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

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Abstract 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. WIREs RNA 2015, 6:47–63. doi: 10.1002/wrna.1255 This article is categorized under: RNA Processing > 3' End Processing RNA Processing > tRNA Processing RNA in Disease and Development > RNA in Disease
The subunit composition of the mRNA 3′ end formation machineries in yeast and mammals. Conserved cis elements (black boxes) are present within the untranslated region of almost every eukaryotic pre‐mRNA that gets polyadenylated, and are crucial for recognition by various cleavage factors and hence for efficient 3′ end processing. (a) Pre‐mRNA 3′ end processing complex in yeast. Sub‐complexes comprise CF IA, cleavage factor IB (CF IB) and cleavage and polyadenylation factor (CPF). CPF is composed of cleavage factor II (CF II) and polyadenylation factor I (PF I). Clp1p, together with Pcf11p, Rna14p, and Rna15p, constitutes CF IA. Dotted arrows indicate known interactions between Clp1p and other subunits within the mRNA 3′ end cleavage complex. The mRNA cleavage occurs at the 3′ side of the ‘Py(A)n’ motif and is executed by the endonucleolytic activity of Brr5p/Ysh1p. Pap1p (Poly(A) polymerase) is the enzymatic activity that adds a poly(A) tail to cleaved pre‐mRNA. Detailed functions of other depicted 3′ end processing factors are reviewed in Refs . (b) Pre‐mRNA 3′ end processing complex in mammals, composed of cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), cleavage factor I (CF Im) and cleavage factor II (CF IIm). CLP1 associates with PCF11 to form CF IIm. CLP1 was further proposed to bridge CF Im with CPSF, and to interact with the cap‐binding complex (CBC) via ARS2 (dotted arrows). The mRNA cleavage occurs at the 3′ side of a consensus CA‐sequence and is mediated by the CPSF‐73 endonuclease. Polyadenylation is executed by the Poly(A) polymerase PAP. A key cis element is the AAUAAA hexamer sequence, present in 80–90% of sequenced mRNAs.
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Scheme summarizing consequences of a mutation in the ATP‐binding site of CLP1 in mouse. A K127A mutation in the Walker A motif at the genomic locus of CLP1 (Clp1k/k mouse) leads to accumulation of tyrosine tRNA (5′pppL‐E) fragments, which are also triggered by exposure of cell lines to oxidative stress agents. We predict that mild oxidative stress conditions are present in Clp1k/k mice responsible for tRNA fragment generation, but it also remains possible that fragments accumulate by other means due to mutation in CLP1. These fragments trigger augmented serine 18 phosphorylation of p53 upon oxidative stress conditions, a possible cause for enhanced motor neuron loss occurring in Clp1k/k mice. This phenotype is rescued in a p53−/− mouse background, which is in line with the assumption that the consequence of CLP1 K127A mutation is mediated via p53‐regulated cell death pathways.
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Potential biogenesis pathways for 5′ leader‐exon tRNA fragments. (a) Schematic outline of 5′ leader‐exon fragments (5′pppL‐E) in the context of a pre‐tRNA. (b) Secondary structure prediction of the two most abundant 5′pppL‐E fragments accumulating in Clp1k/k mice (Mus musculus chr14.trna191‐TyrGTA and M. musculus chr13.trna958‐TyrGTA (mouse July 2007, mm9 genome assembly), as predicted by RNAfold (Vienna RNA Secondary Structure Package). (c) Potential biogenesis pathways to generate the 5′pppL‐E fragments. Depending on whether tRNA exon ligation occurs before or after leader and trailer removal, RNAse P (1) or HSPC117/RTCB (2) activity could be affected in CLP1 kinase‐dead mice or under oxidative stress condition, resulting in the generation and accumulation of 5′ leader‐exon tRNA fragments. tRNA 3′ exon fragments have hitherto remained undetectable by Northern blot analysis and might be subjected to degradation as a result of defective ligation and accumulation of their rather stable 5′pppL‐E counterparts.
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Model for a potential role of CLP1 within the TSEN complex to efficiently generate tRNA exons. Wild type CLP1 is able to bind/hydrolyze ATP, associate with the TSEN complex and efficiently cleave pre‐tRNAs to produce tRNA exon halves (left panel). In contrast, kinase‐dead CLP1 with deficient ATP binding/hydrolysis activity (mutant K127A) binds only weakly to the TSEN complex, correlating with reduced pre‐tRNA cleavage activity (middle panel). A patient‐derived R140H mutation in CLP1 causes an even more severe effect on complex integrity and pre‐tRNA cleavage (right panel). Of note, and in contrast to CLP1 K127A, this mutation does not abolish RNA kinase activity.
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tRNA splicing pathways in yeast and vertebrates. In yeast intron‐containing pre‐tRNAs are cleaved by the tRNA splicing endonuclease Sen (Sen2p, Sen15p, Sen34p, and Sen54p) to produce 5′‐ and 3′‐tRNA exon halves. The multifunctional enzyme Trl1p phosphorylates the 5′ hydroxyl terminus of the 3′ exon by its kinase domain using GTP, followed by hydrolysis of the 2′,3′‐cyclic phosphate and formation of a 5′,3′‐phosphodiester bond. The 2′‐phosphotransferase Tpt1p removes the remaining 2′ phosphate. In vertebrates, with support of CLP1, the tRNA splicing endonuclease (TSEN: TSEN2, TSEN15, TSEN34, and TSEN54) generates exon halves, which are directly ligated by HSPC117/RTCB (‘animal pathway’). The phosphate located in the splice junction originates from the 2′,3′‐cyclic phosphate of the 5′ exon. CLP1 associates with the TSEN complex where it could function in an enigmatic ‘yeast‐like’ tRNA splicing pathway to add an exogenous phosphate from ATP to the 3′ exon, thereby licensing ligation by a still unidentified 5′P—3′OH tRNA ligase. Enzymes potentially exerting activities analogous to the yeast tRNA splicing pathway are discussed in the main text.
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Potential inhibitory mechanism of HSPC117 (RTCB) exon ligation activity by the RNA kinase activity of CLP1. HSPC117 (RTCB) directly joins a 5′ exon ending with a 2′,3′‐cyclic phosphate to the 5′‐hydroxyl group of a 3′ exon. The RNA kinase activity of CLP1 as part of the TSEN complex has been shown to phosphorylate the 5′‐OH of the 3′ exon in vitro. As a consequence, the 5′‐phosphorylated 3′ exon cannot be ligated to the 5′ exon by HSPC117 (RTCB). However, such potential role for CLP1 in regulating tRNA splicing by inhibiting exon ligation has hitherto remained elusive.
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RNA Processing > 3′ End Processing
RNA in Disease and Development > RNA in Disease
RNA Processing > tRNA Processing

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