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Quantifying Protein-mRNA Interactions in Single Live Cells.

ABSTRACT: Specific binding proteins are crucial for the correct spatiotemporal expression of mRNA. To understand this process, a method is required to characterize RNA-protein interactions in single living cells with subcellular resolution. We combined endogenous single RNA and protein detection with two-photon fluorescence fluctuation analysis to measure the average number of proteins bound to mRNA at specific locations within live cells. We applied this to quantify the known binding of zipcode binding protein 1 (ZBP1) and ribosomes to ?-actin mRNA within subcellular compartments of primary fibroblasts and neurons. ZBP1-mRNA binding did not occur in nuclei, contrary to previous conclusions. ZBP1 interaction with ?-actin mRNA was enhanced perinuclearly in neurons compared to fibroblasts. Cytoplasmic ZBP1 and ribosome binding to the mRNA were anti-correlated depending on their location in the cell. These measurements support a mechanism whereby ZBP1 inhibits translation of localizing mRNA until its release from the mRNA peripherally, allowing ribosome binding.

SUBMITTER: Wu B

PROVIDER: S-EPMC4491145 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Quantifying Protein-mRNA Interactions in Single Live Cells.

Wu Bin B Buxbaum Adina R AR Katz Zachary B ZB Yoon Young J YJ Singer Robert H RH

Cell 20150701 1

Specific binding proteins are crucial for the correct spatiotemporal expression of mRNA. To understand this process, a method is required to characterize RNA-protein interactions in single living cells with subcellular resolution. We combined endogenous single RNA and protein detection with two-photon fluorescence fluctuation analysis to measure the average number of proteins bound to mRNA at specific locations within live cells. We applied this to quantify the known binding of zipcode binding p ...[more]

PMID: 26140598

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Project description:BackgroundE2F1 protein, a major effector of the Rb/E2F pathway plays a central role in regulating cell-fate decisions involved in proliferation, apoptosis, and differentiation. Its expression is highly dynamic and tightly modulated through a combination of transcriptional, translational and posttranslational controls. However, the mechanisms by which its expression and activity can promote different cellular outcomes remain to be fully elucidated. To better document E2F1 expression in live cells, we have engineered a series of fluorescent E2F1 protein reporters that quantitatively capture E2F1 protein dynamics.MethodsReporter constructs, under the control of the mouse or human E2F1 proximal promoter, were designed to express an E2F1-Venus fusion protein incapable of binding DNA. In addition, constructs either included or excluded the 3' untranslated region (3'UTR) of the E2F1 gene. These constructs were introduced into fibroblasts and epithelial cells, and expression of the fusion reporter protein was validated and quantified in single cells using live imaging.ResultsIn all cases, expression of the reporter protein effectively recapitulated the behavior of E2F1 under various conditions, including cell cycle progression and genotoxic stress. No or little fluorescent signal of the reporter was detected in G0, but as the cycle progressed, expression of the reporter protein steadily increased in the nucleus, peaking a few hours before cell division, but declining to baseline 2-3 h prior to the onset of mitosis. The absence of the E2F1 3'UTR in the constructs led to considerably higher steady-state levels of the fusion protein, which although normally regulated, exhibited a slightly less complex dynamic profile during the cell cycle or genotoxic stress. Lastly, the presence or absence of Rb failed to impact the overall detection and levels of the reporter proteins.ConclusionsOur validated E2F1 protein reporters complement nicely other reporters of the Rb/E2F pathway and provide a unique tool to follow the complex dynamics of E2F1 expression in real time in single cells.

Dataset Information

Quantifying Protein-mRNA Interactions in Single Live Cells.

Publications

Quantifying Protein-mRNA Interactions in Single Live Cells.

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