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RNA. Feb 2008; 14(2): 204–210.
PMCID: PMC2212240

Mammalian 2′,3′ cyclic nucleotide phosphodiesterase (CNP) can function as a tRNA splicing enzyme in vivo

Abstract

Yeast and plant tRNA splicing entails discrete healing and sealing steps catalyzed by a tRNA ligase that converts the 2′,3′ cyclic phosphate and 5′-OH termini of the broken tRNA exons to 3′-OH/2′-PO4 and 5′-PO4 ends, respectively, then joins the ends to yield a 2′-PO4, 3′-5′ phosphodiester splice junction. The junction 2′-PO4 is removed by a tRNA phosphotransferase, Tpt1. Animal cells have two potential tRNA repair pathways: a yeast-like system plus a distinctive mechanism, also present in archaea, in which the 2′,3′ cyclic phosphate and 5′-OH termini are ligated directly. Here we report that a mammalian 2′,3′ cyclic nucleotide phosphodiesterase (CNP) can perform the essential 3′ end-healing steps of tRNA splicing in yeast and thereby complement growth of strains bearing lethal or temperature-sensitive mutations in the tRNA ligase 3′ end-healing domain. Although this is the first evidence of an RNA processing function in vivo for the mammalian CNP protein, it seems unlikely that the yeast-like pathway is responsible for animal tRNA splicing, insofar as neither CNP nor Tpt1 is essential in mice.

Keywords: 3′ end-healing, RNA 2′,3′, cyclic phosphodiester, tRNA ligase

INTRODUCTION

Intron-containing tRNAs are encoded in all archaeal and eukaryal genomes. The intron is usually located in the anticodon loop of the pre-tRNA, and must be removed precisely for the tRNA to function in protein synthesis. Two incisions of the pre-tRNA at the exon–intron borders yield 2′,3′ cyclic phosphate and 5′-OH termini at both break sites (Peebles et al. 1983). The cleavages are performed by a tRNA splicing endonuclease that recognizes the fold of the pre-tRNA. The structure and mechanism of the splicing endonuclease are conserved among archaea and lower and higher eukaryal species (Trotta et al. 1997; Li et al. 1998; Paushkin et al. 2004; Xue et al. 2006).

By contrast, the tRNA repair phase of the splicing pathway differs in archaea and eukarya. The ends of cleaved archaeal tRNAs are rejoined in a single-step reaction entailing attack of the 5′-OH on the 2′,3′ cyclic phosphodiester to form a 3′,5′ phosphodiester at the splice junction (Zofallova et al. 2000; Salgia et al. 2003). The archaeal protein that catalyzes the tRNA sealing reaction is not known. Yeast tRNA splicing occurs by an entirely different biochemical route. A single multifunctional tRNA ligase enzyme, Trl1, first heals and then seals the ends of the broken tRNA half-molecules. Trl1 performs three reactions: (1) the 2′,3′ cyclic phosphate of the proximal tRNA half-molecule is hydrolyzed to a 3′-OH, 2′-PO4 by a cyclic phosphodiesterase (CPD); (2) the 5′-OH of the distal half-molecule is phosphorylated by a GTP-dependent polynucleotide kinase; and (3) the 3′-OH, 2′-PO4, and 5′-PO4 ends are sealed by an ATP-dependent RNA ligase to form a spliced tRNA containing an unconventional 2′-PO4, 3′-5′ phosphodiester at the splice junction (Greer et al. 1983). The 2′-PO4 at the splice junction is ultimately removed by the 2′-phosphotransferase Tpt1 (Spinelli et al. 1999).

Yeast Trl1 consists of an N-terminal ligase domain (amino acids 1–388) and a C-terminal end-healing domain (amino acids 389–827). The ligase domain is an ATP-dependent strand-sealing enzyme that belongs to the ligase family exemplified by bacteriophage T4 RNA ligase 1. The end-healing domain is composed of distinct kinase and CPD modules (Fig. 1). The kinase module resembles T4 polynucleotide kinase (Wang et al. 2002, 2006). The T4 and yeast kinases belong to the P-loop phosphotransferase superfamily and contain the signature NTP-binding “GKT” motif (Fig. 1). The CPD component belongs to the 2H phosphotransferase superfamily defined by two “HxT” motifs (Mazumder et al. 2002; Nasr and Filipowicz 2000). Trl1-like proteins composed of the same three modules in the same linear order are present in other fungi and in kinetoplastid protozoa (Wang and Shuman 2005). An Arabidopsis tRNA ligase ortholog (AtRNL) is a 1104-amino acid polypeptide with a similar tripartite domain structure (Fig. 1; Englert and Beier 2005; Wang et al. 2006). AtRNL is capable of performing all three essential splicing reactions in vivo in yeast cells that lack Trl1 (Wang et al. 2006).

FIGURE 1.
Rat CNP complements ts mutations in yeast and plant tRNA ligase. (Top panel) The tRNA ligases of yeast (Trl1) and plant (AtRNL) are composed of three discrete catalytic domains: an N-terminal ligase module (magenta), a central 5′-OH polynucleotide ...

A longstanding mystery of tRNA biology concerns the mechanism of tRNA exon ligation in animal cells and the identity of the enzymes involved. Initial studies demonstrated a direct tRNA end-joining reaction in human cell extracts in which the 2′,3′ cyclic phosphate is retained as a 3′-5′ phosphodiester at the splice junction of the mature tRNA (Filipowicz and Shatkin 1983; Laski et al. 1983). Later work suggested that human cells also have a second yeast-like pathway of tRNA splicing (Zillman et al. 1991) in which the junction 3′-5′ phosphate in mature tRNA derives from ATP and the starting 2′,3′ cyclic phosphate is converted to a 2′-PO4 that is ultimately removed. The existence of a yeast-like tRNA repair system in metazoa is consistent with the presence of Tpt1-type phosphotransferase and RNA-specific 5′-OH polynucleotide kinase activities in human cells (Spinelli et al. 1998; Shuman and Hurwitz 1979). However, no Rnl1-type RNA sealing enzyme has been identified in a metazoan organism, and there is no apparent homolog of Rnl1 or the Trl1 ligase domain in any metazoan proteome.

Solving the “division of labor” problem for the two distinct human tRNA exon-joining pathways hinges on identifying the relevant enzymes. A recent advance was the attribution of RNA 5′-OH kinase activity to the human Clp1 protein (Weitzer and Martinez 2007). The crystal structure of yeast Clp1 revealed a central P-loop phosphotransferase module to which ATP was bound (Noble et al. 2007). Available evidence argues against yeast Clp1 being the catalyst of the 5′ end-healing step of yeast tRNA splicing, insofar as Trl1 itself has the requisite kinase function and kinase-inactivating mutations in Trl1 are lethal in vivo (Sawaya et al. 2003). However, the situation in human cells might be different. Human Clp1 can phosphorylate the 5′-OH end of cleaved pre-tRNA and, based on siRNA knockdown experiments, Clp1 is suggested to be an agent of mammalian tRNA splicing (Weitzer and Martinez 2007). This model, although attractive, remains to be tested by generating a mammalian cell line or organism in which Clp1 is either deleted or mutated in its active site.

The observation that RNA repair systems from heterologous sources can complement the catalytic function of one or all of the domains of yeast Trl1 (Schwer et al. 2004; Wang et al. 2006) provides a means to find or validate new proteins that can catalyze tRNA splicing reactions in vivo. Here we apply this approach to show that mammalian 2′,3′ cyclic nucleotide phosphodiesterase (CNP), a protein that has been studied extensively as a major constituent of myelin in the central nervous system (Braun et al. 2004), can perform the 3′ end-healing step of tRNA splicing in yeast. CNP is a member of the 2H phosphotransferase superfamily and is adept at hydrolyzing the O3′–P bond of 2′,3′ cyclic nucleotides to yield a nucleoside 2′-phosphate product.

RESULTS AND DISCUSSION

Mammalian CNP—An enzyme in search of a function

Mammalian CNP is a 400-amino acid polypeptide composed of a C-terminal catalytic fragment (CF) and a 150-amino acid N-terminal extension (Fig. 1; Lee et al. 2001). CNP constitutes 4% of total myelin protein in the central nervous system. CNP is expressed at lower levels outside the central nervous system, and it has been found to associate with mitochondria and cytoskeletal proteins (Bifulco et al. 2002; Lee et al. 2005, 2006). Homozygous CNP-knockout mice were viable, and showed no overt abnormalities up to 4 mo of age (Lappe-Siefke et al. 2003). Myelin in young knockout mice was of normal ultrastructure and protein composition, except for the absence of CNP. Older knockout mice developed progressive motor deficits and died prematurely because of diffuse brain axonal swelling and neurodegeneration leading to hydrocephalus (Lappe-Siefke et al. 2003). It is not known whether the late onset of neural disease is caused by an absence of the CNP protein (e.g., as a structural component of myelin or the cytoskeleton) or an absence of CNPase enzymatic activity. Indeed, there is an outstanding conundrum that 2′,3′ cyclic nucleotides are not present in appreciable amounts in mammalian cells, implying either that the catalytic activity of CNP is a red herring or that 2′,3′ cyclic mononucleotides are not the physiological substrate (Braun et al. 2004).

X-ray and NMR structures of the catalytic fragment (CF) highlighted a bilobar fold composed of two repeated α + β modules related by pseudotwofold symmetry (Kozlov et al. 2003; Sakamoto et al. 2005). The two HxT motifs are located at the active site. Based on these and other 2H protein structures (Hofmann et al. 2000, 2002; Kato et al. 2003; Kozlov et al. 2007), it is proposed that the histidines and threonines coordinate the scissile phosphodiester and promote its hydrolysis by transition-state stabilization and general acid-base catalysis. It has been appreciated for some time that mammalian CNP is biochemically and structurally akin to the CPD domain of yeast tRNA ligase. In fact, CNP can convert a 2′,3′ cyclic phosphodiester RNA terminus to a 3′-OH, 2′-PO4 product in vitro (Filipowicz et al. 1983). The analogy is further strengthened by the fact that the N-terminal extension of CNP includes a prototypal P-loop NTP-binding motif (Fig. 1), much like the kinase-CPD domains of yeast Trl1 and the plant ortholog AtRNL.

Rat CNP performs the 3′ end-healing step of tRNA splicing in vivo

To query whether mammalian CNP can act as a tRNA splicing enzyme, we expressed the full-length CNP protein in Saccharomyces cerevisiae under the control of a constitutive promoter on a 2μ plasmid and asked whether CNP could complement the temperature-sensitive growth defect caused by the H673A mutation in the proximal HxT motif of the CPD module of yeast Trl1 (Sawaya et al. 2003). The H673A yeast strain failed to form colonies at ≥35°C; growth at nonpermissive temperatures was revived by a plasmid borne copy of wild-type TRL1, but not by an empty plasmid vector (Fig. 1). The instructive finding was that rat CNP restored growth at the restrictive temperature, signifying that the mammalian enzyme could act in lieu of Trl1 CPD in tRNA splicing (Fig. 1). Additional experiments showed that rat CNP could not complement a temperature-sensitive mutation in the kinase domain of yeast Trl1 (data not shown; also see below), implying that CNP is unable to perform the 5′ end healing step of the tRNA splicing pathway, notwithstanding the presence of a P-loop motif in its N-terminal segment. Deletion of the N-terminal domain of rat CNP did not affect its ability to complement the CPD function of yeast Trl1, i.e., expression of the C-terminal catalytic fragment (CF) of rat CNP sufficed for growth of the H673A strain at elevated temperatures (Fig. 1).

The plant tRNA ligase AtRNL can provide all of the essential tRNA healing and sealing functions in a yeast trl1Δ null background. The H999A mutation in the first HxT motif of the AtRNL CPD module elicits a temperature-sensitive growth defect in yeast, akin to the equivalent change in the CPD active site of Trl1 (Fig. 1; Wang et al. 2006). This growth defect was reversed by introduction of a 2μ plasmid expressing either full-length CNP or the CNP catalytic fragment, but not by the empty vector control (Fig. 1). A key finding was that a catalytically defective CNP mutant (H230A–T232A–H309A–T311A) containing alanine substitutions for the histidines and threonines of both HxT motifs was unable to complement growth of the AtRNL–H999A strain at restrictive temperature (Fig. 1).

To consolidate the inference from these initial experiments that the 2′,3′ cyclic phosphodiesterase activity of rat CNP can replace the CPD activity of yeast or plant tRNA ligases, we tested the ability of CNP to substitute for lethal mutations in the CPD active site of AtRNL (Wang et al. 2006). Yeast trl1Δ strains carrying TRL1 on a CEN URA3 plasmid were transformed with CEN TRP1 plasmids bearing lethal T1001A and H1060A alleles of AtRNL. We introduced into these strains HIS3 plasmids expressing either wild-type AtRNL, full-length rat CNP, the active CNP catalytic fragment, or the catalytically defective CNP mutant. AtRNL–T1001A and AtRNL–H1060A were unable to sustain growth on medium containing the drug 5-FOA, which selects against the URA3 TRL1 plasmid (Fig. 2, vector). Growth on 5-FOA was restored by a wild-type AtRNL gene, as expected. The salient findings were that CNP and the catalytic fragment complemented the lethal CPD mutations, whereas the catalytically defective mutant did not (Fig. 2).

FIGURE 2.
Rat CNP complements lethal mutations in plant tRNA ligase. The plant tRNA ligase and rat CNP polypeptides are shown in the top panel with the CPD-inactivating alanine mutations at T1001 and H1060 of AtRNL highlighted in medium gray. Yeast trl1Δ ...

CNP complementation of new conditional mutations in the Trl1 healing domain

To obtain additional genetic tools to study the end-healing steps of tRNA splicing, we mutagenized the kinase-CPD segment of the full-length yeast TRL1 gene and screened for alleles that conferred a temperature-sensitive growth phenotype. This resulted in the isolation of five new trl1–ts mutants that grew at 25°C and 30°C, but failed to grow at 35°C or 37°C (Fig. 3). Each of the conditionally defective Trl1 polypeptides contained between two and four amino acid substitutions within the kinase–CPD domain. To delineate which, if any, of the new ts mutants were defective uniquely for CPD activity, we tested them for complementation by mammalian CNP. Growth of three of the five trl1–ts strains at restrictive temperature was fully restored by introduction of a CNP expression plasmid (Fig. 3). The L734P–N773D double mutant that was complemented by CNP contained lesions that mapped exclusively within the CPD module, consistent with a singular defect in CPD activity at restrictive temperature. The N514D–V748E mutant rescued by CNP has one lesion in the CPD domain (V748E) and one in the kinase module (N514D); we surmise that the valine-to-glutamate change is the likely culprit for the growth defect. The K449E–P589L–L780P mutant has one lesion in the CPD domain (L780P) and two lesions in the kinase module (K449E P589L); its complementation by CNP implicates the L780P change in the growth phenotype. The diagnostic value of the complementation test was underscored by the finding that the S44P–I487V mutant, which has two lesions in the kinase domain, was not rescued by CNP (Fig. 3). CNP also failed to complement a quadruple mutant containing two changes in the kinase module (S556L D591G) plus two changes in the CPD module (L730P G786C). We performed additional experiments testing complementation of the new trl1–ts strains by a plasmid encoding the Trl1–H673A protein, which has an isolated defect in the CPD active site and cannot support growth at 37°C. TRL1–H673A complemented only one of the five trl1–ts strains (data not shown), that being S444P–I487V, which has mutations exclusively in the kinase module and could not be rescued by CNP.

FIGURE 3.
Test of CNP complementation of new ts mutations in the Trl1 end-healing domain. Five new trl1–ts alleles bearing the indicated missense mutations in the kinase-CPD domain were isolated as described under Materials and Methods. The amino acid changes ...

Implications for 3′ end healing in tRNA splicing and RNA repair

Here we provided genetic evidence that mammalian CNP is an RNA repair enzyme in vivo and is capable of performing the 3′ end-healing step of yeast tRNA splicing. Mammalian CNP is structurally homologous to the CPD domain of yeast and plant tRNA ligases and the outcomes of the cyclic phosphodiesterase reaction (conversion to a 2′-PO4) are the same. The presence of a 2′-PO4 at the healed 3′-OH RNA end is required in vitro and in vivo for strand sealing by the ligase component of yeast Trl1 (Schwer et al. 2004; Keppetipola et al. 2007).

The RNA end-healing function of CNP could be relevant to either of two well-documented cases of programmed RNA breakage and repair in mammals: (1) tRNA splicing and (2) mRNA splicing during the endoplasmic reticulum unfolded protein response (UPR) (Ron and Walter 2007). tRNA ligase is the enzyme responsible for nonspliceosomal splicing of HAC1 mRNA in the yeast unfolded protein response pathway (Sidrauski et al. 1996; Gonzalez et al. 1999). ER stress-induced cleavage of the HAC1 mRNA by endonuclease Ire1 excises an intron and leaves 2′,3′ cyclic phosphate and 5′-OH termini at the cleavage sites. After healing and sealing by Trl1, the HAC1 translation reading frame is altered so as to replace the C-terminal peptide of the uninduced form of Hac1 (encoded in the intron) with a different segment encoded in the second exon. The induced form of Hac1 is a transcription factor that activates certain nuclear genes in response to ER stress, especially genes that specify ER chaperones. The mammalian UPR also involves induced cleavage and unconventional splicing of an mRNA encoding a transcription factor, XBP1 (Calfon et al. 2002). The identity of the mammalian mRNA repair enzymes in the UPR is not known.

The likely existence of two different RNA repair pathways in mammalian cells (yeast-type and archaeal-type) raises the interesting issue of whether they are functionally redundant or dedicated to a particular physiological setting. For example, one pathway might perform tRNA splicing while the other specializes in mRNA splicing in the UPR. The key feature of the yeast-type pathway is the need for end healing prior to sealing. The benign effects of completely deleting CNP in mice seem to exclude an essential role for the yeast-type splicing pathway, if one assumes that CNP, which is the source of all measurable 2′,3′-cyclic phosphodiesterase activity in mouse brain (Lappe-Siefke et al. 2003), is the only available source of 3′ end-healing for the yeast-like splicing pathway. This scenario would relegate the yeast-type pathway in mammals to either no role, or a functionally redundant role, in both tRNA splicing and the UPR. It is safe to assume that tRNA splicing is required for cell viability and would be lethal if disrupted in the mouse. The UPR is required for proper development of the pancreas and the immune system, neither of which appeared to be affected in CNP-knockout mice. If the yeast-type pathway is redundant, then other mammalian homologs of yeast-type tRNA repair proteins might also not be essential. This is the case for mammalian Tpt1, the ortholog of the yeast phosphotransferase that removes the 2′-PO4 from the tRNA splice junction (Harding et al. 2007). The major challenge now is to identify the protein catalysts of the direct tRNA ligation pathway in mammalian cells (and archaea). Yeast complementation might afford a means to do so by screening mammalian cDNA libraries for functional complementation of a trl1Δ strain.

MATERIALS AND METHODS

Yeast vectors for expression of rat CNP

A 1.2-kb DNA fragment spanning the rat CNP ORF was amplified by PCR to introduce an EcoRI site immediately upstream of the ATG codon and an XhoI restriction site downstream of the TGA stop codon. The fragment was inserted into pYX232 (2μ TRP1), a shuttle vector in which expression of CNP is under the transcriptional control of the constitutive TPI1 promoter to generate the p232–CNP plasmid. The expression plasmids for CF (catalytic fragment, amino acids 151–400) and the catalytically defective CNP mutant (H230A–T232A–H309A–T311A) were generated similarly. All clones were sequenced to exclude the presence of unwanted mutations. The DNA fragments spanning the TPI1–CNP expression cassettes were inserted into pRS423 (HIS3 2μ).

New temperature-sensitive mutations in the Trl1 end-healing domain

We generated a library of mutated TRL1 alleles (TRL1*) by PCR amplification of the gene segment encoding the kinase–CPD domain (amino acids 375–827) under conditions that promote nucleotide misincorporation. In brief, four PCR amplification reactions were performed in parallel in mixtures containing 2 mM MgSO4, 0.1 mM MnCl2, 50 μM of one dNTP, and 200 μM of the three other dNTPs. The PCR-amplified DNAs were pooled, digested with XhoI and SpeI, and then inserted into a CEN TRP1 TRL1 plasmid in lieu of the corresponding wild-type restriction fragment. A p(CEN TRP1 TRL1*) library harvested from a pool of ~100,000 bacterial transformants was introduced into a S. cerevisiae trl1Δ p(CEN URA3 TRL1) strain. Trp+ yeast colonies selected at 25°C were subjected to two rounds of replica-plating at 25°C on medium containing 0.75 mg/mL 5-fluoroorotic acid (FOA) to select for loss of the URA3 TRL1 plasmid. About 1200 FOA-resistant trl1Δ p(CEN TRP1 TRL1*) strains were arrayed on YPD agar master plates. Replica-plates were tested in parallel for growth at 25°C and 35°C. Plasmids were recovered from candidate trl1–ts strains that failed to grow at 35°C and amplified by transformation in Escherichia coli. Individual p(CEN TRP1 TRL1*) plasmids were reintroduced into trl1Δ p(CEN URA3 TRL1) cells, which were subjected to plasmid shuffle and then screened for temperature-sensitive growth. Five confirmed trl1–ts alleles were then sequenced completely to identify the coding changes implicated in conferring the conditional phenotype. In each case, missense mutations were identified within the targeted kinase–CPD segment (Fig. 3).

ACKNOWLEDGMENTS

This work was funded by NIH Grant GM42498. S.S. is an American Cancer Society Research Professor. P.B. is a grantee of the Multiple Sclerosis Society of Canada and the Canadian Institutes for Health Research. B.S. is supported by the William Randolph Hearst Foundation. A.R. is supported by NIH predoctoral training grant T32-GM008539.

Footnotes

Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.858108.

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