Methyltransferase 3 Mediated miRNA m6A Methylation Promotes Stress Granule Formation in the Early Stage of Acute Ischemic Stroke

No fucking clue what this is trying to say.  Even looking up Stress Granule Formation in Google Scholar didn't help. But then this is from integrated medicine and TCM.

Methyltransferase 3 Mediated miRNA m6A Methylation Promotes Stress Granule Formation in the Early Stage of Acute Ischemic Stroke

Wenwen Si¹, Yi Li², Shanyu Ye³, Zhen Li³, Yangping Liu³, Weihong Kuang⁴, Dongfeng Chen^3* and Meiling Zhu^5*

• ¹Shenzhen Bao’an Traditional Chinese Medicine Hospital (Group), Guangzhou University of Chinese Medicine, Shenzhen, China
• ²The First Clinical Medical College, Guangzhou University of Chinese Medicine, Guangzhou, China
• ³Department of Anatomy, The Research Center of Basic Integrative Medicine, Guangzhou University of Chinese Medicine, Guangzhou Higher Education Mega Center, Guangzhou, China
• ⁴The Second Clinical Medical College, Guangdong Medical University, Dongguan, China
• ⁵Shenzhen Hospital of Integrated Traditional Chinese and Western Medicine, Shenzhen, China

The modification of methyltransferase-like (METTL) enzymes plays important roles in various cellular responses by regulating microRNA expression. However, how m6A modification is involved in stress granule (SG) formation in the early stage of acute ischemic stroke by affecting the biogenesis processing of microRNAs remains unclear. Here, we established a middle cerebral artery occlusion (MCAO) model in rats and an oxygen-glucose deprivation/reperfusion (OGD/R) model in primary cortical neurons and PC12 cells to explore the potential mechanism between m6A modification and SG formation. The in vivo results showed that the level of infarction and apoptosis increased while SG formation decreased significantly within the ischemic cortex with improved reperfusion time after 2 h of ischemia. Consistent with the in vivo data, an inverse association between the apoptosis level and SG formation was observed in PC12 cells during the reperfusion period after 6 h of OGD stimulation. Both in vivo and in vitro results showed that the expression of METTL3 protein, m6A and miR-335 was significantly decreased with the reperfusion period. Overexpression of the METTL3 and METTL3 gene-knockdown in PC12 cells were achieved via plasmid transfection and CRISPR-Cas9 technology, respectively. Overexpression or knockdown of METTL3 in oxygen-glucose deprivation of PC12 cells resulted in functional maturation of miR-335, SG formation and apoptosis levels. In addition, we found that miR-335 enhanced SG formation through degradation of the mRNA of the eukaryotic translation termination factor (Erf1). In conclusion, we found that METTL3-mediated m6A methylation increases the maturation of miR-335, which promotes SG formation and reduces the apoptosis level of injury neurons and cells, and provides a potential therapeutic strategy for AIS.

Introduction

The acute ischemic stroke (AIS) is a severe neurological disease with high incidence, disability and mortality, which brings enormous psychological and economic burden to patients and the social economy (Manning et al., 2014; Fisher and Saver, 2015; Fraser, 2016). Intravenous recombinant tissue plasminogen activator (tPA) and endovascular thrombectomy (EVT) are the only effective treatments available for AIS (Lee et al., 2018). However, due to the narrow therapeutic window, patients with AIS may fail to gain the benefits of acute treatments (Nogueira et al., 2018). Cerebral ischemia/reperfusion (I/R) injury rapidly triggers different types of programmed cell death in neurons, such as apoptosis, autophagy and programmed necrosis, and this injury is inevitable and irreversible (Li et al., 2018). Therefore, it is vital to increase the resistance of neurons to ischemic conditions in the early stage of AIS when mildly injured neurons have not entered the process of apoptosis or necrosis (Ovesen et al., 2014).

Since the pathogenesis of AIS involves complex physiological and pathological processes, it is challenging to develop effective therapies and biological markers for the treatment in the early phase of the disease (Khandelwal et al., 2016; Zerna et al., 2016; Catanese et al., 2017). When neurons are exposed to ischemic and hypoxic microenvironments surrounding the ischemic brain tissue, the transient translation arrest occurs in the ischemic region, which reduces the misfolding of proteins and mistranslation of mRNA, thus inhibiting the necrosis of brain tissue and neuronal death (DeGracia and Hu, 2007; DeGracia et al., 2008; Vosler et al., 2011). Interestingly, the translation arrest of proteins is also an essential condition for the production of stress granules (SGs) (Beckham and Parker, 2008; Anderson and Kedersha, 2009). SGs are non-membrane aggregates of proteins and mRNAs, produced in the cytoplasm under various harmful environmental stimuli (Bhattacharyya et al., 2006; Buchan and Parker, 2009). SGs can stall immediately and transiently mRNA translation to protect valuable mRNAs and proteins from the injury of the harmful environment, thus improving the survival rate of cells in the early stage of AIS (Takahashi et al., 2013; Waris et al., 2014; Arimoto-Matsuzaki et al., 2016). However, the mechanism of SG formation in the early stage of AIS remains unclear.

Mature miRNAs are produced from primary microRNAs (pri-miRNAs) of several 100 base lengths through digestion with a series of nucleases (Murchison and Hannon, 2004; Winter et al., 2009). Recent evidence suggests that methyltransferase 3 (METTL3) increases the production of mature miRNA by adding a m6A modification to the primary miRNAs (pri-miRNAs), which makes it easier to be identified and digested by the DiGeorge syndrome critical region 8 (DGCR8) and Drosha enzyme (Alarcon et al., 2015; Han et al., 2019). In addition, it was found that METTL3 knockout decreased the binding activity between DGCR8 and pri-miRNA, leading to decreased expression of mature miRNAs (Alarcon et al., 2015). Our previous studies showed that miR-335 promotes the production of stress granules by targeting the Rho-associated coiled-coil forming protein kinase 2 (ROCK2), and its expression is reduced in AIS specificity (Si et al., 2019). Therefore, we hypothesized that m6A modification is involved in SG formation in the early stage of AIS by affecting the biogenesis processing of miR-335.

In this study, we used a rat MCAO (middle cerebral artery occlusion) model and an OGD/R model of PC12 cells and primary cortical neurons, to investigate METTL3-mediated m6A methylation of miR-335 in SG formation at the early stage of AIS.