Persistent redistribution of poly-adenylated mRNAs correlates with translation arrest and cell death following global brain ischemia and reperfusion

Abstract

Although persistent translation arrest correlates with the selective vulnerability of post-ischemic hippocampal CA1 neurons, the mechanism of persistent translation arrest is not fully understood. Using fluorescent in situ hybridization and immunofluorescence histochemistry, we studied colocalization of polyadenylated mRNAs [poly(A)] with the following mRNA binding factors: eIF4G (translation initiation factor), HuR (ARE-containing mRNA stabilizing protein), poly(A) binding protein (PABP), S6 (small ribosomal subunit marker), TIA-1 (stress granule marker), and TTP (processing body marker). We compared staining in vulnerable CA1 and resistant CA3 from 1 to 48 hr reperfusion, following 10 min global ischemia in the rat. In both CA1 and CA3 neurons, cytoplasmic poly(A) mRNAs redistributed from a homogenous staining pattern seen in controls to granular structures we term mRNA granules. The mRNA granules abated after 16 hr reperfusion in CA3, but persisted in CA1 neurons to 48 hr reperfusion. Protein synthesis inhibition correlated precisely with the presence of the mRNA granules. In both CA1 and CA3, the mRNA granules colocalized with eIF4G and PABP, but not S6, TIA-1 or TTP, indicating that they were neither stress granules nor processing bodies. Colocalization of HuR in the mRNA granules correlated with translation of HSP70, which occurred early in CA3 (8hr) and was delayed in CA1 (36 hr). Thus, differential compartmentalization of mRNA away from the 40S subunit correlated with translation arrest in post-ischemic neurons, providing a concise mechanism of persistent translation arrest in post-ischemic CA1.

Mammalian Staufen 1 is recruited to stress granules and impairs their assembly

“…The formation of stress granules is usually transient and stress granules and related silencing foci containing polyadenylated mRNA form persistently when cell viability is compromised (Kayali et al, 2005;Jamison et al, 2008;Anderson and Kedersha, 2008). We found that cell survival upon stress is affected in Stau1-depleted cells and this might be connected to the differential persistence of stress granules.…”

“…Mammalian Staufen molecules contain several putative phosphorylation sites, and it has been shown that phosphorylation of Xenopus Staufen modulates its binding to intracellular structures (Allison et al, 2004). Whether differences in the expression levels, splicing variants or phosphorylation state of Stau1 and Stau2 modulate stress granule formation remains an open question.The formation of stress granules is usually transient and stress granules and related silencing foci containing polyadenylated mRNA form persistently when cell viability is compromised (Kayali et al, 2005;Jamison et al, 2008;Anderson and Kedersha, 2008). We found that cell survival upon stress is affected in Stau1-depleted cells and this might be connected to the differential persistence of stress granules.…”

Role of oxygen consumption in hypoxia protection by translation factor depletion

“…Components of the translation machinery have been found to be aggregated after hypoxia and this aggregation may promote or reduce cell death depending on the experimental model (Jamison et al, 2008;Buchan and Parker, 2009). Further, depletion of specific translation factors has differential effects on translation machinery aggregation (Mokas et al, 2009).…”

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] …”

“…12 We previously showed that poly-adenylated mRNAs form granular structures we termed ''mRNA granules, '' whose presence correlated precisely with depressed translation rates. 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.…”