tRNA Splicing Endonuclease Research Articles

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

Cleavage factor polyribonucleotide kinase subunit 1 (CLP1), an RNA kinase, plays essential roles in protein complexes involved in the 3′-end formation and polyadenylation of mRNA and the tRNA splicing endonuclease complex, which is involved in precursor tRNA splicing. The mutation R140H in human CLP1 causes pontocerebellar hypoplasia type 10 (PCH10), which is characterized by microcephaly and axonal peripheral neuropathy. Previously, we reported that RNA fragments derived from isoleucine pre-tRNA introns (Ile-introns) accumulate in fibroblasts of patients with PCH10. Therefore, it has been suggested that this intronic RNA fragment accumulation may trigger PCH10 onset. However, the molecular mechanism underlying PCH10 pathogenesis remains elusive. Thus, we generated knock-in mutant mice that harbored a CLP1 mutation consistent with R140H. As expected, these mice showed progressive loss of the upper motor neurons, resulting in impaired locomotor activity, although the phenotype was milder than that of the human variant. Mechanistically, we found that the R140H mutation causes intracellular accumulation of Ile-introns derived from isoleucine pre-tRNAs and 5′ tRNA fragments derived from tyrosine pre-tRNAs, suggesting that these two types of RNA fragments were cooperatively or independently involved in the onset and progression of the disease. Taken together, the CLP1-R140H mouse model provided new insights into the pathogenesis of neurodegenerative diseases, such as PCH10, caused by genetic mutations in tRNA metabolism-related molecules.

Reconstitution of Human tRNA Intron Cleavage Leads to an Updated Model of tRNA Splicing

Transfer RNAs (tRNAs) are critical for translation. tRNAs require extensive post-transcriptional maturation, including the removal of an intron for a subset of tRNAs. tRNA splicing is a multi-step process that involves the cleavage of introns and the ligation of tRNA exons back together. In eukaryotes, tRNA introns are removed by the heterotetrametric tRNA splicing endonuclease (TSEN) complex. In metazoans, ligation is carried out by the tRNA ligase, RTCB, which also ligates introns into tRNA intronic circular RNAs (tricRNAs). Furthermore, the metazoan TSEN complex associates with an accessory protein 5’-hydroxyl polyribonucleotide kinase CLP1. Until recently, the function of CLP1 in tRNA splicing was thought to be in supporting TSEN function/stability as mutations in CLP1 and the TSEN proteins are known to lead to a family of neurological and neurodegenerative diseases, pontocerebellar hypoplasia (PCH). Despite these connections, non-metazoan eukaryotes do not utilize CLP1 in tRNA splicing. To address the role of CLP1 in TSEN function and tRNA splicing, we developed the first system for the production of recombinant active TSEN complex from E. coli and complemented this approach with immunoprecipitations from HEK cells. We showed for the first time that CLP1 is not required for the TSEN complex to remove tRNA introns, uniting how eukaryotes cleave tRNA introns. We then utilized a reporter tRNA to monitor tRNA splicing in cells. We overexpressed CLP1 and kinase-dead CLP1 in HEK cells and monitored splicing of our reporter tRNA. We found that kinase-active but not kinase-dead CLP1 reduced the production of tricRNAs, suggesting that CLP1 might negatively regulate the ligation step of tRNA splicing. In parallel, we knocked down CLP1's homolog, cbc, in Drosophila cells and looked at the maturation of a Drosophila-specific tRNA reporter. We found that depletion of cbc led to a significant increase in both mature tRNA as well as tricRNAs for our reporter construct. Together, our data support a new model of tRNA splicing in which CLP1 is a negative regulator of tRNA and tricRNAs maturation. Hayne, et al. Reconstitution of the human tRNA splicing endonuclease complex: insight into the regulation of pre-tRNA cleavage (2020), Nucleic Acids Research.

TRNA: Splicing and Intron Turnover and Possible Functions

A subset of tRNA genes in eukaryotes contains an intron. tRNAs introns are short sequences located 1 nt. 3′ of the anticodon. Introns are removed from precursor tRNAs (pre-tRNA) by the conserved heterotetrameric tRNA splicing endonuclease (SEN), that recognizes pre-tRNA structure rather than sequence motifs at splice junctions. In vertebrate cells intron removal from pre-tRNAs occurs in the nucleoplasm; in contrast, pre-tRNA splicing in budding yeast and fission yeast occurs on the mitochondrial surface (Yoshihisa et al. 2003; Wan & Hopper, 2018); therefore, pre-tRNA splicing on the mitochondrial surface has been conserved for greater than 500 million years. Free tRNA introns are rarely detected in cells because they are rapidly and efficiently destroyed. We identified at least 5 separate, family-specific mechanisms that function in tRNA intron destruction (Wu & Hopper, 2014; Bao, Metcalf & Hopper, unpublished). Interestingly, under particular stress conditions, specific tRNA introns accumulate to high levels, possibly indicative of their functions in stress responses (Peltier, Metcalf, & Hopper, unpublished). Moreover, we discovered that tRNA introns possess long stretches of perfect complementarity to particular budding yeast mRNA ORFs (Bao, Nostramo, & Hopper, unpublished). Deletion of the introns from the 2 copies of the genes encoding tRNAIleUAU results in elevated levels of mRNAs with complementarity, but not mRNAs lacking complementarity; further, transformation of the cells lacking the tRNAIleUAU introns with extrachromosomal intron-containing tRNAIleUAU suppresses the mRNA enhanced levels. These results support the hypothesis that tRNA introns may function as a novel type of noncoding regulatory RNA, perhaps functioning in stress response (Nostramo & Hopper, unpublished).

Comparative parallel analysis of RNA ends identifies mRNA substrates of a tRNA splicing endonuclease-initiated mRNA decay pathway

Eukaryotes share a conserved messenger RNA (mRNA) decay pathway in which bulk mRNA is degraded by exoribonucleases. In addition, it has become clear that more specialized mRNA decay pathways are initiated by endonucleolytic cleavage at particular sites. The transfer RNA (tRNA) splicing endonuclease (TSEN) has been studied for its ability to remove introns from pre-tRNAs. More recently it has been shown that single amino acid mutations in TSEN cause pontocerebellar hypoplasia. Other recent studies indicate that TSEN has other functions, but the nature of these functions has remained obscure. Here we show that yeast TSEN cleaves a specific subset of mRNAs that encode mitochondrial proteins, and that the cleavage sites are in part determined by their sequence. This provides an explanation for the counterintuitive mitochondrial localization of yeast TSEN. To identify these mRNA target sites, we developed a "comPARE" (comparative parallel analysis of RNA ends) bioinformatic approach that should be easily implemented and widely applicable to the study of endoribonucleases. The similarity of tRNA endonuclease-initiated decay to regulated IRE1-dependent decay of mRNA suggests that mRNA specificity by colocalization may be an important determinant for the degradation of localized mRNAs in a variety of eukaryotic cells.

Reconstitution of the human tRNA splicing endonuclease complex: insight into the regulation of pre-tRNA cleavage.

The splicing of tRNA introns is a critical step in pre-tRNA maturation. In archaea and eukaryotes, tRNA intron removal is catalyzed by the tRNA splicing endonuclease (TSEN) complex. Eukaryotic TSEN is comprised of four core subunits (TSEN54, TSEN2, TSEN34and TSEN15). The human TSEN complex additionally co-purifies with the polynucleotide kinase CLP1; however, CLP1's role in tRNA splicing remains unclear. Mutations in genes encoding all four TSEN subunits, as well as CLP1, are known to cause neurodegenerative disorders, yet the mechanisms underlying the pathogenesis of these disorders are unknown. Here, we developed a recombinant system that produces active TSEN complex. Co-expression of all four TSEN subunits is required for efficient formation and function of the complex. We show that human CLP1 associates with the active TSEN complex, but is not required for tRNA intron cleavage in vitro. Moreover, RNAi knockdown of the Drosophila CLP1 orthologue, cbc, promotes biogenesis of mature tRNAs and circularized tRNA introns (tricRNAs) in vivo. Collectively, these and other findings suggest that CLP1/cbc plays a regulatory role in tRNA splicing by serving as a negative modulator of the direct tRNA ligation pathway in animal cells.

TRNA: Splicing and Intron Turnover

In most eukaryotic genomes a subset of tRNA genes contains introns. Introns in tRNAs genes are short sequences located 1 nt. 3′ of the anticodon. Introns are removed from precursor tRNAs (pre‐tRNA) by a conserved heterotetrameric protein enzyme, tRNA splicing endonuclease (SEN), that recognizes pre‐tRNA structure rather than sequence motifs at splice junctions. In vertebrate cells intron removal from pre‐tRNAs occurs in the nucleoplasm; in contrast, pre‐tRNA splicing in budding yeast was surprisingly discovered to occur on the mitochondrial surface (Yoshihisa et al. 2003). We recently reported that the SEN complex also locates to the mitochondrial surface in fission yeast (Wan & Hopper 2018) and, therefore, pre‐tRNA splicing on the mitochondrial surface has been conserved for at least 500 million years. The mitochondrial outer membrane protein, Tom70, is required for efficient SEN subunit location to mitochondria in both budding and fission yeast (Wan & Hopper 2018). The freed introns are rarely detected in cells and until recently it was unknown how are they are turned over. We identified at least 5 separate, species‐specific mechanisms that function in tRNA intron destruction (Wu & Hopper, 2014; Bao, unpublished). Interestingly, under particular stress conditions, specific tRNA introns accumulate, possibly indicating their functions in stress response (Peltier, unpublished). Moreover, we discovered that tRNA introns possess long stretches of perfect complementarity to particular budding yeast mRNAs (Bao, unpublished), also indicative of their possible roles in gene regulation. Our initial experiments support the hypothesis that tRNA introns may function as a novel type of noncoding regulatory RNA (Nostramo, unpublished).Support or Funding InformationNIH GMS 122884

CLP1 acts as the main RNA kinase in mice

CLP1 plays an essential role in the protein complex involved in mRNA 3′-end formation and polyadenylation as well as in the tRNA splicing endonuclease (TSEN) complex involved in the splicing of precursor tRNAs. NOL9 localizes in the nucleolus of cells and plays an essential role in ribosomal RNA maturation. Both CLP1 and NOL9 are RNA kinases that phosphorylate the 5′ end of RNAs. From the evidence that phosphorylation of the 5′ end of a siRNA is essential for its efficient RNA cleavage, it was expected that CLP1 and NOL9 would be corresponding molecules. However, there had been no direct evidence that this is the case. In this study, murine NOL9 showed no apparent RNA kinase activity in cells or even in an RNA kinase assay using recombinant murine NOL9 protein. Although siRNA efficiency was decreased in CLP1 kinase-dead (Clp1K/K) cells, it was not influenced by NOL9 overexpression. These findings indicate that in mouse cells it is CLP1 that mainly acts to phosphorylate the 5′ end of RNAs in the siRNA pathway, with no apparent involvement of NOL9.

TSEN54 missense variant in Standard Schnauzers with leukodystrophy.

We report a hereditary leukodystrophy in Standard Schnauzer puppies. Clinical signs occurred shortly after birth or started at an age of under 4 weeks and included apathy, dysphoric vocalization, hypermetric ataxia, intension tremor, head tilt, circling, proprioceptive deficits, seizures and ventral strabismus consistent with a diffuse intracranial lesion. Magnetic resonance imaging revealed a diffuse white matter disease without mass effect. Macroscopically, the cerebral white matter showed a gelatinous texture in the centrum semiovale. A mild hydrocephalus internus was noted. Histopathologically, a severe multifocal reduction of myelin formation and moderate diffuse edema without inflammation was detected leading to the diagnosis of leukodystrophy. Combined linkage analysis and homozygosity mapping in two related families delineated critical intervals of approximately 29 Mb. The comparison of whole genome sequence data of one affected Standard Schnauzer to 221 control genomes revealed a single private homozygous protein changing variant in the critical intervals, TSEN54:c.371G>A or p.(Gly124Asp). TSEN54 encodes the tRNA splicing endonuclease subunit 54. In humans, several variants in TSEN54 were reported to cause different types of pontocerebellar hypoplasia. The genotypes at the c.371G>A variant were perfectly associated with the leukodystrophy phenotype in 12 affected Standard Schnauzers and almost 1000 control dogs from different breeds. These results suggest that TSEN54:c.371G>A causes the leukodystrophy. The identification of a candidate causative variant enables genetic testing so that the unintentional breeding of affected Standard Schnauzers can be avoided in the future. Our findings extend the known genotype-phenotype correlation for TSEN54 variants.

Low mutational load in pediatric medulloblastoma still translates into neoantigens as targets for specific T-cell immunotherapy

BackgroundMedulloblastoma is the most common malignant brain tumor in childhood and adolescence. Although some patients present with distinct genetic alterations, such as mutated TP53 or MYC amplification, pediatric medulloblastoma is a tumor entity with minimal mutational load and low immunogenicity. MethodsWe identified tumor-specific mutations using next-generation sequencing of medulloblastoma DNA and RNA derived from primary tumor samples from pediatric patients. Tumor-specific mutations were confirmed using deep sequencing and in silico analyses predicted high binding affinity of the neoantigen-derived peptides to the patients’ human leukocyte antigen molecules. Tumor-specific peptides were synthesized and used to induce a de novo T-cell response characterized by interferon gamma and tumor necrosis factor alpha release of CD8+ cytotoxic T cells in vitro. ResultsDespite low mutational tumor burden, at least two immunogenic tumor-specific peptides were identified in each patient. T cells showed a balanced CD4/CD8 ratio and mostly effector memory phenotype. Induction of a CD8-specific T-cell response was achieved for the neoepitopes derived from Histidine Ammonia-Lyase (HAL), Neuraminidase 2 (NEU2), Proprotein Convertase Subtilisin (PCSK9), Programmed Cell Death 10 (PDCD10), Supervillin (SVIL) and tRNA Splicing Endonuclease Subunit 54 (TSEN54) variants. ConclusionDetection of patient-specific, tumor-derived neoantigens confirms that even in tumors with low mutational load a molecular design of targets for specific T-cell immunotherapy is possible. The identified neoantigens may guide future approaches of adoptive T-cell transfer, transgenic T-cell receptor transfer or tumor vaccination.

From powerhouse to processing plant: conserved roles of mitochondrial outer membrane proteins in tRNA splicing.

The mitochondrial cytoplasmic surface serves as a processing site for numerous RNAs from budding yeast to metazoans. We report that budding yeast mitochondrial outer membrane (MOM) proteins that are subunits of the translocase of the outer mitochondrial membrane (Tom70 and Tom 22) and sorting and assembly machinery (Sam37) are required for efficient pretransfer RNA (pre-tRNA) splicing. Defective pre-tRNA splicing in MOM mutants is due not to loss of respiratory metabolism but instead inefficient targeting/tethering of tRNA splicing endonuclease (SEN) subunits to mitochondria. Schizosaccharomyces pombe SEN subunits also localize to mitochondria, and Tom70 is required for this localization and pre-tRNA splicing. Thus, the role of MOM protein in targeting/tethering SEN subunits to mitochondria has been conserved for >500 million years.

Three-dimensional microCT imaging of murine embryonic development from immediate post-implantation to organogenesis: application for phenotyping analysis of early embryonic lethality in mutant animals

In this work, we applied three-dimensional microCT imaging to study murine embryogenesis in the range from immediate post-implantation period (embryonic day 5.5) to mid-gestation (embryonic day 12.5) with the resolution up to 1.4 µm/voxel. Also, we introduce an imaging procedure for non-invasive volumetric estimation of an entire litter of embryos within the maternal uterine structures. This method allows for an accurate, detailed and systematic morphometric analysis of both embryonic and extra-embryonic components during embryogenesis. Three-dimensional imaging of unperturbed embryos was performed to visualize the egg cylinder, primitive streak, gastrulation and early organogenesis stages of murine development in the C57Bl6/N mouse reference strain. Further, we applied our microCT imaging protocol to determine the earliest point when embryonic development is arrested in a mouse line with knockout for tRNA splicing endonuclease subunit Tsen54 gene. Our analysis determined that the embryonic development in Tsen54 null embryos does not proceed beyond implantation. We demonstrated that application of microCT imaging to entire litter of non-perturbed embryos greatly facilitate studies to unravel gene function during early embryogenesis and to determine the precise point at which embryonic development is arrested in mutant animals. The described method is inexpensive, does not require lengthy embryos dissection and can be applicable for detailed analysis of mutant mice at laboratory scale as well as for high-throughput projects.

Homozygous Truncating Variants in TBC1D23 Cause Pontocerebellar Hypoplasia and Alter Cortical Development

Pontocerebellar hypoplasia (PCH) is a heterogeneous group of rare recessive disorders with prenatal onset, characterized by hypoplasia of pons and cerebellum. Mutations in a small number of genes have been reported to cause PCH, and the vast majority of PCH cases are explained by mutations in TSEN54, which encodes a subunit of the tRNA splicing endonuclease complex. Here we report three families with homozygous truncating mutations in TBC1D23 who display moderate to severe intellectual disability and microcephaly. MRI data from available affected subjects revealed PCH, small normally proportioned cerebellum, and corpus callosum anomalies. Furthermore, through in utero electroporation, we show that downregulation of TBC1D23 affects cortical neuron positioning. TBC1D23 is a member of the Tre2-Bub2-Cdc16 (TBC) domain-containing RAB-specific GTPase-activating proteins (TBC/RABGAPs). Members of this protein family negatively regulate RAB proteins and modulate the signaling between RABs and other small GTPases, some of which have a crucial role in the trafficking of intracellular vesicles and are involved in neurological disorders. Here, we demonstrate that dense core vesicles and lysosomal trafficking dynamics are affected in fibroblasts harboring TBC1D23 mutation. We propose that mutations in TBC1D23 are responsible for a form of PCH with small, normally proportioned cerebellum and should be screened in individuals with syndromic pontocereballar hypoplasia.

Autosomal-Recessive Mutations in the tRNA Splicing Endonuclease Subunit TSEN15 Cause Pontocerebellar Hypoplasia and Progressive Microcephaly

The tRNA splicing endonuclease is a highly evolutionarily conserved protein complex, involved in the cleavage of intron-containing tRNAs. In human it consists of the catalytic subunits TSEN2 and TSEN34, as well as the non-catalytic TSEN54 and TSEN15. Recessive mutations in the corresponding genes of the first three are known to cause pontocerebellar hypoplasia (PCH) types 2A-C, 4, and 5. Here, we report three homozygous TSEN15 variants that cause a milder version of PCH2. The affected individuals showed progressive microcephaly, delayed developmental milestones, intellectual disability, and, in two out of four cases, epilepsy. None, however, displayed the central visual failure seen in PCH case subjects where other subunits of the TSEN are mutated, and only one was affected by the extensive motor defects that are typical in other forms of PCH2. The three amino acid substitutions impacted the protein level of TSEN15 and the stoichiometry of the interacting subunits in different ways, but all resulted in an almost complete loss of invitro tRNA cleavage activity. Taken together, our results demonstrate that mutations in any known subunit of the TSEN complex can cause PCH and progressive microcephaly, emphasizing the importance of its function during brain development.

The essential function of the Trypanosoma brucei Trl1 homolog in procyclic cells is maturation of the intron-containing tRNATyr.

Trypanosoma brucei, the etiologic agent of sleeping sickness, encodes a single intron-containing tRNA, tRNATyr, and splicing is essential for its viability. In Archaea and Eukarya, tRNA splicing requires a series of enzymatic steps that begin with intron cleavage by a tRNA-splicing endonuclease and culminates with joining the resulting tRNA exons by a splicing tRNA ligase. Here we explored the function of TbTrl1, the T. brucei homolog of the yeast Trl1 tRNA ligase. We used a combination of RNA interference and molecular biology approaches to show that down-regulation of TbTrl1 expression leads to accumulation of intron-containing tRNATyr and a concomitant growth arrest at the G1 phase. These defects were efficiently rescued by expression of an “intronless” version of tRNATyr in the same RNAi cell line. Taken together, these experiments highlight the crucial importance of the TbTrl1 for tRNATyr maturation and viability, while revealing tRNA splicing as its only essential function.

Identification of Alternative Variants and Insertion of the Novel Polymorphic AluYl17 in TSEN54 Gene during Primate Evolution.

TSEN54 encodes a subunit of the tRNA-splicing endonuclease complex, which catalyzes the identification and cleavage of introns from precursor tRNAs. Previously, we identified an AluSx-derived alternative transcript in TSEN54 of cynomolgus monkey. Reverse transcription-polymerase chain reaction (RT-PCR) amplification and TSEN54 sequence analysis of primate and human samples identified five novel alternative transcripts, including the AluSx exonized transcript. Additionally, we performed comparative expression analysis via RT-qPCR in various cynomolgus, rhesus monkey, and human tissues. RT-qPCR amplification revealed differential expression patterns. Furthermore, genomic PCR amplification and sequencing of primate and human DNA samples revealed that AluSx elements were integrated in human and all of the primate samples tested. Intriguingly, in langur genomic DNA, an additional AluY element was inserted into AluSx of intron eight of TSEN54. The new AluY element showed polymorphic insertion. Using standardized nomenclature for Alu repeats, the polymorphic AluY of the langur TSEN54 was designated as being of the AluYl17 subfamily. Our results suggest that integration of the AluSx element in TSEN54 contributed to diversity in transcripts and induced lineage- or species-specific evolutionary events such as alternative splicing and polymorphic insertion during primate evolution.

Circularization restores signal recognition particle RNA functionality in Thermoproteus.

Signal recognition particles (SRPs) are universal ribonucleoprotein complexes found in all three domains of life that direct the cellular traffic and secretion of proteins. These complexes consist of SRP proteins and a single, highly structured SRP RNA. Canonical SRP RNA genes have not been identified for some Thermoproteus species even though they contain SRP19 and SRP54 proteins. Here, we show that genome rearrangement events in Thermoproteus tenax created a permuted SRP RNA gene. The 5'- and 3'-termini of this SRP RNA are located close to a functionally important loop present in all known SRP RNAs. RNA-Seq analyses revealed that these termini are ligated together to generate circular SRP RNA molecules that can bind to SRP19 and SRP54. The circularization site is processed by the tRNA splicing endonuclease. This moonlighting activity of the tRNA splicing machinery permits the permutation of the SRP RNA and creates highly stable and functional circular RNA molecules.

The tRNA Splicing Endonuclease Complex Cleaves the Mitochondria-localized CBP1 mRNA

The tRNA splicing endonuclease (Sen) complex is located on the mitochondrial outer membrane and splices precursor tRNAs in Saccharomyces cerevisiae. Here, we demonstrate that the Sen complex cleaves the mitochondria-localized mRNA encoding Cbp1 (cytochrome b mRNA processing 1). Endonucleolytic cleavage of this mRNA required two cis-elements: the mitochondrial targeting signal and the stem-loop 652-726-nt region. Mitochondrial localization of the Sen complex was required for cleavage of the CBP1 mRNA, and the Sen complex cleaved this mRNA directly in vitro. We propose that the Sen complex cleaves the CBP1 mRNA, which is co-translationally localized to mitochondria via its mitochondrial targeting signal.

Global analysis of RNA cleavage by 5'-hydroxyl RNA sequencing.

RNA cleavage by some endoribonucleases and self-cleaving ribozymes produces RNA fragments with 5′-hydroxyl (5′-OH) and 2′,3′-cyclic phosphate termini. To identify 5′-OH RNA fragments produced by these cleavage events, we exploited the unique ligation mechanism of Escherichia coli RtcB RNA ligase to attach an oligonucleotide linker to RNAs with 5′-OH termini, followed by steps for library construction and analysis by massively parallel DNA sequencing. We applied the method to RNA from budding yeast and captured known 5′-OH fragments produced by tRNA Splicing Endonuclease (SEN) during processing of intron-containing pre-tRNAs and by Ire1 cleavage of HAC1 mRNA following induction of the unfolded protein response (UPR). We identified numerous novel 5′-OH fragments derived from mRNAs: some 5′-OH mRNA fragments were derived from single, localized cleavages, while others were likely produced by multiple, distributed cleavages. Many 5′-OH fragments derived from mRNAs were produced upstream of codons for highly electrostatic peptides, suggesting that the fragments may be generated by co-translational mRNA decay. Several 5′-OH RNA fragments accumulated during the induction of the UPR, some of which share a common sequence motif that may direct cleavage of these mRNAs. This method enables specific capture of 5′-OH termini and complements existing methods for identifying RNAs with 2′,3′-cyclic phosphate termini.

CLP1 as a novel player in linking tRNA splicing to neurodegenerative disorders.

Defects in RNA metabolic pathways are well-established causes for neurodegenerative disorders. Several mutations in genes involved in pre-messenger RNA (pre-mRNA) and tRNA metabolism, RNA stability and protein translation have been linked to motor neuron diseases. Our study on a mouse carrying a catalytically inactive version of the RNA kinase CLP1, a component of the tRNA splicing endonuclease complex, revealed a neurological disorder characterized by progressive loss of lower spinal motor neurons. Surprisingly, mutant mice accumulate a novel class of tRNA-derived fragments. In addition, patients with homozygous missense mutations in CLP1 (R140H) were recently identified who suffer from severe motor-sensory defects, cortical dysgenesis and microcephaly, and exhibit alterations in transfer RNA (tRNA) splicing. Here, we review functions of CLP1 in different RNA pathways and provide hypotheses on the role of the tRNA splicing machinery in the generation of tRNA fragments and the molecular links to neurodegenerative disorders. We further immerse the biology of tRNA splicing into topics of (t)RNA metabolism and oxidative stress, putting forward the idea that defects in tRNA processing leading to tRNA fragment accumulation might trigger the development of neurodegenerative diseases.

Human CLP1 Mutations Alter tRNA Biogenesis, Affecting Both Peripheral and Central Nervous System Function

CLP1 is a RNA kinase involved in tRNA splicing. Recently, CLP1 kinase-dead mice were shown to display a neuromuscular disorder with loss of motor neurons and muscle paralysis. Human genome analyses now identified a CLP1 homozygous missense mutation (p.R140H) in five unrelated families, leading to a loss of CLP1 interaction with the tRNA splicing endonuclease (TSEN) complex, largely reduced pre-tRNA cleavage activity, and accumulation of linear tRNA introns. The affected individuals develop severe motor-sensory defects, cortical dysgenesis, and microcephaly. Mice carrying kinase-dead CLP1 also displayed microcephaly and reduced cortical brain volume due to the enhanced cell death of neuronal progenitors that is associated with reduced numbers of cortical neurons. Our data elucidate a neurological syndrome defined by CLP1 mutations that impair tRNA splicing. Reduction of a founder mutation to homozygosity illustrates the importance of rare variations in disease and supports the clan genomics hypothesis.