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Asymmetric mRNA localization contributes to fidelity and sensitivity of spatially localized systems

Nature Structural & Molecular Biology volume 21, pages 833–839 (2014)Cite this article

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Abstract

Although many proteins are localized after translation, asymmetric protein distribution is also achieved by translation after mRNA localization. Why are certain mRNA transported to a distal location and translated on-site? Here we undertake a systematic, genome-scale study of asymmetrically distributed protein and mRNA in mammalian cells. Our findings suggest that asymmetric protein distribution by mRNA localization enhances interaction fidelity and signaling sensitivity. Proteins synthesized at distal locations frequently contain intrinsically disordered segments. These regions are generally rich in assembly-promoting modules and are often regulated by post-translational modifications. Such proteins are tightly regulated but display distinct temporal dynamics upon stimulation with growth factors. Thus, proteins synthesized on-site may rapidly alter proteome composition and act as dynamically regulated scaffolds to promote the formation of reversible cellular assemblies. Our observations are consistent across multiple mammalian species, cell types and developmental stages, suggesting that localized translation is a recurring feature of cell signaling and regulation.

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Figure 1: Classification and characterization of TAS and DSS proteins.
Figure 2: Structural analysis of DSS proteins reveals an enrichment in disordered regions.
Figure 3: Analysis of DSS proteins reveals an enrichment for linear motifs, phase-transition (i.e., higher-order assembly) promoting segments and PTM sites that act as molecular switches.
Figure 4: Dynamic regulation of DSS transcripts and proteins.
Figure 5: An overview of the potential advantages conferred by distal-site protein synthesis, inferred from our analysis.

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Acknowledgements

We thank S. Bullock, S. Balaji, C. Chothia, M. Buljan, B. Lang, M. Hegde, G. Chalancon, K. Van Roey, T. Flock, N. Latysheva, A.J. Venkatakrishnan and G. Toedt for stimulating discussions and their comments on this work. This work was supported by the Medical Research Council (MC_U105185859; R.J.W.; M.M.B.), Human Frontiers Science Program (RGY0073/2010; M.M.B.), the European Molecular Biology Organization Young Investigator Program (M.M.B.), ERASysBio+ (GRAPPLE; R.J.W. and M.M.B.), the European Molecular Biology Laboratory International PhD Program (R.J.W.) and a Canadian Institute of Health Research Postdoctoral Fellowship (R.J.W.).

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  1. Robert J Weatheritt

    Present address: Present address: The Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.,

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  1. MRC Laboratory of Molecular Biology, Cambridge, UK

    Robert J Weatheritt & M Madan Babu

  2. Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany

    Toby J Gibson

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Contributions

R.J.W. collected the data sets and performed the analysis; R.J.W. and M.M.B. designed the study; R.J.W., T.J.G. and M.M.B. conceived the idea and wrote the paper.

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Integrated supplementary information

Supplementary Figure 1 The identification of asymmetrically localized proteins and transcripts.

An illustrative explanation of the resolution of the study and the concept of asymmetric localization of proteins and mRNA. In this example, on the left a neuron is divided into its cell body and axon terminal, and transcriptome/proteomic analysis is undertaken on both samples. The Venn diagram describes the Cell Body (CB) and Axon Terminal (AT) regions with A and B representing protein/mRNA found within the proteomic/transcriptomics analyses respectively. AB proteins/mRNA are found in both screens. The proteins/mRNA found exclusively in set B are considered as asymmetrically localized to the axon terminals. A similar principle holds for the right-hand figure showing the fibroblast-like cells being divided into its cell body and pseudopodia. These studies employ a similar experimental set-up. In the case of fibroblast-like cells a gradient of the chemoattractant l-α-lysophosphatidic acid (LPA) induces pseudopodia formation in cell culture on a polycarbonate microporous filter. These are then scraped from the filter surface into lysis buffer for biochemical analysis30.

Supplementary Figure 2 Graphs and boxplots of functional and structural properties for distal site synthesis (DSS) proteins (red) and transport after synthesis (TAS) proteins (gray).

See Online Methods for details and legend of Figure 2 for a description of boxplots and statistical tests.

Supplementary Figure 3 Boxplots and graphs of functional and structural properties of distal site synthesis (DSS) proteins (red) and transport after synthesis (TAS) proteins (gray) when proteins with transmembrane regions were removed from the analysis.

See legend of Figure 2 for a description of boxplots and statistical tests.

Supplementary Figure 4 Graphs of cytoplasmic polyadenylation elements (CPE) and genomic differences between genes in the pseudopodia of mouse fibroblast cells comparing DSS transcripts (red) to TAS transcripts (gray).

The CPE interacts with CPEB1 and depending on the combination of HEX and/or CPSF elements can lead to either repression or activation of translation (See Supplementary Results). No difference is identified for the numbers of occurrence of alternative promoters, presence of splice variants or exons numbers between the transcripts from the two datasets (see Supplementary Results).

Supplementary Figure 5 Graphs and boxplots of translational repression elements and sequence differences between distal site synthesis (DSS) transcripts (red) and transport after synthesis (TAS) transcripts (gray).

See Online Methods for details and legend of Figure 2 for a description of boxplots and statistical tests (see Supplementary Results).

Supplementary Figure 6 Boxplots comparing the change in phosphorylation in distal site synthesis proteins as compared to transport after synthesis proteins across different time points in HeLa cells and SCC-9 cells after activation of signaling with EGF and LPA, respectively.

a. all phosphorylation sites and b. only tyrosine phosphorylation sites. The steady increase in phosphorylation is maintained over a longer time-course in DSS proteins than in TAS proteins. Between time point 10-20 minutes the DSS protein display a significant increase in tyrosine phosphorylation whereas the TAS proteins display a significant decrease in tyrosine phosphorylation (see Main Text for discussion). c changes in the phosphorylation state of regions close to putative linear motifs in SCC-9 cells compared to previous time point upon stimulation with lysophosphatidic acid. d, DSS proteins show higher levels of phosphorylation and maintain sustained phosphorylation after stimulation compared to TAS proteins in HEK-293 cells after stimulation with angiotensin (3 and 15 minutes time point). See legend of Figure 2 for a description of boxplots and statistical tests. d. changes in the phosphorylation states in HEK-293 cells. The abundance of phosphopeptides in DSS cells in maintained 15 minutes after stimulations. The abundance of TAS phosphopeptides drops significantly between 3 and 15 minutes.

Supplementary Figure 7 Boxplots comparing the genome-wide quantitative measurements of gene expression of mRNAs associated with free (in this case TAS) and mitochondrion-bound (in this case DSS) polysomes in yeast.

The top row of boxplots describes the genome-wide features associated with the transcriptional rate (left), mRNA abundance (center) and mRNA half-life (right), with the first two reflecting those results found for localized mRNA in fibroblast-like cells in mouse. The middle row of boxplots describe the genome-wide features associated with protein translation rate (left), protein abundance (center) and protein half-life (right) and similarly reflect the aforementioned results found in mouse. Finally, the bottom row describes molecular features of mRNA found associated with mitochondrion-bound polysomes. These results are different from our own aforementioned results. This is in-line with these proteins being of bacterial origin. The datasets used in each case are referenced in the boxplot title. For descriptions of datasets see Supplementary Table 1. See legend of Figure 2 for a description of boxplots and statistical tests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Tables 1 and 2, and Supplementary Note (PDF 3712 kb)

Supplementary Table 3

The results for the genome-wide analysis of asymmetrically localized proteins and transcripts from a mouse neuroblastoma cell line. (DOCX 469 kb)

Supplementary Table 4

The fibroblast-like cell line conversion table concatenated with results from genome-wide gene expression studies Ensembl_human IPI number EnsEMB. (DOCX 496 kb)

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Weatheritt, R., Gibson, T. & Babu, M. Asymmetric mRNA localization contributes to fidelity and sensitivity of spatially localized systems. Nat Struct Mol Biol 21, 833–839 (2014). https://doi.org/10.1038/nsmb.2876

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