Composition of Staufen2 ribonucleoprotein particles in neurons

In neurons, many mRNAs are transported along neuronal dendrites to their site of translation by ribonucleoprotein complexes (mRNPs). Numerous mRNA-binding, motor and regulatory proteins within mRNPs finely regulate the fate of each of these mRNAs and their specific combination is likely to define different types of mRNA complexes and their neuronal functions. Staufen2 is a well known mRNA-binding protein present in distinct population of mRNPs in neurons and is implicated in the transport of mRNAs along the dendritic microtubules. However, little is known about the associated mRNAs in Staufen2 neuronal mRNPs complexes. As a means to understand its role in neuronal processes, a genome wide approach has been used to identify mRNAs that are co-immunoprecipitated with Staufen2 complexes from embryonic rat brains. The genome wide approach identified 1780 mRNAs associated with Staufen2 mRNPs that code for proteins involved in cellular processes such as post-translational protein modifications, RNA metabolism, intracellular transport and translation. All these mRNAs are consistent with the role of Staufen2 in synaptogenesis. Immunoprecipitation of Staufen2-containing mRNPs was performed on cell extracts prepared from whole brains of E17-E18 Sprague Dawley rat embryos. Cells were dissociated with a manual putter and lysed in 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.5% Triton X-100, 15 mM EGTA, 1 mM DTT and complete EDTA-free protease inhibitor cocktail (Roche). Cell lysates were centrifuged at 10 000 rpm for 10 min. After centrifugation, the supernatants were incubated with a rabbit polyclonal anti-Staufen2 or preimmune antibodies for 2 hours at 4°C, then with a 50% protein A-sepharose slurry for 2 hours at 4°C. Immune complexes were washed five times with the lysis buffer and eluted from the resin by heating at 95°C for 5 minutes in elution buffer (100 mM Tris-HCl pH 7.4, 200 mM DTT and 4% SDS). RNAs were isolated by Trizol (Invitrogen) extraction. Experiments were repeated several times (Anti-Stau2: n = 6; control preimmune: n = 6).