

2024-04-16 | PXD049316 | Pride

The RBPome of influenza A virus mRNA reveals a role for TDP-43 in viral replication

2024-04-16 | PXD040783 | Pride

RNase-mediated protein footprint sequencing reveals protein-binding sites throughout the human transcriptome

Project description:RNAs are continuously associated with RNA-binding proteins (RBPs), and these interactions are necessary for many key cellular processes ranging from splicing to chromatin regulation. Although numerous approaches have been developed to map RNA-binding sites of individual RBPs, few methods exist that allow assessment of global RBP-RNA interactions. Here, we describe a universal, high-throughput, ribonuclease-mediated protein footprint sequencing approach that reveals RNA-protein interaction sites throughout a transcriptome of interest. We apply this method to the HeLa transcriptome and compare RBP binding sites found using different cross-linkers and ribonucleases. From this analysis, we identify numerous putative RBP binding motifs, reveal novel insights into co-binding by RBPs, and uncover a significant enrichment for disease-associated polymorphisms within RBP interaction sites. Protein interaction profile sequencing (PIP-seq) in HeLa cells. Two crosslinkers (formaldehyde and UV) with two RNases (dsRNase and ssRNase) each, as well as a no-crosslink sample. Performed with and without proteins. Three replicates for formaldehyde, two replicates for UV, single replicate for no crosslinker.

2013-11-06 | E-GEOD-49309 | biostudies-arrayexpress

Mechanochemical tuning of a kinesin motor essential for malaria parasite transmission

Project description:Kinesins are a superfamily of molecular motors that undertake ATP-dependent microtubule-based movement or regulate microtubule dynamics. Plasmodium species, which cause malaria and kill hundreds of thousands annually, encode ~9 kinesins in their genomes. Of these, two – kinesin-8B and kinesin-8X - are canonically classified as kinesin-8s, which in other eukaryotes typically modulate microtubule dynamics. Unexpectedly, Plasmodium kinesin-8B is required for development of the flagellated male gamete in the mosquito host, and its absence completely blocks parasite transmission. To understand the molecular basis of kinesin-8B’s essential role, we characterised the in vitro properties of the kinesin-8B motor domains from P. berghei and P. falciparum. Both motors drive plus-end directed ATP-dependent microtubule gliding, but also catalyse ATP-dependent microtubule depolymerisation. We determined the microtubule-bound structures of these motors using cryo-electron microscopy. P. berghei and P. falciparum kinesin-8B exhibit a very similar mode of microtubule interaction, in which Plasmodium-distinct sequences at the microtubule-kinesin interface influence motor function. Intriguingly, however, P. berghei kinesin-8B exhibits a non-canonical structural response to ATP analogue binding such that neck linker docking is not induced. Nevertheless, the neck linker region is absolutely required for motility and depolymerisation activities of these motors. Taken together, these data suggest that the mechanochemistry of Plasmodium kinesin-8Bs has been functionally tuned to efficiently contribute to flagella formation.

2022-12-05 | PXD037474 | Pride

Specific kinesin expression profiles associated with taxane resistance in breast cancer

2012-12-01 | GSE22796 | GEO

Organelle-specific RNA Tagging Probes for Spatially Resolved Profiling of RNA Binding Proteome and Investigation upon Ferroptosis Induction

Project description:The key role of RNA-binding proteins (RBPs) in posttranscriptional regulation of gene expression is intimately tied to their subcellular localization. Dysregulated localization may severely disrupt the biological functions of RBPs. To reveal the regulatory mechanisms of RBPs, methods for systematically mapping the subcellular localized RBPs and monitoring their translocations under physiological conditions are in high demand. Herein, a subcellular-specific RNA labeling method was developed for efficient enrichment and deep profiling of nuclear and cytoplasmic RBPs. A total of 1221 nuclear RBPs and 1333 cytoplasmic RBPs were enriched and identified using nuclear/cytoplasm targeting enrichment probes, which represented an increase of 54.4% and 85.7% compared with previous reports. The probes were further applied in the first omics-level investigation of subcellular-specific RBP-RNA interactions upon ferroptosis induction. Interestingly, large-scale RBPs displayed enhanced interaction with RNAs in nucleus but reduced association with RNAs in cytoplasm during ferroptosis process. Among these RBPs with regulated RNA-binding, translation was found as one of the commonly enriched GO functions by different ferroptosis inducers, indicating ferroptosis could disturb protein translation via different pathways. Furthermore, we discovered dozens of nucleoplasmic translocation candidate RBPs upon ferroptosis induction and validated representative ones by immunofluorescence imaging. The enrichment of TCA cycle in the translocation candidate RBPs may provide new insights for investigating their possible roles in ferroptosis induced metabolism dysregulation. The above findings suggest the potential of our enrichment method for high-throughput RBP mapping with organelle-level spatial resolution.

2022-12-01 | GSE205553 | GEO

Global analysis of the RNA-protein interaction and RNA secondary structure landscapes of the Arabidopsis nucleus

Project description:Post-transcriptional regulation in eukaryotes requires cis- and trans-acting features and factors including RNA secondary structure, and RNA-binding proteins (RBPs). However, a comprehensive view of the structural and RBP interaction landscape of RNAs in the nucleus has yet to be compiled for any organism. Here, we use our ribonuclease-mediated structure and RBP binding site mapping approach on Arabidopsis seedling nuclei in vivo to globally profile these features within the nuclear compartment. We reveal opposing patterns of secondary structure and RBP binding levels throughout native messenger RNAs that demarcate alternative splicing and polyadenylation. We also uncover a collection of protein bound sequence motifs, and identify their structural contexts, co-occurrences in transcripts encoding functionally related proteins, and interactions with putative RBPs. Finally, we identify a nuclear role for the chloroplast RBP, CP29A. In total, we provide the first simultaneous view of the RNA secondary structure and RBP interaction landscapes in a eukaryotic nucleus. Protein interaction profile sequencing (PIP-seq) in Arabidopsis seedling nuclei. These are crosslinked with formaldehyde and treated with two RNases (ssRNase and dsRNase) with two replicates

2014-12-16 | E-GEOD-58974 | biostudies-arrayexpress

Quantifying RNA-Protein Interactions Characterizes Recurrent Mutationsin RNA Binding Proteins in Cancer

Project description:Quantitative criteria to identify proteins as RNA-binding proteins (RBPs) are presently lacking, as are criteria to define RBP target RNAs. Here, we develop an ultraviolet (UV) cross-linking immunoprecipitation (CLIP)-sequencing method, easyCLIP. easyCLIP provides absolute cross-link rates, as well as increased simplicity, efficiency, and capacity to visualize RNA libraries. Measurement of >200 independent cross-link experiments across >35 proteins identifies an RNA cross-link rate threshold that distinguishes RBPs from non-RBPs and defines target RNAs as those with a complex frequency unlikely for a random protein. We apply easyCLIP to the 33 most recurrent cancer mutations across 28 RBPs, finding increased RNA binding per RBP molecule for PCBP1 L100P/Q cancer mutations. Quantitating RBP-RNA interactions can thus nominate proteins as RBPs and define the impact of specific disease-associated RBP mutations on RNA association.

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