Similar Datasets
Project description:The transport of mRNAs to distal subcellular compartments is an important component of spatial gene expression control in neurons. However, the mechanisms that control mRNA localization in neurons are not completely understood. Here, we identify the abundant base modification, m6A, as a novel regulator of this process. Transcriptome-wide analysis following genetic loss of m6A reveals hundreds of transcripts that exhibit altered subcellular localization in hippocampal neurons. Additionally, using a reporter system, we show that mutation of specific m6A sites in select neuronal transcripts diminishes their localization to neurites. Single molecule fluorescent in situ hybridization experiments further confirm our findings and identify the m6A reader proteins YTHDF2 and YTHDF3 as mediators of this effect. Our findings reveal a novel function for m6A in controlling mRNA localization in neurons and enable a better understanding of the mechanisms through which m6A influences gene expression in the brain.
| S-EPMC9071445 | biostudies-literature
Project description:The transport of mRNAs to distal subcellular compartments is an important component of spatial gene expression control in neurons. However, the mechanisms that control mRNA localization in neurons are not completely understood. Here, we identify the abundant base modification, m6A, as a novel regulator of this process. Transcriptome-wide analysis following genetic loss of m6A reveals hundreds of transcripts that exhibit altered subcellular localization in hippocampal neurons. Additionally, using a reporter system, we show that mutation of specific m6A sites in select neuronal transcripts diminishes their localization to neurites. Single molecule fluorescent in situ hybridization experiments further confirm our findings and identify the m6A reader proteins YTHDF2 and YTHDF3 as mediators of this effect. Our findings reveal a novel function for m6A in controlling mRNA localization in neurons and enable a better understanding of the mechanisms through which m6A influences gene expression in the brain.
2022-04-26 | GSE194207 | GEO
Project description:The transport of mRNAs to distal subcellular compartments is an important component of spatial gene expression control in neurons. However, the mechanisms that control mRNA localization in neurons are not completely understood. Here, we identify the abundant base modification, m6A, as a regulator of mRNA localization in neurons. Transcriptome-wide analysis of mRNA localization in neurons following genetic loss of m6A reveals hundreds of transcripts that exhibit altered subcellular localization. Additionally, using a reporter system, we show that mutation of specific m6A sites in select neuronal transcripts diminishes their localization to neurites. Single molecule fluorescent in situ hybridization experiments further confirm our findings and identify the m6A reader proteins YTHDF2 and YTHDF3 as mediators of this effect. Our findings reveal a novel function for m6A in controlling mRNA localization in neurons and enable a better understanding of the mechanisms through which m6A influences gene expression in the brain.
2022-04-26 | GSE171199 | GEO
Project description:m6A and YTHDF proteins control the subcellular localization of select neuronal mRNAs
| PRJNA799732 | ENA
Project description:m6A and YTHDF proteins control the subcellular localization of select neuronal mRNAs [RIP- and RNA-seq]
| PRJNA799736 | ENA
Project description:m6A and YTHDF proteins control the subcellular localization of select neuronal mRNAs [RNA-seq and DART-seq]
| PRJNA718617 | ENA
Project description:Diverse RNAs and RNA-binding proteins form phase-separated, membraneless granules in cells under stress conditions. However, the role of the prevalent mRNA methylation, m6A, and its binding proteins in stress granule (SG) assembly remain unclear. Here, we show that m6A-modified mRNAs are enriched in SGs, and that m6A-binding YTHDF proteins are critical for SG formation. Depletion of YTHDF1/3 inhibits SG formation and recruitment of mRNAs to SGs. Both the N-terminal intrinsically disordered region and the C-terminal m6A-binding YTH domain of YTHDF proteins are important for SG formation. Super-resolution imaging further reveals that YTHDF proteins appear to be in a super-saturated state, forming clusters that often reside in the periphery of or at the junctions between SG core clusters, and potentially promote SG formation by reducing the activation energy barrier and critical size for SG condensate formation. Our results suggest a new function of the m6A-binding YTHDF proteins in regulating SG formation.
| S-EPMC7442727 | biostudies-literature
Project description:Full-length unspliced human immunodeficiency virus type 1 (HIV-1) RNAs serve dual roles in the cytoplasm as mRNAs encoding the Gag and Gag-Pol capsid proteins as well as genomic RNAs (gRNAs) packaged by Gag into virions undergoing assembly at the plasma membrane (PM). Because Gag is sufficient to drive the assembly of virus-like particles even in the absence of gRNA binding, whether viral RNA trafficking plays an active role in the native assembly pathway is unknown. In this study, we tested the effects of modulating the cytoplasmic abundance or distribution of full-length viral RNAs on Gag trafficking and assembly in the context of single cells. Increasing full-length viral RNA abundance or distribution had little-to-no net effect on Gag assembly competency when provided in trans In contrast, artificially tethering full-length viral RNAs or surrogate gag-pol mRNAs competent for Gag synthesis to non-PM membranes or the actin cytoskeleton severely reduced net virus particle production. These effects were explained, in large part, by RNA-directed changes to Gag's distribution in the cytoplasm, yielding aberrant subcellular sites of virion assembly. Interestingly, RNA-dependent disruption of Gag trafficking required either of two cis-acting RNA regulatory elements: the 5' packaging signal (Psi) bound by Gag during genome encapsidation or, unexpectedly, the Rev response element (RRE), which regulates the nuclear export of gRNAs and other intron-retaining viral RNAs. Taken together, these data support a model for native infection wherein structural features of the gag-pol mRNA actively compartmentalize Gag to preferred sites within the cytoplasm and/or PM.IMPORTANCE The spatial distribution of viral mRNAs within the cytoplasm can be a crucial determinant of efficient translation and successful virion production. Here we provide direct evidence that mRNA subcellular trafficking plays an important role in regulating the assembly of human immunodeficiency virus type 1 (HIV-1) virus particles at the plasma membrane (PM). Artificially tethering viral mRNAs encoding Gag capsid proteins (gag-pol mRNAs) to distinct non-PM subcellular locales, such as cytoplasmic vesicles or the actin cytoskeleton, markedly alters Gag subcellular distribution, relocates sites of assembly, and reduces net virus particle production. These observations support a model for native HIV-1 assembly wherein HIV-1 gag-pol mRNA localization helps to confine interactions between Gag, viral RNAs, and host determinants in order to ensure virion production at the right place and right time. Direct perturbation of HIV-1 mRNA subcellular localization may represent a novel antiviral strategy.
| S-EPMC5331792 | biostudies-literature
Project description:BackgroundThe computational prediction of mycobacterial proteins' subcellular localization is of key importance for proteome annotation and for the identification of new drug targets and vaccine candidates. Several subcellular localization classifiers have been developed over the past few years, which have comprised both general localization and feature-based classifiers. Here, we have validated the ability of different bioinformatics approaches, through the use of SignalP 2.0, TatP 1.0, LipoP 1.0, Phobius, PA-SUB 2.5, PSORTb v.2.0.4 and Gpos-PLoc, to predict secreted bacterial proteins. These computational tools were compared in terms of sensitivity, specificity and Matthew's correlation coefficient (MCC) using a set of mycobacterial proteins having less than 40% identity, none of which are included in the training data sets of the validated tools and whose subcellular localization have been experimentally confirmed. These proteins belong to the TBpred training data set, a computational tool specifically designed to predict mycobacterial proteins.ResultsA final validation set of 272 mycobacterial proteins was obtained from the initial set of 852 mycobacterial proteins. According to the results of the validation metrics, all tools presented specificity above 0.90, while dispersion sensitivity and MCC values were above 0.22. PA-SUB 2.5 presented the highest values; however, these results might be biased due to the methodology used by this tool. PSORTb v.2.0.4 left 56 proteins out of the classification, while Gpos-PLoc left just one protein out.ConclusionBoth subcellular localization approaches had high predictive specificity and high recognition of true negatives for the tested data set. Among those tools whose predictions are not based on homology searches against SWISS-PROT, Gpos-PLoc was the general localization tool with the best predictive performance, while SignalP 2.0 was the best tool among the ones using a feature-based approach. Even though PA-SUB 2.5 presented the highest metrics, it should be taken into account that this tool was trained using all proteins reported in SWISS-PROT, which includes the protein set tested in this study, either as a BLAST search or as a training model.
| S-EPMC2685389 | biostudies-literature
Project description:We describe here a method, termed immunoFISH, for simultaneous in situ analysis of the composition and distribution of proteins and individual RNA transcripts in single cells. Individual RNA molecules are labeled by hybridization and target proteins are concurrently stained using immunofluorescence. Multicolor fluorescence images are acquired and analyzed to determine the abundance, composition, and distribution of hybridized probes and immunofluorescence. We assessed the ability of immunoFISH to simultaneous quantify protein and transcript levels and distribution in cultured HER2 positive breast cancer cells and human breast tumor samples. We demonstrated the utility of this assay in several applications including demonstration of the existence of a layer of normal myoepithelial KRT14 expressing cells that separate HER2+ cancer cells from the stromal and immune microenvironment in HER2+ invasive breast cancer. Our studies show that immunoFISH provides quantitative information about the spatial heterogeneity in transcriptional and proteomic features that exist between and within cells.
| S-EPMC5705767 | biostudies-literature