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 hr 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.

HuR Function and Translational State Analysis Following Global Brain Ischemia and Reperfusion

“…Colocalization studies [11, 12] support the conclusion that mRNA granules are a form of HuR granule [13, 14]. When HuR colocalized with mRNA granules during reperfusion, hsp70 translation occurred [11].…”

“…Two mechanisms of prolonged TA have been demonstrated in post-ischemic CA1: sequestration of ribosomes in protein aggregates [9, 10] and the sequestration of mRNA away from ribosomal subunits in the form of mRNA granules [11, 12] (see Lewis et al this issue). Colocalization studies [11, 12] support the conclusion that mRNA granules are a form of HuR granule [13, 14].…”

Embryonic lethal abnormal vision proteins and adenine and uridine-rich element mRNAs after global cerebral ischemia and reperfusion in the rat

“…Normothermic global forebrain ischemia in male Long Evans rats, weighing 275-300 g, used the twovessel bilateral carotid artery occlusion and hypovolemic hypotension (2VO/HT) model of Smith et al, 30 as we previously described. 13,14,[31][32][33] …”

“…All antisera were previously validated for single band reactivity on Western blot. 14 Western blot procedures were as previously described. 31 …”

“…13 The mRNA granules colocalized with eukaryotic initiation factor (eIF) 4E, eIF4G, and poly-A-binding protein (PABP) but not with ribosomal subunit markers or organelle markers. [13][14][15] The embryonic lethal abnormal vision (ELAV) proteins colocalized with mRNA granules immediately in resistant neurons, but was delayed substantially in vulnerable neurons after global brain I/R, 13 suggesting that ELAV proteins contribute to outcome after brain I/R.…”