Genome-wide screen reveals APC-associated RNAs enriched in cell protrusions

Abstract

RNA localization is important for the establishment and maintenance of polarity in multiple cell types. Localized RNAs are usually transported along microtubules or actin filaments1 and become anchored at their destination to some underlying subcellular structure. Retention commonly involves actin or actin-associated proteins2,3,4,5,6,7, although cytokeratin filaments and dynein anchor certain RNAs8,9. RNA localization is important for diverse processes ranging from cell fate determination to synaptic plasticity; however, so far there have been few comprehensive studies of localized RNAs in mammalian cells. Here we have addressed this issue, focusing on migrating fibroblasts that polarize to form a leading edge and a tail in a process that involves asymmetric distribution of RNAs10,11,12. We used a fractionation scheme13 combined with microarrays to identify, on a genome-wide scale, RNAs that localize in protruding pseudopodia of mouse fibroblasts in response to migratory stimuli. We find that a diverse group of RNAs accumulates in such pseudopodial protrusions. Through their 3′ untranslated regions these transcripts are anchored in granules concentrated at the plus ends of detyrosinated microtubules. RNAs in the granules associate with the adenomatous polyposis coli (APC) tumour suppressor and the fragile X mental retardation protein (FMRP). APC is required for the accumulation of transcripts in protrusions. Our results suggest a new type of RNA anchoring mechanism as well as a new, unanticipated function for APC in localizing RNAs.

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Figure 1: Several RNAs are enriched in protruding pseudopodia of migrating cells.
Figure 2: The 3′ UTRs direct RNAs to accumulate in granules at tips of protrusions.
Figure 3: Localized RNA granules are anchored at the plus ends of detyrosinated microtubules.
Figure 4: APC associates with RNP complexes containing FMRP and is required for localization of RNAs in protrusions.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

The microarray data have been deposited in NCBI's Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE10230.

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Acknowledgements

We thank Y. Bao for the microarray and real-time PCR analysis, and J. Lykke-Andersen, I. Nathke, J. T. Parsons, R. Darnell and G. Gundersen for plasmids and antibodies. S.M. is a fellow of the Leukemia and Lymphoma Society. This work was supported by a grant from the NIH to I.G.M., and by the James and Rebecca Craig Foundation.

Author Contributions S.M. performed the experiments. S.M., K.M. and I.G.M. designed the experiments and analysed the data. K.M. provided reagents. S.M. and I.G.M. wrote the manuscript.

Author information

Correspondence to Stavroula Mili or Ian G. Macara.

Supplementary information

Supplementary Information

The file contains Supplementary Figures 1-14 with Legends. and Legends to Supplementary Movies 1-5. (PDF 10564 kb)

Supplementary Table S1

The file contains Supplementary Table S1. This file contains a list of RNAs significantly enriched in pseudopodia in response to both LPA and fibronectin (FN) stimulation. (XLS 25 kb)

Supplementary Table S2

The file contains Supplementary Table S2. This file contains a list of RNAs significantly enriched in pseudopodia (Ps) or cell bodies (CB) in response to LPA. (XLS 136 kb)

Supplementary Table S3

The file contains Supplementary Table S3. This file contains a list of RNAs significantly enriched in pseudopodia (Ps) or cell bodies (CB) in response to Fibronectin. (XLS 248 kb)

Supplementary Movie 1

The file contains Supplementary Movie 1. Confocal fluorescence time lapse imaging of a cell expressing mRFP (red), MS2-GFP (green) and the β globin-24bs/pkp4 mRNA. Overlay images of the two channels are shown. Arrows point to protrusions with localized RNA granules. Note that the RNA granules remain stationary over the course of observation. Time is indicated on the upper left corner in seconds. (MOV 2009 kb)

Supplementary Movie 2

The file contains Supplementary Movie 2. FRAP experiment of a localized RNA granule. Shown is a cell expressing MS2-GFP and the β globin-24bs/pkp4 mRNA. A localized RNA granule at the end of a protrusion is indicated by an arrow. Fluorescence at this protrusion was bleached and its recovery recorded over ca 60 seconds. Note that fluorescence recovers only minimally during the course of observation. Time is indicated on the upper left corner in seconds. (MOV 211 kb)

Supplementary Movie 3

The file contains Supplementary Movie 3. FRAP experiment of a localized RNA granule. Shown is a cell expressing MS2-GFP and the β globin-24bs/pkp4 mRNA and which exhibits two localized RNA granules at the ends of two protrusions. Fluorescence at one of the granules (arrow) was bleached and its recovery recorded over ca 4 minutes. Note that fluorescence recovers only minimally during the course of observation while the second granule (arrowhead) remains stationary. Time is indicated on the upper left corner in seconds. (MOV 194 kb)

Supplementary Movie 4

The file contains Supplementary Movie 4. FRAP experiment of a cell expressing EB1-GFP. Fluorescence at a protrusion (arrow) was bleached and its recovery recorded over ca 60 seconds. Note that EB1-GFP comets (representing +ends of dynamic MTs) rapidly move throughout the cell body and several of them enter into protrusions over the course of observation. Time is indicated on the upper left corner in seconds. (MOV 2488 kb)

Supplementary Movie 5

The file contains Supplementary Movie 5. FRAP experiment of a cell expressing GFP-APC which exhibits APC granules at the tips of different protrusions. Fluorescence at the tip of one protrusion (arrow) was bleached and its recovery recorded over ca 60 seconds. Note that fluorescence recovers only minimally during the course of observation while another APC granule (arrowhead) remains stationary. Time is indicated on the upper left corner in seconds. (MOV 601 kb)

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Mili, S., Moissoglu, K. & Macara, I. Genome-wide screen reveals APC-associated RNAs enriched in cell protrusions. Nature 453, 115–119 (2008). https://doi.org/10.1038/nature06888

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