Quantification of Protein Levels in Single Living Cells

Lo, Chiu-An; Kays, Ibrahim; Emran, Farida; Lin, Tung-Yi; Cvetkovska, Vedrana; Chen, Brian Edwin

doi:10.1016/j.celrep.2019.02.112

Cited by 25 publications

(41 citation statements)

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“…Additionally, these results are significant because they allow us to examine intrinsic noise at the protein level, complementing RNA based studies. The in vivo protein work is a necessary complement to RNA studies because, in some scenarios, there can be little correlation between protein and RNA levels, as in (39), and reviewed in 2012 here (40) and in 2016 here (41). Additionally, studies of allele bias at the RNA level can sometimes have trouble distinguishing between transcriptional bursting (42) and actual allele bias that manifests at the protein level (13), because they often lack means to quantify the distinct allelic protein products.…”

Section: Discussionmentioning

confidence: 99%

Introns Control Stochasticity in Metazoan Gene Expression

Sands

Shi

2019

Preprint

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Understanding sources of variation in gene expression is important because they underlie variation in traits. Studies in microbes and cell culture have revealed significant, intrinsic nongenetic, nonenvironmental axes of variation in gene expression. Stochastic autosomal allele bias (including monoallelic expression), which can be quantified as intrinsic noise, is one of these natural axes. Higher intrinsic noise means a higher chance of observing a cell with allelically biased expression. Here, we surveyed intrinsic noise in the tissues of C. elegans using fluorescent reporter alleles controlled by the hsp-90 promoter. We saw visually striking allele bias present in several distinct cell types, including nerves, muscles and intestines. Because intron sequences can both alter chromatin markings and increase expression level, we hypothesized that introns increase the probability of fully expressing both alleles of a gene, thereby decreasing intrinsic noise. We found that intrinsic noise decreases by an order of magnitude when alleles contain synthetic or natural introns. We found that this is true in both diploid muscle cells and polyploid intestine cells. Our results show introns control intrinsic noise for other distinctly regulated genes (vit-2 and hsp-16.2). As in prior studies in yeast, we found these distinct promoters also impact intrinsic noise. Finally, we found that introns control intrinsic noise using a 5'-position dependent mechanism. Intron control of intrinsic noise may help explain why some genes have lost introns, why so many genes have introns, and why deep intronic mutations can result in allele silencing and disease.3

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Section: Discussionmentioning

confidence: 99%

Introns Control Stochasticity in Metazoan Gene Expression

Sands

Shi

2019

Preprint

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“…The first being that the two plasmid system required for TRIBE (one plasmid carrying the RBP fused to ADAR, the other carrying GFP as a marker) was combined onto a single multicistronic vector by using the p2A system. The equimolar expression for the upstream RBP-ADAR and downstream GFP cistrons, allowed for accurate quantification RBP expression levels during FACS sorting (Lo et al, 2015). Additionally, competition from the endogenous RBP was removed by performing all experiments in KO backgrounds.…”

Section: Modifications From Previously Published Tribe Protocolmentioning

confidence: 99%

MS2-TRIBE evaluates protein-RNA interactions and nuclear organization of transcription by RNA editing

Biswas

Rahman

Gupta³

et al. 2019

Preprint

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Nearly every step of RNA regulation is mediated by binding proteins (RBPs). The most common method to identify specific RBP target transcripts in vivo is by crosslinking ("CLIP" and its variants), which rely on protein-RNA crosslinking and specific antibodies. Another recently introduced method exploits RNA editing, with the catalytic domain of ADAR covalently attached to a specific RBP ("TRIBE"). Both approaches suffer from difficulties in distinguishing real RNA targets from false negative and especially false positive signals. To critically evaluate this problem, we used fibroblasts from a mouse where every endogenous β -actin mRNA molecule was tagged with the bacteriophage MS2 RNA stem loops; hence there is only a single bona fide target mRNA for the MS2 capsid protein (MCP). CLIP and TRIBE could both detect the single RNA target, albeit with some false positives (transcripts lacking the MS2 stem loops). Consistent false positive CLIP signals could be attributed to nonspecific antibody crosslinking. To our surprise, the supposed false positive TRIBE targets correlated with the location of genes spatially proximal to the β -actin gene. This result indicates that MCP-ADAR bound to β -actin mRNA contacted and edited nearby nascent transcripts, as evidenced by frequent intronic editing. Importantly, nascent transcripts on nearby chromosomes were also edited, agreeing with the interchromosomal contacts observed in chromosome paint and Hi-C. The identification of nascent RNA-RNA contacts imply that RNA-regulatory proteins such as splicing factors can associate with multiple nascent transcripts and thereby form domains of post-transcriptional activity, which increase their local concentrations. These results more generally indicate that TRIBE combined with the MS2 system, MS2-TRIBE, is a new tool to study nuclear RNA organization and regulation.3 Introduction:

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“…Thus, in order to track and quantify the dynamics of protein synthesis in single cells in animals, we used the Protein Quantitation Ratioing (PQR) technique (Figure 1A) (Lo et al 2015). Using CRISPR-Cas9 genome editing of Drosophila melanogaster, we inserted a PQR DNA construct with a red fluorescent protein (RFP) reporter at the end of the coding sequence for the Ribosomal protein L13a (Rpl13a) gene (Lo et al 2015) (Figure 1A). This animal produces one molecule of RFP for every one molecule of Rpl13a protein that is made during protein synthesis.…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, the red fluorescence intensity in a cell is proportional to the Rpl13a concentration and can be used to track and measure Rpl13a dynamics over time in every cell in the animal. We chose Rpl13a because, as a ribosomal subunit, it is itself involved in protein translation and has a long turnover rate similar to fluorescent proteins (Mazumder et al 2003;Chaudhuri et al 2007;Kapasi et al 2007;Boisvert et al 2012;Jia et al 2012;Lo et al 2015). Rpl13a is expressed in all cells at moderately high levels with several hundreds of mRNA copies per cell (Lo et al 2015).…”

Section: Introductionmentioning

confidence: 99%

“…We chose Rpl13a because, as a ribosomal subunit, it is itself involved in protein translation and has a long turnover rate similar to fluorescent proteins (Mazumder et al 2003;Chaudhuri et al 2007;Kapasi et al 2007;Boisvert et al 2012;Jia et al 2012;Lo et al 2015). Rpl13a is expressed in all cells at moderately high levels with several hundreds of mRNA copies per cell (Lo et al 2015). Rpl13a mRNA expression dynamics in the brain and in the whole organism are not circadian driven in flies and mice (Keegan et al 2007;Hughes et al 2010;Hughes et al 2012).…”

Section: Introductionmentioning

confidence: 99%

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Parental Allele-Specific Protein Expression in Single CellsIn Vivo

Chen

2018

Preprint

Self Cite

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Allelic expression from each parent-of-origin is important as a backup and to ensure that enough protein products of a gene are produced. Thus far, it is not known how each cell throughout a tissue differs in parental allele expression at the level of protein synthesis. Here, we measure the expression of the Ribosomal protein L13a (Rpl13a) from both parental alleles simultaneously in single cells in the living animal. We use genome-edited Drosophila that have a quantitative reporter of protein synthesis inserted into the endogenous Rpl13a locus. We find that individual cells can have large (>10-fold) differences in protein expression between the two parental alleles. Cells can produce protein from only one allele oftentimes, and time-lapse imaging of protein production from each parental allele in each cell showed that the imbalance in expression from one parental allele over the other can invert over time. One sentence summaryParental allele-specific protein expression varies widely across cells and over time. Highlights1. We used genome editing to insert a quantifiable protein translation reporter into the endogenous Ribosomal protein L13a gene and thus track the protein expression of both parental alleles simultaneously in every single cell in the awake animal.2. Cells can have a large difference in protein expression for one parental allele over the other, and this can invert over time, and can occur in clusters of cells within a tissue.3. We demonstrate the highly variable nature of heterozygous and homozygous definitions across single cells, and over time.4. Our study demonstrates a new paradigm that can be used to examine inherited epigenetic control of expression from a specific parent-of-origin allele across single clonal cells from a common progenitor in vivo.

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Quantification of Protein Levels in Single Living Cells

Cited by 25 publications

References 0 publications

Introns Control Stochasticity in Metazoan Gene Expression

Introns Control Stochasticity in Metazoan Gene Expression

MS2-TRIBE evaluates protein-RNA interactions and nuclear organization of transcription by RNA editing

Parental Allele-Specific Protein Expression in Single CellsIn Vivo

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