Tyrosine pre-transfer RNA fragments are linked to p53-dependent neuronal cell death via PKM2

https://doi.org/10.1016/j.bbrc.2020.02.157Get rights and content

Highlights

  • CLP1 mutation induces the accumulation of pre-tRNA fragments in mice and humans.

  • 5′ Tyr-tRF leads to p53-dependent cell death in SH-SY5Y cell line.

  • Microinjection of 5′ Tyr-tRF into zebrafish embryos leads to p53-dependent neuronal defects.

  • 5′ Tyr-tRF directly binds to PKM2.

  • Neuronal defects induced by 5′ Tyr-tRF are ameliorated by PKM2 overexpression in zebrafish.

Abstract

Fragments of transfer RNA (tRNA), derived either from pre-tRNA or mature tRNA, have been discovered to play an essential role in the pathogenesis of various disorders such as neurodegenerative disease. CLP1 is an RNA kinase involved in tRNA biogenesis, and mutations in its encoding gene are responsible for pontocerebellar hypoplasia type-10. Mutation of the CLP1 gene results in the accumulation of tRNA fragments of several different kinds. These tRNA fragments are expected to be associated with the disease pathogenesis. However, it is still unclear which of the tRNA fragments arising from the CLP1 gene mutation has the greatest impact on the onset of neuronal disease. We found that 5′ tRNA fragments derived from tyrosine pre-tRNA (5′ Tyr-tRF) caused p53-dependent neuronal cell death predominantly more than other types of tRNA fragment. We also showed that 5′ Tyr-tRF bound directly to pyruvate kinase M2 (PKM2). Injection of zebrafish embryos with PKM2 mRNA ameliorated the neuronal defects induced in zebrafish embryos by 5′ Tyr-tRF. Our findings partially uncovered a mechanistic link between 5′ Tyr-tRF and neuronal cell death that is regulated by 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

References (24)

  • S. Weitzer et al.

    The human RNA kinase hClp1 is active on 3’ transfer RNA exons and short interfering RNAs

    Nature

    (2007)
  • A. Ramirez et al.

    Human RNA 5’-kinase (hClp1) can function as a tRNA splicing enzyme in vivo

    RNA

    (2008)
  • Cited by (15)

    • Modeling a human CLP1 mutation in mouse identifies an accumulation of tyrosine pre-tRNA fragments causing pontocerebellar hypoplasia type 10

      2021, Biochemical and Biophysical Research Communications
      Citation Excerpt :

      Hence, we evaluated the effects of these tRNA fragments on neuronal cell death in vitro using the human neuroblastoma cell line SH-SY5Y and in vivo using a zebrafish (Danio rerio) model. We demonstrated that 5′ Tyr-tRF leads to neuronal defects during vertebrate neurogenesis by suppressing pyruvate kinase M2 function [13]. Here, we report the generation and phenotype of knock-in mice carrying a homozygous missense mutation (p.R140H) in CLP1.

    • Intrinsic disorder and phase transitions: Pieces in the puzzling role of the prion protein in health and disease

      2021, Progress in Molecular Biology and Translational Science
      Citation Excerpt :

      The anti-apoptotic role of some tRFs that complex with cytochrome c, ultimately preventing apoptosome formation, has also been described.173 In another context, some tRFs have been associated with p53-dependent neuronal cell death.174,175 Further studies dissecting tRNAs and the interaction between their fragments and PrP would be interesting, considering in particular two studies that identified the interaction of PrP with argonaute proteins (AGO1 and AGO2).176,177

    • Roles and regulation of tRNA-derived small RNAs in animals

      2024, Nature Reviews Molecular Cell Biology
    View all citing articles on Scopus
    View full text