Increased expression of fragmented tRNA promoted neuronal necrosis

Yanyan Cao,Ying Xiong,Lei Liu,Chunyue Zhao,Kai Liu

doi:10.1038/s41419-021-04108-6

Abstract

Neuronal necrosis induced by excessive glutamate release is well known to contribute morbidity and mortality in ischemic stroke. Over the past decades, strategies on targeting glutamate receptor did not achieve desirable clinical outcomes. Finding the downstream mechanism of the glutamate receptor activation may provide new targets to suppress the cell death. Previously, our study demonstrated that the increase of H3K4 trimethylation (H3K4me3) played a key detrimental role on neuronal necrosis; however, the mechanism of this histone modification is unclear. Through a genome-wide small RNA sequencing, we identified several tRNA-derived fragments (tRFs) and piwi-interacting RNA (piRNAs) species were enriched in glutamate-induced neuronal necrosis in rat primary neuron cultures, and this enrichment was dependent on the H3K4me3 increase. Strikingly, when we transfected several synthesized tRFs and piRNA species into neurons, the tRFs but not the piRNAs induced neuron swelling and death. The cell death morphology recapitulated neuronal necrosis induced by glutamate. For the cytotoxic effect of tRFs, our data suggested that protein synthesis was inhibited likely through induction of ribosomal stalling. By proteomic analysis of tRFs effect, the most affected pathway was enriched in the mitochondrial metabolism. Consistently, mitochondrial fragmentation was increased in neuronal necrosis, and suppression of mitochondrial fission by genetic manipulation or drug rescued neuronal necrosis. Using our previously established Drosophila model of neuronal necrosis, we found that inhibition of small RNA transcription, blocking RNA transport from nucleus to cytosol, or knocking down Ago1/2 to suppress the RNA interference effect, all rescued the fly death, suggesting transcription and processing of small RNAs contribute to neuronal necrosis. Together, these results indicate that the abnormal transcription of tRFs may play a key role downstream of the H3K4me3 increase. This provides a potential new strategy to suppress neuronal necrosis.

Highlights

Excitotoxicity is induced by the excessive synaptic release of glutamate, which binds with various glutamate receptors, especially the N-methyl-D-aspartate (NMDA), to induce calcium overload and subsequent neuronal cell death
Our RNAseq data suggested that certain small noncoding RNAs (ncRNAs) (14−35 nucleotides) especially the tRNA-derived fragments (tRFs) and piRNAs were enriched in the mammalian neuron necrosis model
A Compared to the control (AG flies without induction of necrosis), the top 10 enriched Gene Ontology (GO) annotations and KEGG pathway analysis of downregulated proteins are listed

Summary

INTRODUCTION

Excitotoxicity is induced by the excessive synaptic release of glutamate, which binds with various glutamate receptors, especially the N-methyl-D-aspartate (NMDA), to induce calcium overload and subsequent neuronal cell death. A double-mutant peptide (DM pep) that was unable to interfere with the WDR5/MLL1 complex, showed no rescue effect on neuronal necrosis [5]. Considering the H3K4me increase is a common marker of active transcription [8] and the inhibition of H3K4 methylation could rescue neuronal necrosis [5], we hypothesize that active transcription may produce some abnormal small RNAs which are cytotoxic. The rtStarTM tRF & tiRNA pretreatment kit (Arraystar, USA) was used to remove tRNAs with diverse modifications such as pseudouridine, 4-thiouridine, 1-methyladenosine, and queuosine. These modifications may interfere with the construction of the cDNA library. The libraries were denatured and diluted to a loading volume of 1.3 ml with a concentration of 1.8 pM

MATERIALS AND METHODS

RESULT

Findings

DISCUSSION