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

Abstract

RNA localization is important for establishment and maintenance of polarity in multiple cell types. Localized RNAs are usually transported along microtubules or actin filaments 1 , and become anchored at their destination to some underlying subcellular structure. Retention commonly involves actin or actin-associated proteins 2-7 , although cytokeratin filaments and dynein anchor certain RNAs 8,9 . RNA polarization is important for diverse processes ranging from cell fate determination to synaptic plasticity. However, there has been to date no comprehensive identification of localized RNAs in any mammalian cell type. Here we have addressed this issue, focusing on migrating fibroblasts, which polarize to form a leading edge and a tail, in a process that involves asymmetric distribution of RNAs 10-14 . We used a fractionation scheme 15 combined with microarrays to identify for the first time, on a genome-wide scale, localized RNAs in mammalian cells. We find that a diverse group of RNAs accumulates in pseudopodial protrusions during cell migration. Through their 3'UTRs these transcripts are anchored in granules concentrated at the plus ends of detyrosinated microtubules. RNAs in the granules associate with the APC tumor suppressor and the Fragile X Mental Retardation protein (FMRP). APC is required for accumulation of transcripts in protrusions. Our results reveal a new type of RNA anchoring mechanism as well as a novel, unanticipated function for APC in localizing RNAs.To identify on a genome-wide scale RNAs that are enriched at the leading edge of migrating cells, we employed a fractionation method in which cells are plated on a microporous filter and are induced to polarize and extend pseudopodial protrusions in response to a migratory stimulus. Pseudopodia and cell bodies are then physically isolated and their contents compared 15 . We isolated pseudopodia (Ps) and cell body (CB) fractions from NIH/3T3 cells migrating towards a chemotactic (lysophosphatidic acid, LPA) or a haptotactic (fibronectin, FN) stimulus (Fig. 1a, b and S1a). The quality of the fractionation was assessed by immunoblotting for activated FAK (phosphorylated at Y397), which is concentrated at the leading edge during migration 15,16 . Activated FAK was enriched in the pseudopodial fraction (Fig. S1b) (see table 1 and S1). However, comparison of the primary sequences of these transcripts did not reveal any readily identifiable motifs shared among them, suggesting that potential localization signals are likely defined by a combination of primary sequence and higher order structure, as is common for most localized RNAs 17 .To dissect the localization mechanisms, we focused on the rab13 and plakophilin 4 (pkp4) mRNAs, which both showed robust localization. First, we expressed in NIH/3T3 cells the rab13 gene encompassing the whole open reading frame and 3'UTR (Fig. 2a). A sequence encoding the FLAG tag was introduced at the 5'end of the first coding exon to distinguish the exogenous mRNA from endogenous transcript. Transfected cells plate...

Computer vision for image-based transcriptomics

“…6A): transcripts, which become translated at the endoplasmic reticulum (ER), should have a shorter distance to the nucleus compared to transcripts with a cytoplasmic translation. For instance, we noticed that transcripts of RAB13, which have previously been described to enrich in filopodia [35], tended to be furthest from the nucleus (Fig. 6B).…”

Teachings from the egg: New and unexpected functions of RNAs

“…3;Jansen, 2001;Czaplinski and Singer, 2006;Bramham and Wells, 2007;Singer, 2008). Recent genome-wide screen of RNAs localized in leading edge of migrating mammalian fibroblasts showed that in response to migratory stimuli, 50 different mRNAs (besides previously identified b-actin mRNA) accumulated, via microtubule mediated transport, in the fibroblast protrusions rich in actin cytoskeletal elements (Mili et al, 2008). In epithelial cells, the apical cytoskeleton containing cytokeratin, actin, and microtubules (Salas et al, 1997;Sugimoto et al, 2008) is enriched in localized wingless mRNA (Simmonds et al, 2001;Najand and Simmonds, 2007).…”

Beyond ‘furballs’ and ‘dumpling soups’ – towards a molecular architecture of signaling complexes and networks

“…More than 70% of mRNAs of 3370 genes analyzed were asymmetrically expressed in Drosophila [52]. The first systematic investigation of mRNA localization in mammalian cells found that more than 50 species of mRNA preferentially accumulate in the cellular protrusions of migrating mouse NIH3T3 fibroblasts [53].…”