Tyrosine pre-transfer RNA fragments are linked to p53-dependent neuronal cell death via PKM2
Introduction
Transfer RNAs (tRNAs) are one of the most abundant types of non-coding RNA, and are essential for protein synthesis by bringing amino acids to the translating ribosome. Current deep-sequencing technologies have revealed that various types of small RNA fragments derived from tRNAs are present in most organisms. Recent studies have shown that some tRNA fragments directly contribute to the pathogenesis of some human diseases [[1], [2], [3]].
CLP1 is an RNA kinase that phosphorylates the 5′ hydroxyl ends of RNA [[4], [5], [6]]. Human CLP1 is a component of the messenger RNA 3′-end cleavage and polyadenylation machinery [7]. Human CLP1 is also a component of the tRNA splicing endonuclease (TSEN) complex, which removes the intron present within the anticodon loop of several pre-transfer RNAs and generates tRNA exon halves [8]. Previously, we have shown that CLP1 kinase-dead knock-in mice developed progressive neurodegenerative disease [9]. We also showed that the cause of the neuronal pathogenesis is the accumulation of the 5′ exon of tyrosine pre-tRNA fragments (5′ Tyr-tRF), which comprises a 5′ leader sequence followed by the 5′ exon Tyr-tRNA. The 5′ Tyr-tRF augments the activation of p53 resulting in neuronal cell death.
Human CLP1 mutation (p.R140H) has been identified in four Turkish families [10,11]. As was also shown in the CLP1 kinase-dead knock-in mice, human CLP1 mutation causes a neurological syndrome. This syndrome is called pontocerebellar hypoplasia type-10 (PCH10) and involves microcephaly and axonal peripheral neuropathy. We detected an accumulation of introns derived from isoleucine pre-tRNAs in patient’s fibroblasts, indicating that these tRNA introns may be a cause of the neurodevelopmental and neurodegenerative disorder. Further, the 3′ exon halves of Tyr-tRNA, in which the 5′ end was unphosphorylated, have been reported to be the most toxic in human cells [11]. However, the pathological association between these tRNA fragments and neurodegeneration remained unclear.
Here, we report the pathological significance of 5′ Tyr-tRF in neuronal development by using a zebrafish (Danio rerio) model. We also demonstrated by using a biochemical method that 5′ Tyr-tRF directly binds to PKM2. Neuronal defects induced in zebrafish by the accumulation of 5′ Tyr-tRF were ameliorated by microinjection of PKM2 mRNA into zebrafish embryos. These results suggest that the neuronal defects are initiated by the interaction between 5′ Tyr-tRF and PKM2 during neurogenesis.
Section snippets
Cell culture
SH-SY5Y cells and HEK293T cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) and Ham’s F-12 Nutrient Mixture (DMEM/Ham’s F12) with l-glutamine (Wako, Osaka, Japan), containing 10% FBS (Life Technologies, Grand Island, NY, USA) and 1% penicillin/streptomycin (Nacalai Tesque, Kyoto, Japan). Cells were maintained at 37 °C in a saturated humidity atmosphere containing 95% air and 5% CO2. For differentiation into neuronal cells, SH-SY5Y cells were cultured with 15 μM all-trans-retinoic
Toxicity of 5′ Tyr-tRF in human SH-SY5Y cells
To assess the potential roles of RNA fragments derived from tyrosine pre-tRNA in neuronal cells, 5′ Tyr-tRF and 3′ Tyr-tRF as well as control-tRF (5′ arginine-tRF [5′ Arg-tRF]) were transfected into the SH-SY5Y human neuroblastoma cell line. The concentrations of each of the tRFs used for the cell transfection were those used in previous experiments examining tRF toxicity in human fibroblasts [11].
Subsequently, neuronal differentiation of the cells was induced by retinoic acid. Although none of
Discussion
In this study, we investigated the role of tRNA fragments produced by CLP1 mutation in mice and human patients. 5′ Tyr-tRF was more toxic for SH-SY5Y cells differentiation by retinoic acid stimulation than the other tRNA fragments. 5′ Tyr-tRF injection into one-cell zebrafish embryos caused more severe neuronal abnormalities than did other tRNA fragments. Furthermore, we identified PKM2 as the target molecule for 5′ Tyr-tRF. PKM2 mRNA injection prevented the developmental abnormalities induced
Declaration of competing interest
The authors declare no conflict of interests.
Acknowledgments
We thank M. Nakamura-Ota, M. Oda, E. Koba, and T. Nitta for their excellent technical assistance. T.H. was supported by the Japan Society for the Promotion of Science [17K19919], Takeda Science Foundation, Astellas Foundation for Research on Metabolic Disorders, The Uehara Memorial Foundation, Japan Foundation for Applied Enzymology, Mitsubishi Foundation, and Mizoguchi Urology Clinic. Parts of this work were performed as part of the Cooperative Research Project Program of the Medical Institute
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