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Identification of mRNAS enriched in endothelial cell protrusions

ABSTRACT: Deciphering the complex biology of cell migration is key to understand human-related disorders. During angiogenesis, endothelial cells engage in coordinated migration events to form new blood vessels from parental counterparts. However, while subcellular localisation of mRNAs and localised translation are fundamental steps between gene transcription and protein activity, whether local regulation of gene expression controls the complex morphogenetic process of angiogenesis has never been addressed. Here, we set out to investigate the cytoplasmic distribution of mRNAs in migratory endothelial cells. We isolated RNA from endothelial cell protrusions from their cell bodies and used RNA-sequencing to identify asymmetrically distributed transcripts. These studies identified a set of transcripts enriched in endothelial cell protrusions over cell bodies, which included classically protrusion-enriched mRNAs and other intriguing transcripts encoding proteins implicated in cell migration.

ORGANISM(S): Homo sapiens

PROVIDER: GSE133055 | GEO | 2020/09/07

REPOSITORIES: GEO

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Effect of overexpressed UTR constructs on RNA enrichment at protrusions

Project description:We generated fibroblast lines overexpressing beta-globin constructs under a doxycycline-inducible promoter. These constructs contain the beta-globin coding sequence followed by either a control 3'UTR (HBB) or the Pkp4 3'UTR. The Pkp4 3'UTR carries a protrusion-targeting element and is expected to compete with localization of endogenous protrusion-localized mRNAs. We used a fractionation scheme to isolate protrusions and cell bodies from HBB and Pkp4 UTR-expressing fibroblasts, which were induced to extend protrusions in response to addition of lysophosphatidic acid (LPA). 4 replicate protrusion and cell body samples for each cell type were analyzed by RNA-Seq. To identify RNAs enriched in protrusions, a protrusion/cell body ratio was calculated. We find that ca. 4.5% of detected RNAs were enriched in protrusions of control HBB cells. Overexpression of the Pkp4 UTR reduced the protrusion enrichement of a subset of these RNAs, while the degree of localization of the remaining RNAs was not significantly affected.

2017-10-11 | GSE82230 | GEO

RNAs enriched at protrusions of control or APC knockdown fibroblasts

Project description:We used a fractionation scheme to isolate protrusions and cell bodies from control or APC knockdown fibroblasts, which were induced to extend protrusions in response to addition of lysophosphatidic acid (LPA). Four replicate protrusion and cell body samples for each cell type were analyzed by RNA-Seq. To identify RNAs enriched in protrusions, a protrusion/cell body ratio was calculated. We find that ca. 7% of detected RNAs were enriched in protrusions of control cells. Knockdown of APC reduced the protrusion enrichement of a subset of these RNAs, indicating that they are dependent on APC for their localization. Localization of the remaining RNAs was not significantly affected upon APC knockdown suggesting that their localization is mediated by an APC-independent mechanism.

2017-10-11 | GSE82229 | GEO

RNA localization to protrusions of mouse melanoma YUMM1.7 cells.

Project description:Subcellular localization of messenger RNA (mRNA) is a widespread phenomenon that can impact the regulation and function of the encoded protein. In non-neuronal cells, a subset of mRNAs localize to cell protrusions and proper mRNA localization is required for cell migration. However, the mechanisms by which mRNA localization regulates protein function in this setting remain unclear. Here, we examined the functional consequences of localization of the mRNA encoding KIF1C. KIF1C is a kinesin motor protein required for cell migration and mRNA trafficking, including trafficking of its own mRNA. We show that Kif1c mRNA localization does not regulate KIF1C protein abundance, distribution, οr ability to traffic other mRNAs. Conversely, robust Kif1c mRNA localization is required for directed cell migration. We used mass spectrometry to identify binding partners of endogenous KIF1C, which revealed dramatic dysregulation of the number and identity of KIF1C interactors in response to Kif1c mRNA mis-localization. These results therefore uncovered a mechanistic connection between mRNA localization to cell protrusions and the specificity of protein-protein interactions. We anticipate that this mechanism is not limited to Kif1c and is likely to be a general principle used by protrusion-enriched mRNAs in non-neuronal cells.

2023-02-24 | GSE219089 | GEO

The oligodendrocyte "protrusiosome": Identification of compartmentalized enrichment of transcripts in newly-formed protrusions of oligodendrocyte precursor cells (OPC) by RNAseq

Project description:Like neurons, oligodendrocytes (OL) are cells with elaborate morphology that probably require asymmetrical spatial regulation of biological processes. Formation of membrane protrusions is critical for OL development and interaction with axons. We hypothesized that the enrichment of specific mRNAs in protrusions of oligodendrocyte precursor cells (OPC) is important for morphological differentiation, thus having an impact in myelination. To explore this hypothesis, we established a modified Boyden chamber system to physically separate soma from membrane protrusions of rat primary OPC cultured in vitro for 24h. We performed a whole transcriptome analysis (RNAseq) of primary rat OPC soma and membrane protrusion fractions and found a subcellular enrichment of mRNAs in these structures during initial protrusion formation. At the very initial stage of OPC protrusion extension, there is a significant subcellular enrichment of transcripts encoding proteins related to cellular component assembly and cytoskeleton organization, particularly of actin-related molecules. This suggests that the regulation of the cytoskeleton dynamics may be locally controlled in OPCs and probably relevant for their differentiation program.

2018-05-07 | GSE106862 | GEO

QUANTSEQ 3' sequencing of protrusions and cell bodies of control (NT) or LARP6 siRNA (LARP6 KD) transfected MDA-MB231 breast cancer cells

Project description:To assess how LARP6 affects mRNA localisation to cell-protrusions, we transfected MDA-MB231 cells with either non-targeting control siRNA or LARP6 siRNA for 72 hrs, before fractionating the cells into protrusion and cell-bodies, and analysed total RNA from each fraction via Lexogen QUANTSEQ FWD 3' mRNA sequencing. Protrusion induction was done for 2 hrs on 3 micron transwell filters (Corning). The experiment was performed twice using two independent batches of cells, and sequencing was carried out on an Illumina NextSeq 500 sequencer.

2020-10-30 | E-MTAB-9520 | biostudies-arrayexpress

Identification of mRNAS enriched in endothelial cell protrusions

Project description:Identification of mRNAS enriched in endothelial cell protrusions

| PRJNA549849 | ENA

Fam49/CYRI interacts with Rac1 and locally suppresses protrusions

Project description:Actin-based protrusions driving cell migration are reinforced through positive feedback, but it is unclear how the cell restricts the eventual size of protrusions, or limits positive signals to allow them to split or retract. We have identified an evolutionarily conserved regulator of the protrusion machinery, which we name CYRI (CYFIP-related Rac interactor). CYRI binds specifically to activated Rac1 via a common motif that is also found in CYFIP, the Domain of Unknown Function DUF1394; we demonstrate that DUF1394 as a new class of Rac1 binding module. CYRI-depleted cells have broad, Scar/WAVE-enriched lamellipodia and display an enhanced duration and extent of pseudopod extension in response to optogenetic Rac1 activation. Conversely, CYRI overexpression suppresses recruitment of active Scar/WAVE complex to the cell edge and results in short-lived, unproductive protrusions. CYRI dynamically inhibits Scar/WAVE induced actin to focus positive protrusion signals and regulate pseudopod complexity. It therefore behaves like a “local inhibitor” predicted and described in widely accepted mathematical models, but not previously identified in living cells. CYRI is important for processes requiring polarity and plasticity of protrusions, including directional chemotactic migration and polarization of epithelial cysts.

2018-10-08 | PXD010460 | Pride

3' mRNA-seq of protrusions and cell bodies of non-transfected (nt) or LARP6 siRNA transfected MDA-MB231 cells

Project description:3' mRNA-seq of protrusions and cell bodies of nt or LARP6 siRNA transfected MDA-MB231 cells. These data show that Ribosomal protein (RP) -mRNAs localization to cell protrusions is mediated by LARP6.

2022-10-03 | E-MTAB-8472 | biostudies-arrayexpress

3' mRNA-seq of protrusions and cell bodies of BJ, PC-3M, RPE-1, U-87 and WM-266.4 cells

Project description:3' mRNA-seq of protrusions and cell bodies of a panel of normal and malignant cell lines. These data show that Ribosomal protein (RP) -mRNAs localization to cell protrusions is a conserved phenomenon.

2020-10-30 | E-MTAB-8470 | biostudies-arrayexpress

Quantitative proteomics analysis of actin-rich protrusions and cell-bodies in migratory cells

Project description:This upload concerns 2 independent but related experiments that were searched together: a) To reveal proteins that consistently localise to actin-rich protrusions across different human migratory cell-lines, we profiled the distribution of cellular proteins between protrusions and cell-bodies in a panel of 5 established human cell-lines (see experimental design). Cell lines were seeded on top of collagen-I coated 3 micron transwell filters (corning). No media was added to the bottom chamber and cells were allowed to adhere to the filter overnight. The next day, the media on top of the cells was refreshed, and media was also added to the bottom chamber in order to open the pores and allow formation of protrusions. Cells were allowed to form protrusions for 2 hrs before being fixed with ice-cold methanol. Cells were then rehydrated in PBS, followed by independent lysis of protrusions and cell-bodies on opposite sides of the filters in 2% SDS, 100mM Tris pH 7.5 b) To analyse the temporal dynamics of protein localisation to cell protrusions, we carried out a time-course spatial proteomics analysis of protrusions and cell-bodies in MDA-MB231 cells. Cells were seeded on top of collagen-I coated 3 micron transwell filters (corning). No media was added to the bottom chamber and cells were allowed to adhere to filter overnight. The next day, the media on top of cells was refreshed, and the filters were either left with no media in the bottom chamber (closed pores), or media added to the bottom chamber (open pores) for different lengths of times (1, 2, 4 , & 8 hrs) to induce protrusion formation. Cells were then fixed with ice-cold methanol, rehydrated in PBS, followed by independent lysis of protrusions and cell-bodies on opposite sides of each filter by 2% SDS, 100mM Tris pH 7.5.

2020-10-20 | PXD021239 | Pride

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