Project description:SARS-CoV-2 is a positive single-stranded RNA virus which interacts at different stages with the host proteins of infected cells. These interactions are necessary for the host to recognize and block the replication of the virus. But, at the same time, the virus requires host proteins to translate, transcribe and replicate its genetic material. In order to identify the host proteins that interact with SARS-CoV-2 RNA, we adopted the RNA-protein interaction detection coupled to mass spectrometry (RaPID-MS) technology, which allows the purification and identification by MS-based proteomics of the proteins associated to a specific RNA of interest expressed in mammalian cells. In particular, we conducted the analysis on the more structured regions of SARS-CoV-2 RNA and retrieved several proteins specifically associated with each region. Overall, our data revealed a list of proteins associated to SARS-CoV-2 RNA that will be further characterized to understand their role in SARS-CoV-2 infection and viral replication.
Project description:During Drosophila oogenesis, the localization and translational regulation of maternal transcripts relies on RNA-binding proteins (RBPs). Many of these RBPs localize several mRNAs and may have additional direct interaction partners to regulate their functions. Using immunoprecipitation from whole Drosophila ovaries coupled to mass spectrometry, we examined protein-protein associations of 6 GFP-tagged RBPs expressed at physiological levels. Analysis of the interaction network and further validation in human cells allowed us to identify 26 previously unknown associations, besides recovering several well characterized interactions.
Project description:During Drosophila oogenesis, the localization and translational regulation of maternal transcripts relies on RNA-binding proteins (RBPs). Many of these RBPs localize several mRNAs and may have additional direct interaction partners to regulate their functions. Using immunoprecipitation from whole Drosophila ovaries coupled to mass spectrometry, we examined protein-protein associations of 6 GFP-tagged RBPs expressed at physiological levels. Analysis of the interaction network and further validation in human cells allowed us to identify 26 previously unknown associations, besides recovering several well characterized interactions. We identified interactions between RBPs and several splicing factors, providing links between nuclear and cytoplasmic events of mRNA regulation. Additionally, components of the translational and RNA decay machineries were selectively co-purified with some baits, suggesting a mechanism for how RBPs may regulate maternal transcripts. Given the evolutionary conservation of the studied RBPs, the interaction network presented here provides the foundation for future functional and structural studies of mRNA localization across metazoans.
Project description:RNA-binding proteins play essential roles in the regulation of gene expression. Many have modular structures and combine relatively few common domains in various arrangements to recognize RNA sequences and/or structures. Recent progress in engineering the specificity of the PUF class RNA-binding proteins has shown that RNA-binding domains may be combined with various effector or functional domains to regulate the metabolism of targeted RNAs. Designer RNA-binding proteins with tailored sequence specificity will provide valuable tools for biochemical research as well as potential therapeutic applications. In this review, we discuss the suitability of various RNA-binding domains for engineering RNA-binding specificity, based on the structural basis for their recognition. We also compare various protein engineering and design methods applied to RNA-binding proteins, and discuss future applications of these proteins.
Project description:Sequencing was performed to assess the ability of Nanopore direct cDNA and native RNA sequencing to characterise human transcriptomes. Total RNA was extracted from either HAP1 or HEK293 cells, and the polyA+ fraction isolated using oligodT dynabeads. Libraries were prepared using Oxford Nanopore Technologies (ONT) kits according to manufacturers instructions. Samples were then sequenced on ONT R9.4 flow cells to generate fast5 raw reads in the ONT MinKNOW software. Fast5 reads were then base-called using the ONT Albacore software to generate Fastq reads.
Project description:EATRO1125 bloodstream forms grown to about 5 x 10e5 per ml. Lister 427 procyclic forms at no more than 2 million per ml. Direct nanopore sequencing on cells approaching their expiration date.
Project description:The mechanisms controlling the abundance and sub-cellular distribution of caveolae are not well described. A first step towards determining such mechanisms would be identification of relevant proteins that interact with known components of caveolae. Here, we applied proximity biotinylation (BioID) to identify a list of proteins that may interact with the caveolar protein cavin1. Screening of these candidates using siRNA to reduce their expression revealed that one of them, CSDE1, regulates the levels of mRNAs and protein expression for multiple components of caveolae. A second candidate, CD2AP, co-precipitated with cavin1. Caveolar proteins were observed in characteristic and previously un-described linear arrays adjacent to cell-cell junctions in both MDCK cells, and in HeLa cells overexpressing an active form of the small GTPase Rac1. CD2AP was required for the recruitment of caveolar proteins to these linear arrays. We conclude that BioID will be useful in identification of new proteins involved in the cell biology of caveolae, and that interaction between CD2AP and cavin1 may have an important role in regulating the sub-cellular distribution of caveolae.
Project description:Despite the enormous proliferation of bacterial genome data, surprisingly persistent collections of bacterial proteins have resisted functional annotation. In a typical genome, roughly 30% of genes have no assigned function. Many of these proteins are conserved across a large number of bacterial genomes. To assign a putative function to these conserved proteins of unknown function, we created a physical interaction map by measuring biophysical interaction of these proteins. Binary protein--protein interactions in the model organism Streptococcus pneumoniae (TIGR4) are measured with a microfluidic high-throughput assay technology. In some cases, informatic analysis was used to restrict the space of potential binding partners. In other cases, we performed in vitro proteome-wide interaction screens. We were able to assign putative functions to 50 conserved proteins of unknown function that we studied with this approach.
Project description:The combination of high-throughput sequencing and in vivo crosslinking approaches leads to the progressive uncovering of the complex interdependence between cellular transcriptome and proteome. Yet, the molecular determinants governing interactions in protein-RNA networks are not well understood. Here we investigated the relationship between the structure of an RNA and its ability to interact with proteins. Analysing in silico, in vitro and in vivo experiments, we find that the amount of double-stranded regions in an RNA correlates with the number of protein contacts. This relationship -which we call structure-driven protein interactivity- allows classification of RNA types, plays a role in gene regulation and could have implications for the formation of phase-separated ribonucleoprotein assemblies. We validate our hypothesis by showing that a highly structured RNA can rearrange the composition of a protein aggregate. We report that the tendency of proteins to phase-separate is reduced by interactions with specific RNAs.