Live cell co-transfected with plasmids encoding mRFP, MS2-GFP and beta-globin constructs with Pkp4 3' UTRs. The beta-globin mRNA localizes in granules at tips of protrusions (green).

From Nature 453, 115-119 (2008).

Although RNA localization is known to be important for establishing and maintaining cell polarity, there have been few comprehensive studies of localized RNAs in mammalian cells. As reported in Nature, Ian Macara and colleagues have now identified the RNAs that accumulate at cell protrusions via an unanticipated mechanism involving the APC protein.

Using a fractionation method combined with Affymetrix GeneChip arrays, the authors identified approximately 50 RNAs that preferentially localized to the pseudopodial protrusions of fibroblasts exposed to migratory stimuli. Two of these mRNAs - Rab13 and Pkp4 (plakophilin 4) - were chosen to study the mechanism of localization.

The Rab13 mRNAs containing the open reading frame and the 3'-untranslated region (3'-UTR) were enriched in pseudopodia, but lost this localization when the 3'-UTR was replaced with that of another non-localized gene. Moreover, fusion of the Rab13 3'-UTR to the non-localized -globin mRNA resulted in -globin being recruit ed to pseudopodia, showing that Rab13 3'-UTR is necessary and sufficient to direct RNA accumulation in pseudopodia.

To analyse the localization of these RNAs in live cells, the authors inserted 24 repeats of the MS2 phage coat protein-binding sites between the -globin region and the Rab13 3'-UTR. This reporter RNA could be indirectly visualised upon co-expression with an MS2-green fluorescent protein (GFP) fusion that binds to the 24 repeats, and thus accompanied the RNA to the cytoplasm. The authors observed that the GFP signal concentrated in granules at the tips of protrusions and that the Pkp4 3'-UTR conferred a similar localization. FRAP experiments indicated that the RNAs were stably sequestered in these granules.

But what are the cellular structures that anchor transcripts at pseudopodial tips? Expression of fluorescently tagged markers for various structures revealed that the granules were attached to microtubule plus ends. This association was specific for stable microtubules that did not exhibit dynamic instability - a finding consistent with the granules being relatively stationary.

The APC tumour suppressor is known to bind to plus ends of stable microtubules that grow into migrating cell protrusions. The authors found that APC co-localized with the RNA granules, and that both Rab13 and Pkp4 transcripts associated with endogenous APC. Furthermore, APC knockdown reduced the number of these transcripts in pseudopodia, showing that APC mediates RNA accumulation and anchoring in protrusions.

This work identifies on a genome-wide scale the RNAs that are asymmetrically distributed in migrating cells, and shows that APC anchors RNAs with specific 3'-UTRs to microtubule plus ends at pseudopodial tips. APC was also found to bind the translational regulator FMRP (fragile X mental retardation protein) and a poly(A)-binding protein (PABP1), suggesting that APC is a component of RNP complexes that contain these proteins. However, whether the effects of the APC tumour suppressor on cell polarization and migration are due to its RNA anchoring function remains to be determined.

Author: Kim Baumann