Novel insights into RNP granules by employing the trypanosome’s microtubule skeleton as a molecular sieve

We have exploited a unique feature of trypanosomes to prepare a cellular fraction highly enriched in starvation stress granules. First, granules remain trapped within the subpellicular microtubule array of the trypanosome cytoskeleton while soluble proteins are washed away. Second, the microtubules are depolymerized and the granules are released. RNA sequencing combined with single mRNA FISH revealed that mRNA localization to granules positively correlates with mRNA size and negatively with mRNA abundance. Only 25% of all mRNA molecules localize to granules. mRNAs encoding ribosomal proteins remain excluded from granules. We have identified 463 stress granule candidate proteins. For 18 proteins we have validated the localization to granules, including two phosphatases and two proteins with a function in life-cycle regulation. Our protocol is rapid, granule dissociation and mRNA degradation are minimized and high throughput experiments are possible. Our data provide novel insight into the possible mechanism of mRNA localization to RNP granules.