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 Z(L) processes a nuclear-encoded pre-tRNA approximately 1600-fold more efficiently than tRNase Z(S) and is predicted to have a strong mitochondrial transport signal. tRNase Z(L) 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 tRNA(Ser(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.     

tRNASer(UCN)7472delC可能是耳聋与癫痫相关的线粒体基因新突变

线粒体DNA突变与许多人类疾病的发病机制相关。文章报道1例典型的患有耳聋与癫痫症状的具有母系遗传特征的中国家系。该家系共3代人, 其中14名母系成员中有3名耳聋患者, 3名癫痫患者, 而其他成员则无临床症状。线粒体全基因组序列分析表明, tRNA^Ser(UCN)基因7472delC新突变和33个多态位点属于东亚单体型B4b1a2。7472delC突变位于tRNA^Ser(UCN)高度保守的T-arm上。而在该区域的相同位点7472insC突变已在多个无遗传相关的家系中被发现与耳聋和癫痫相关。7472insC突变使tRNA代谢和线粒体功能产生缺陷。这样与7472insC突变相近的7472delC突变可能也会以相似机制引起线粒体功能障碍。同时, 在该家系中未发现GJB2基因及其他线粒体基因突变。因此, ^tRNASer(UCN) 7472delC可能是耳聋与癫痫相关的线粒体基因新突变。     

Crystal structure of the tRNA 3' processing endoribonuclease tRNase Z from Thermotoga maritima

The maturation of the tRNA 3' end is catalyzed by a tRNA 3' processing endoribonuclease named tRNase Z (RNase Z or 3'-tRNase) in eukaryotes, Archaea, and some bacteria. The tRNase Z generally cuts the 3' extra sequence from the precursor tRNA after the discriminator nucleotide. In contrast, Thermotoga maritima tRNase Z cleaves the precursor tRNA precisely after the CCA sequence. In this study, we determined the crystal structure of T. maritima tRNase Z at 2.6-A resolution. The tRNase Z has a four-layer alphabeta/betaalpha sandwich fold, which is classified as a metallo-beta-lactamase fold, and forms a dimer. The active site is located at one edge of the beta-sandwich and is composed of conserved motifs. Based on the structure, we constructed a docking model with the tRNAs that suggests how tRNase Z may recognize the substrate tRNAs.     

Two archaeal tRNase Z enzymes: similar but different

The endoribonuclease tRNase Z plays an essential role in tRNA metabolism by removal of the 3' trailer element of precursor RNAs. To investigate tRNA processing in archaea, we identified and expressed the tRNase Z from Haloferax volcanii, a halophilic archaeon. The recombinant enzyme is a homodimer and efficiently processes precursor tRNAs. Although the protein is active in vivo at 2-4 M KCl, it is inhibited by high KCl concentrations in vitro, whereas 2-3 M (NH(4))(2)SO(4) do not inhibit tRNA processing. Analysis of the metal content of the metal depleted tRNase Z revealed that it still contains 0.4 Zn(2+) ions per dimer. In addition tRNase Z requires Mn(2+) ions for processing activity. We compared the halophilic tRNase Z to the homologous one from Pyrococcus furiosus, a thermophilic archaeon. Although both enzymes have 46% sequence similarity, they differ in their optimal reaction conditions. Both archaeal tRNase Z proteins process mitochondrial pre-tRNAs. Only the thermophilic tRNase Z shows in addition activity toward intron containing pre-tRNAs, 5' extended precursors, the phosphodiester bis(p-nitrophenyl)phosphate (bpNPP) and the glyoxalase II substrate S-D: -lactoylglutathion (SLG).     

Specific cleavage of target RNAs from HIV-1 with 5' half tRNA by mammalian tRNA 3' processing endoribonuclease.

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) can be converted to an RNA cutter that recognizes four bases, with about a 65-nt 3'-truncated tRNA(Arg) or tRNA(Ala). The 3'-truncated tRNA recognizes the target RNA via four base pairings between the 5'terminal sequence and a sequence 1-nt upstream of the cleavage site, resulting in a pre-tRNA-like complex (Nashimoto M, 1995, Nucleic Acids Res 23:3642-3647). Here I developed a general method for more specific RNA cleavage using 3' tRNase. In the presence of a 36-nt 5' half tRNA(Arg) truncated after the anticodon, 3' tRNase cleaved the remaining 56-nt 3' half tRNA(Arg) with a 19-nt 3' trailer after the discriminator. This enzyme also cleaved its derivatives with a 5' extra sequence or nucleotide changes or deletions in the T stem-loop and extra loop regions, although the cleavage efficiency decreases as the degree of structural change increases. This suggests that any target RNA can be cleaved site-specifically by 3'tRNase in the presence of a 5' half tRNA modified to form a pre-tRNA-like complex with the target. Using this method, two partial HIV-1 RNA targets were cleaved site-specifically in vitro. These results also indicate that the sequence and structure of the T stem-loop domain are important, but not essential, for the recognition of pre-tRNAs by 3' tRNase.     

Long 5' leaders inhibit removal of a 3' trailer from a precursor tRNA by mammalian tRNA 3' processing endoribonuclease.

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) can remove a 3' trailer from various pre-tRNAs without 5' leader nucleotides. To examine how 5[prime] leader sequences affect 3' processing efficiency, we performed in vitro 3' processing reactions with purified pig 3' tRNase and pre-tRNAArgs containing a 13-nt 3' trailer and a 5[prime] leader of various lengths. The 3' processing was slightly stimulated by 5[prime] leaders containing up to 7 nt, whereas leaders of 9 nt or longer severely inhibited the reaction. Structure probing indicated that the 5' leader sequences had little effect on pre-tRNA folding. Similar results were obtained using pre-tRNA(Val)s containing a 5' leader of various lengths. We also investigated whether 3'tRNase can remove 3' trailers that are stably base-paired with 5' leaders to form an extended acceptor stem. Even such small 5' leaders as 3 and 6 nt, when base-paired with a 3' trailer, severely hindered removal of the 3' trailer by 3' tRNase.     

Arabidopsis Encodes Four tRNase Z Enzymes

Functional transfer RNA (tRNA) molecules are a prerequisite for protein biosynthesis. Several processing steps are required to generate the mature functional tRNA from precursor molecules. Two of the early processing steps involve cleavage at the tRNA 5′ end and the tRNA 3′ end. While processing at the tRNA 5′ end is performed by RNase P, cleavage at the 3′ end is catalyzed by the endonuclease tRNase Z. In eukaryotes, tRNase Z enzymes are found in two versions: a short form of about 250 to 300 amino acids and a long form of about 700 to 900 amino acids. All eukaryotic genomes analyzed to date encode at least one long tRNase Z protein. Of those, Arabidopsis (Arabidopsis thaliana) is the only organism that encodes four tRNase Z proteins, two short forms and two long forms. We show here that the four proteins are distributed to different subcellular compartments in the plant cell: the nucleus, the cytoplasm, the mitochondrion, and the chloroplast. One tRNase Z is present only in the cytoplasm, one protein is found exclusively in mitochondria, while the third one has dual locations: nucleus and mitochondria. None of these three tRNase Z proteins is essential. The fourth tRNase Z protein is present in chloroplasts, and deletion of its gene results in an embryo-lethal phenotype. In vitro analysis with the recombinant proteins showed that all four tRNase Z enzymes have tRNA 3′ processing activity. In addition, the mitochondrial tRNase Z proteins cleave tRNA-like elements that serve as processing signals in mitochondrial mRNA maturation.     

Conversion of mammalian tRNA 3' processing endoribonuclease to four-base-recognizing RNA cutters.

The spermidine-dependent, sequence-specific endoribonuclease (RNase 65) activities in mammalian cell extracts require both protein and 3' truncated tRNA, species of which direct their substrate sequence specificity. Computer analysis for searching possible base pairing between substrate RNAs and their corresponding 3' truncated tRNA, suggested a unified model for substrate recognition mechanism, in which a four-nucleotide (nt) sequence in the target tRNAs 1 nt upstream of their cleavage site, base pairs with the 5' terminal 4 nt sequence of their corresponding 3' truncated tRNA. This model was supported by experiments with several RNA substrates containing a substituted nucleotide in the target 4 nt sequence. In this model, the tRNA substrates and their corresponding 3' truncated tRNA form a complex resembling a 5' processed tRNA precursor containing a 3' trailer, suggesting that the protein component of RNase 65 is identical to tRNA 3' processing endoribonuclease (3' tRNase). Actually, 3' tRNase purified from pig liver cleaved the target RNAs at the expected sites only in the presence of their corresponding 3' truncated tRNA. These results show that the 3' tRNase can be converted to 4 nt specific RNA cutters using the 3' truncated tRNAs.     

Endonucleolytic processing of CCA-less tRNA precursors by RNase Z in Bacillus subtilis

In contrast to Escherichia coli, where the 3' ends of tRNAs are primarily generated by exoribonucleases, maturation of the 3' end of tRNAs is catalysed by an endoribonuclease, known as RNase Z (or 3' tRNase), in many eukaryotic and archaeal systems. RNase Z cleaves tRNA precursors 3' to the discriminator base. Here we show that this activity, previously unsuspected in bacteria, is encoded by the yqjK gene of Bacillus subtilis. Decreased yqjK expression leads to an accumulation of a population of B.subtilis tRNAs in vivo, none of which have a CCA motif encoded in their genes, and YqjK cleaves tRNA precursors with the same specificity as plant RNase Z in vitro. We have thus renamed the gene rnz. A CCA motif downstream of the discriminator base inhibits RNase Z activity in vitro, with most of the inhibition due to the first C residue. Lastly, tRNAs with long 5' extensions are poor substrates for cleavage, suggesting that for some tRNAs, processing of the 5' end by RNase P may have to precede RNase Z cleavage.     

Anomalous RNA substrates for mammalian tRNA 3' processing endoribonuclease

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) is an enzyme responsible for the removal of a 3' trailer from pre-tRNA. The enzyme can also recognize and cleave any target RNA that forms a pre-tRNA-like complex with another RNA. To investigate the interaction between 3' tRNase and substrates, we tested various anomalous pre-tRNA-like complexes for cleavage by pig 3' tRNase. We examined how base mismatches in the acceptor stem affect 3' tRNase cleavage of RNA complexes, and found that even one base mismatch in the acceptor stem drastically reduces the cleavage efficiency. Mammalian 3' tRNase was able to recognize complexes between target RNAs and 5'-half tDNAs, and cleave the target RNAs, although inefficiently, whereas the enzyme had no activity to cleave phosphodiester bonds of DNA. A relatively long RNA target, the Escherichia coli chloramphenicol acetyltransferase (CAT) mRNA, was cleaved by 3' tRNase in the presence of appropriate 5'-half tRNAs. We also demonstrated that an RNA complex of lin-4 and lin-14 from Caenorhabditis elegans can be recognized and cleaved by pig 3' tRNase.     

Mutations at position 7445 in the precursor of mitochondrial tRNA(Ser(UCN)) gene in three maternal Chinese pedigrees with sensorineural hearing loss

We report here the clinical, genetic and molecular characterization of three Chinese pedigrees with nonsyndromic bilateral hearing loss. Clinical and genetic evaluations revealed the variable severity and age-of-onset in hearing impairment in these families. Strikingly, there were extremely low penetrances of hearing impairment in these Chinese families. Sequence analysis of the complete mitochondrial DNA (mtDNA) showed the known A7445C mutation in two pedigrees and the novel A7445T mutation in another pedigree, in addition to distinct sets of mtDNA polymorphisms belong to Asian haplogroups D4j and F4. Indeed, the A7445C or A7445T mutation in the CO1 and the precursor of tRNA(Ser(UCN)) genes was present in homoplasmy only in the maternal lineage of those pedigrees but not other members of these families and 164 Chinese controls. In fact, the A7445C or A7445T mutation results in a read-through of the stop condon AGA of the CO1 message on the H strand of mtDNA, thereby adding three amino acids (Ser-Gln-Lys) to the C-terminal of the polypeptide. However, the mutated polypeptide may retain a partial function. Alternatively, the A7445C or A7445T mutation is adjacent to the site of 3' end endonucleolytic processing of L-strand RNA precursor, spanning tRNA(Ser(UCN)) and ND6 mRNA. Thus, the A7445C or A7445T mutation may also cause a defect in the processing of the L-strand RNA precursor, thus causing mitochondrial dysfunctions. Furthermore, the occurrence of the mutations at position 7445 in these genetically unrelated subjects affected by hearing impairment strongly indicates that mutations at the position 7445 are involved in the pathogenesis of hearing impairment.     

tRNA 3' end maturation in archaea has eukaryotic features: the RNase Z from Haloferax volcanii

Here, we report the first characterization and partial purification of an archaeal tRNA 3' processing activity, the RNase Z from Haloferax volcanii. The activity identified here is an endonuclease, which cleaves tRNA precursors 3' to the discriminator. Thus tRNA 3' processing in archaea resembles the eukaryotic 3' processing pathway. The archaeal RNase Z has a KCl optimum at 5mM, which is in contrast to the intracellular KCl concentration being as high as 4M KCl.The archaeal RNase Z does process 5' extended and intron-containing pretRNAs but with a much lower efficiency than 5' matured, intronless pretRNAs. At least in vitro there is thus no defined order for 5' and 3' processing and splicing. A heterologous precursor tRNA is cleaved efficiently by the archaeal RNase Z. Experiments with precursors containing mutated tRNAs revealed that removal of the anticodon arm reduces cleavage efficiency only slightly, while removal of D and T arm reduces processing effciency drastically, even down to complete inhibition. Comparison with its nuclear and mitochondrial homologs revealed that the substrate specificity of the archaeal RNase Z is narrower than that of the nuclear RNase Z but broader than that of the mitochondrial RNase Z.     

RNA heptamers that direct RNA cleavage by mammalian tRNA 3' processing endoribonuclease.

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) can recognize and cleave any target RNA that forms a precursor tRNA-like complex with another RNA. Various sets of RNA molecules were tested to identify the smallest RNA that can direct target RNA cleavage by 3' tRNase. A 3' half tRNAArgwas cleaved efficiently by 3' tRNase in the presence of small 5' half tRNAArgvariants, the D stem-loop region of which was partially deleted. Remarkably, 3' tRNase also cleaved the 3' half tRNAArgin the presence of a 7 nt 5' tRNAArg composed only of the acceptor stem region with a catalytic efficiency comparable with that of cleavage directed by an intact 5' half tRNAArg. The catalytic efficiency of cleavage directed by the heptamer decreased as the stability of the T stem-loop structures of 3' half tRNAArg variants decreased. No heptamer-directed cleavage of a 3' half tRNAArg without T stem base pairs was detected. A heptamer also directed cleavage of an HIV-1 RNA containing a stable hairpin structure. These findings suggest that in the presence of an RNA heptamer, 3' tRNase can discriminate and eliminate target RNAs that possess a stable hairpin adjacent to the heptamer binding sequence from a large complex RNA pool.     

The Deafness-Associated Mitochondrial DNA Mutation at Position 7445, Which Affects tRNASer(UCN) Precursor Processing, Has Long-Range Effects on NADH Dehydrogenase Subunit ND6 Gene Expression

The pathogenetic mechanism of the deafness-associated mitochondrial DNA (mtDNA) T7445C mutation has been investigated in several lymphoblastoid cell lines from members of a New Zealand pedigree exhibiting the mutation in homoplasmic form and from control individuals. We show here that the mutation flanks the 3′ end of the tRNA^Ser(UCN) gene sequence and affects the rate but not the sites of processing of the tRNA precursor. This causes an average reduction of ~70% in the tRNA^Ser(UCN) level and a decrease of ~45% in protein synthesis rate in the cell lines analyzed. The data show a sharp threshold in the capacity of tRNA^Ser(UCN) to support the wild-type protein synthesis rate, which corresponds to ~40% of the control level of this tRNA. Strikingly, a 7445 mutation-associated marked reduction has been observed in the level of the mRNA for the NADH dehydrogenase (complex I) ND6 subunit gene, which is located ~7 kbp upstream and is cotranscribed with the tRNA^Ser(UCN) gene, with strong evidence pointing to a mechanistic link with the tRNA precursor processing defect. Such reduction significantly affects the rate of synthesis of the ND6 subunit and plays a determinant role in the deafness-associated respiratory phenotype of the mutant cell lines. In particular, it accounts for their specific, very significant decrease in glutamate- or malate-dependent O₂ consumption. Furthermore, several homoplasmic mtDNA mutations affecting subunits of NADH dehydrogenase may play a synergistic role in the establishment of the respiratory phenotype of the mutant cells.