Louis Levinger

Matrices of Paired Substitutions Show the Effects of tRNA D/T Loop Sequence on Drosophila RNase P and 3*-tRNase Processing*

Drosophila RNase P and 3′-tRNase endonucleolytically process the 5′ and 3′ ends of tRNA precursors. We examined the processing kinetics of normal substrates and the inhibitory effect of the tRNA product on both processing reactions. The product is not a good RNase P inhibitor, with a K I approximately 7 times greater than the substrate K M of ∼200 nm and is a better inhibitor of 3′-tRNase, with aK I approximately two times theK M of ∼80 nm. We generated matrices of substitutions at positions G18/U55 and G19/C56(two contiguous universally conserved D/T loop base pairs) inDrosophila tRNAHis precursors. More than half the variants display a significant reduction in their ability to be processed by RNase P and 3′-tRNase. Minimal substrates with deleted D and anticodon stems could be processed by RNase P and 3′-tRNase much like full-length substrates, indicating that D/T loop contacts and D arm/enzyme contacts are not required by either enzyme. Selected tRNAs that were poor substrates for one or both enzymes were further analyzed using Michaelis-Menten kinetics and by structure probing. Processing reductions arise principally due to an increase inK M with relatively little change inV max, consistent with the remote location of the sequence and structure changes from the processing site for both enzymes. Local changes in variant tRNA susceptibility to RNase T1 and RNase A did not coincide with processing disabilities.

Naturally Occurring Mutations in Human Mitochondrial Pre-tRNASer(UCN) Can Affect the Transfer Ribonuclease Z Cleavage Site, Processing Kinetics, and Substrate Secondary Structure

tRNAs are transcribed as precursors with a 5′ end leader and a 3′ end trailer. The 5′ end leader is processed by RNase P, and in most organisms in all three kingdoms, transfer ribonuclease (tRNase) Z can endonucleolytically remove the 3′ end trailer. Long (L) and short (S) forms of the tRNase Z gene are present in the human genome. tRNase ZL processes a nuclear-encoded pre-tRNA ∼1600-fold more efficiently than tRNase ZS and is predicted to have a strong mitochondrial transport signal. tRNase ZL could, thus, process both nuclear- and mitochondrially encoded pre-tRNAs. More than 150 pathogenesis-associated mutations have been found in the mitochondrial genome, most of them in the 22 mitochondrially encoded tRNAs. All the mutations investigated in human mitochondrial tRNASer(UCN) affect processing efficiency, and some affect the cleavage site and secondary structure. These changes could affect tRNase Z processing of mutant pre-tRNAs, perhaps contributing to mitochondrial disease.

Effect of Changes in the Flexible Arm on tRNase Z Processing Kinetics

tRNAs are transcribed as precursors and processed in a series of reactions culminating in aminoacylation and translation. Central to tRNA maturation, the 3′ end trailer can be endonucleolytically removed by tRNase Z. A flexible arm (FA) extruded from the body of tRNase Z consists of a structured ααββ hand that binds the elbow of pre-tRNA. Deleting the FA hand causes an almost 100-fold increase in Km with little change in kcat, establishing its contribution to substrate recognition/binding. Remarkably, a 40-residue Ala scan through the FA hand reveals a conserved leucine at the ascending stalk/hand boundary that causes practically the same increase in Km as the hand deletion, thus nearly eliminating its ability to bind substrate. Km also increases with substitutions in the GP (α4–α5) loop and at other conserved residues in the FA hand predicted to contact substrate based on the co-crystal structure. Substitutions that reduce kcat are clustered in the β10–β11 loop.

Pathogenesis-related mutations in the T-loops of human mitochondrial tRNAs affect 3′ end processing and tRNA structure

Numerous mutations in the mitochondrial genome are associated with maternally transmitted diseases and syndromes that affect muscle and other high energy-demand tissues. The mitochondrial genome encodes 13 polypeptides, 2 rRNAs and 22 interspersed tRNAs via long bidirectional polycistronic primary transcripts, requiring precise excision of the tRNAs. Despite making up only ~10% of the mitochondrial genome, tRNA genes harbor most of the pathogenesis-related mutations. tRNase Z endonucleolytically removes the pre-tRNA 3′ trailer. The flexible arm of tRNase Z recognizes and binds the elbow (including the T-loop) of pre-tRNA. Pathogenesis-related T-loop mutations in mitochondrial tRNAs could thus affect tRNA structure, reduce tRNase Z binding and 3′ processing, and consequently slow mitochondrial protein synthesis. Here we inspect the effects of pathogenesis-related mutations in the T-loops of mitochondrial tRNAs on pre-tRNA structure and tRNase Z processing. Increases in KM arising from 59A > G substitutions in mitochondrial tRNAGly and tRNAIle accompany changes in T-loop structure, suggesting impaired substrate binding to enzyme.

Mutations in ELAC2 associated with hypertrophic cardiomyopathy impair mitochondrial tRNA 3'-end processing

Dysfunction of mitochondrial gene expression, caused by mutations in either the mitochondrial or nuclear genomes, is associated with a diverse group of human disorders characterized by impaired mitochondrial respiration. Within this group, an increasing number of mutations have been identified in nuclear genes involved in mitochondrial RNA metabolism. For instance, pathogenic mutations have been identified in the genes encoding enzymes involved in the precursor transcript processing, including ELAC2. The ELAC2 gene codes for the mitochondrial RNase Z, which is responsible for endonucleolytic cleavage of the 3’ ends of mitochondrial pre-tRNAs. Here, we report the identification of sixteen novel ELAC2 variants in individuals presenting with mitochondrial respiratory chain deficiency, hypertrophic cardiomyopathy and lactic acidosis. We provided further evidence for the pathogenicity of the three previously reported variants by studying the RNase Z activity in an in vitro system and applied this recombinant system to investigate all novel missense variants, confirming the pathogenic role of these new ELAC2 mutations. We also modelled the residues affected by missense mutation in solved RNase Z structures, providing insight into enzyme structure and function. Finally, we show that primary fibroblasts from the individuals with novel ELAC2 variants have elevated levels of unprocessed mitochondrial RNA precursors. Our study thus broadly confirms the correlation of ELAC2 variants with severe infantile-onset forms of hypertrophic cardiomyopathy and mitochondrial respiratory chain dysfunction. One rare missense variant associated with the occurrence of prostate cancer (p.Arg781His) impairs the mitochondrial RNase Z activity of ELAC2, possibly indicating a functional link between tumorigenesis and mitochondrial RNA metabolism.

Substitutions in conserved regions preceding and within the linker affect activity and flexibility of tRNase ZL, the long form of tRNase Z

The enzyme tRNase Z, a member of the metallo-β-lactamase family, endonucleolytically removes 3’ trailers from precursor tRNAs, preparing them for CCA addition and aminoacylation. The short form of tRNase Z, tRNase ZS, functions as a homodimer and is found in all prokaryotes and some eukaryotes. The long form, tRNase ZL, related to tRNase ZS through tandem duplication and found only in eukaryotes, possesses ~2,000-fold greater catalytic efficiency than tRNase ZS. tRNase ZL consists of related but diverged amino and carboxy domains connected by a flexible linker (also referred to as a flexible tether) and functions as a monomer. The amino domain retains the flexible arm responsible for substrate recognition and binding while the carboxy domain retains the active site. The linker region was explored by Ala-scanning through two conserved regions of D. melanogaster tRNase Z: NdomTprox, located at the carboxy end of the amino domain proximal to the linker, and Tflex, a flexible site in the linker. Periodic substitutions in a hydrophobic patch (F329 and L332) at the carboxy end of NdomTprox show 2,700 and 670-fold impairment relative to wild type, respectively, accompanied by reduced linker flexibility at N-T inside the Ndom- linker boundary. The Ala substitution for N378 in the Tflex region has 10-fold higher catalytic efficiency than wild type and locally decreased flexibility, while the Ala substitution at R382 reduces catalytic efficiency ~50-fold. These changes in pre-tRNA processing kinetics and protein flexibility are interpreted in light of a recent crystal structure for S. cerevisiae tRNase Z, suggesting transmission of local changes in hydrophobicity into the skeleton of the amino domain.