Organelles do not colocalize with mRNA granules in post-ischemic neurons

Abstract

Following global brain ischemia and reperfusion, it is well-established that neurons undergo a translation arrest that is reversible in surviving neurons, but irreversible in vulnerable neurons. We previously showed a correlation between translation arrest in reperfused neurons and the presence of granular mRNA-containing structures we termed "mRNA granules." Here we further characterized the mRNA granules in reperfused neurons by performing colocalization studies using fluorescent in situ hybridization for poly(A) mRNAs and immunofluorescence histochemistry for markers of organelles and mRNA-binding proteins. There was no colocalization between the mRNA granules and markers of endoplasmic reticulum, cis- or trans-Golgi apparatus, mitochondria, microtubules, intermediate filaments, 60S ribosomal subunits, or the HuR ligands APRIL and pp32. The mRNA granules colocalized with the neuronal marker NeuN regardless of the relative vulnerability of the neuron type. RNA immunoprecipitation of HuR from the cytoplasmic fraction of 8 h reperfused forebrains selectively isolated hsp70 mRNA suggesting the mRNA granules are soluble structures. Together, these results rule out several organelle systems and a known HuR pathway as being directly involved in mRNA granule function.