Polyadenylation and degradation of structurally abnormal mitochondrial tRNAs in human cells.

RNA 3' polyadenylation is known to serve diverse purposes in biology, in particular, regulating mRNA stability and translation. Here we determined that, upon exposure to high levels of the intercalating agent ethidium bromide (EtBr), greater than those required to suppress mitochondrial transcription, mitochondrial tRNAs in human cells became polyadenylated. Relaxation of the inducing stress led to rapid turnover of the polyadenylated tRNAs. The extent, kinetics and duration of tRNA polyadenylation were EtBr dose-dependent, with mitochondrial tRNAs differentially sensitive to the stress. RNA interference and inhibitor studies indicated that ongoing mitochondrial ATP synthesis, plus the mitochondrial poly(A) polymerase and SUV3 helicase were required for tRNA polyadenylation, while polynucleotide phosphorylase counteracted the process and was needed, along with SUV3, for degradation of the polyadenylated tRNAs. Doxycycline treatment inhibited both tRNA polyadenylation and turnover, suggesting a possible involvement of the mitoribosome, although other translational inhibitors had only minor effects. The dysfunctional tRNALeu(UUR) bearing the pathological A3243G mutation was constitutively polyadenylated at a low level, but this was markedly enhanced after doxycycline treatment. We propose that polyadenylation of structurally and functionally abnormal mitochondrial tRNAs entrains their PNPase/SUV3-mediated destruction, and that this pathway could play an important role in mitochondrial diseases associated with tRNA mutations.

ABCE1 is essential for S phase progression in human cells.

ABCE1 is a highly conserved protein universally present in eukaryotes and archaea, which is crucial for the viability of different organisms. First identified as RNase L inhibitor, ABCE1 is currently recognized as an essential translation factor involved in several stages of eukaryotic translation and ribosome biogenesis. The nature of vital functions of ABCE1, however, remains unexplained. Here, we study the role of ABCE1 in human cell proliferation and its possible connection to translation. We show that ABCE1 depletion by siRNA results in a decreased rate of cell growth due to accumulation of cells in S phase, which is accompanied by inefficient DNA synthesis and reduced histone mRNA and protein levels. We infer that in addition to the role in general translation, ABCE1 is involved in histone biosynthesis and DNA replication and therefore is essential for normal S phase progression. In addition, we analyze whether ABCE1 is implicated in transcript-specific translation via its association with the eIF3 complex subunits known to control the synthesis of cell proliferation-related proteins. The expression levels of a few such targets regulated by eIF3A, however, were not consistently affected by ABCE1 depletion.

Antisense oligonucleotide corrects splice abnormality in hereditary myopathy with lactic acidosis.

Hereditary myopathy with lactic acidosis (HML) (OMIM #255125) presents in childhood with exercise intolerance and muscle pain on trivial exercise, lactic acidosis, dyspnoea, palpitations, and rhabdomyolysis which can be fatal. The disease is recessively inherited and caused by a deep intronic, single base transition in the iron-sulfur cluster scaffold, ISCU gene that causes retention of a pseudoexon and introduction of a premature termination codon. IscU protein deficiency causes secondary defects in several iron-sulfur dependant proteins, including enzymes involved in aerobic energy metabolism. We have shown in a previous study that the splice abnormality affects skeletal muscle more than other tissues, leading to the purely muscular phenotype. Antisense oligonucleotides (AOs) have been able to redirect mRNA splicing in a number of disease models, and show promise in clinical studies. We designed 2'O-methyl phosphorothioate AOs targeting either splice site of the detrimental HML pseudoexon. The acceptor site AO effectively redirected splicing towards the normal state in cultured muscle fibroblasts, whilst the donor site AO promoted pseudoexon inclusion in both patient and control cells. Our results show that AO therapy seems feasible in HML, but care must be taken to avoid adverse splicing effects.

Differences in RNA processing underlie the tissue specific phenotype of ISCU myopathy.

Hereditary myopathy with lactic acidosis, or myopathy with exercise intolerance, Swedish type (OMIM #255125) is caused by mutations in the iron-sulfur cluster scaffold (ISCU) gene. The g.7044G>C ISCU mutation induces a splicing error in the pre-mRNA that strengthens a weak intronic splice site leading to inclusion of a new exon and subsequent loss of mRNA and protein. While ISCU is widely expressed, homozygosity for this particular intronic mutation gives rise to a pure myopathy. In order to investigate tissue specificity and disease mechanism, we studied muscle, myoblasts, fibroblasts and blood cells from the first non-Swedish case of this disease. Consistent with the recognised role of ISCU, we found abnormal activities of respiratory chain complexes containing iron-sulfur clusters in patient muscle. We confirmed that, in the presence of the g.7044G>C mutation, splicing produces both abnormally and normally spliced mRNA in all tissues. The ratio of these products varies dramatically between tissues, being most abnormal in mature skeletal muscle that also has the lowest relative starting levels of ISCU mRNA compared with other tissues. Myoblasts and fibroblasts have more of the normally spliced variant as well as higher starting levels of ISCU mRNA. Up-regulation of mtDNA copy number was found in skeletal muscle and myoblasts, but not fibroblasts, and is thought to represent a compensatory response. Tissue specificity in this disorder appears therefore to be dependent on the mRNA starting level, the amount of remaining normally spliced RNA, and the degree to which compensatory mechanisms can respond.

The 7472insC mtDNA mutation impairs 5' and 3' processing of tRNA(Ser(UCN)).

The deafness-associated 7472insC mtDNA mutation was previously shown to decrease the steady-state level of tRNA(Ser(UCN)) post-transcriptionally. To identify the affected tRNA maturation step(s) we analysed the effects of the mutation on processing in vivo and in vitro. tRNA(Ser(UCN)) from cybrid cells homoplasmic for 7472insC contained a high frequency (>11%) of molecules misprocessed at one or both termini. In vitro assays using partially purified HeLa cell RNase P and mitochondrial tRNA 3' processing endonuclease (tRNase Z) confirmed that the efficiency of both 5' and 3' processing was impaired. A mutant precursor not already processed at the 5' end was poorly processed in vitro by tRNase Z. Misprocessing at the 3' end further impaired the efficiency and accuracy of 5' processing of the mutant substrate. The mutation thus appears to affect several distinct, but interdependent, RNA processing steps, with the predicted outcome dependent on the exact processing pathway operating in vivo.

The 7472insC mitochondrial DNA mutation impairs the synthesis and extent of aminoacylation of tRNASer(UCN) but not its structure or rate of turnover.

The 7472insC mitochondrial DNA mutation in the tRNA(Ser(UCN)) gene is associated with sensorineural deafness combined, in some patients, with a wider neurological syndrome. In cultured cybrid cells it causes a 70% decrease in tRNA(Ser(UCN)) abundance and mild respiratory impairment, previously suggested to be due to decreased tRNA stability. When mitochondrial transcription was blocked by ethidium bromide treatment, the half-life of the mutant tRNA was not significantly different from that of wild-type tRNA(Ser(UCN)). Over-expression of mitochondrial translational elongation factor EF-Tu also had no effect on the mutant phenotype. However, during recovery from prolonged ethidium bromide treatment, the synthesis of the mutant tRNA(Ser(UCN)) was specifically impaired, without polarity effects on downstream tRNAs of the light strand transcription unit. We infer that the mutation acts posttranscriptionally to decrease tRNA(Ser(UCN)) abundance by affecting its synthesis rather than its stability. The extent of aminoacylation of the mutant tRNA was also decreased by approximately 25%. In contrast, the mutation had no detectable effect on tRNA(Ser(UCN)) base modification or structure other than the insertion of an extra guanosine templated by the mutation, which was structurally protected from nuclease digestion like the surrounding nucleotides. These findings indicate a common molecular process underlying sensorineural deafness caused by mitochondrial tRNA(Ser(UCN)) mutations.