Impairment of mitochondrial tRNAIle processing by a novel mutation associated with chronic progressive external ophthalmoplegia.

We report a sporadic case of chronic progressive external ophthalmoplegia associated with ragged red fibers. The patient presented with enlarged mitochondria with deranged internal architecture and crystalline inclusions. Biochemical studies showed reduced activities of complex I, III and IV in skeletal muscle. Molecular genetic analysis of all mitochondrial tRNAs revealed a G to A transition at nt 4308; the G is a highly conserved nucleotide that participates in a GC base-pair in the T-stem of mammalian mitochondrial tRNA(Ile). The mutation was detected at a high level (approx. 50%) in muscle but not in blood. The mutation co-segregated with the phenotype, as the mutation was absent from blood and muscle in the patient's healthy mother. Functional characterization of the mutation revealed a six-fold reduced rate of tRNA(Ile) precursor 3' end maturation in vitro by tRNAse Z. Furthermore, the mutated tRNA(Ile) displays local structural differences from wild-type. These results suggest that structural perturbations reduce efficiency of tRNA(Ile) precursor 3' end processing and contribute to the molecular pathomechanism of this mutation.

In vitro 3'-end endonucleolytic processing defect in a human mitochondrial tRNA(Ser(UCN)) precursor with the U7445C substitution, which causes non-syndromic deafness.

Eukaryotic tRNAs are transcribed as precursors. A 5'-end leader and 3'-end trailer are endonucleolytically removed by RNase P and 3'-tRNase before 3'-end CCA addition, aminoacylation, nuclear export and translation. 3'-End -CC can be a 3'-tRNase anti-determinant with the ability to prevent mature tRNA from recycling through 3'-tRNase. Twenty-two tRNAs punctuate the two rRNAs and 13 mRNAs in long, bidirectional mitochondrial transcripts. Accurate mitochondrial gene expression thus depends on endonucleolytic excision of tRNAs. Various mitochondrial diseases and syndromes could arise from defective tRNA end processing. The U7445C substitution in the human mitochondrial L-strand transcript (U74C directly following the discriminator base of tRNA(Ser(UCN))) causes non-syndromic deafness. The sequence of the precursor (G/UCU) becomes G/CCU, resembling a 3'-tRNase anti-determinant. We demonstrate that a tRNA(Ser(UCN)) precursor with the U7445C substitution cannot be processed in vitro by 3'-tRNase from human mitochondria. A 3'-end processing defect in this tRNA precursor could thus be responsible for mitochondrial disease.

The effects of matrices of paired substitutions in mid-acceptor stem on Drosophila tRNA(His) structure and end-processing.

End-maturation reactions, in which the 5' end leader and 3' end trailer of precursor tRNA are removed by RNase P and 3'-tRNase, respectively, are early, essential steps in eukaryotic precursor tRNA processing. End-processing enzymes may be expected to contact the acceptor stem of tRNA due to its proximity to both cleavage sites. We constructed matrices of pair-wise substitutions in mid-acceptor stem at nt 3/70 and 4/69 of Drosophila tRNA(His) and analyzed their ability to be processed by Drosophila RNase P and 3'-tRNase. In accord with our earlier study of D/T loop processing matrices, we find that tRNA end processing enzymes respond to sequence changes differently. More processing defects were observed with 3'-tRNase than with RNase P, and substitutions at 4/69 reduced processing more than those at 3/70. We evaluated tRNA folding using structure probing nucleases and investigated the contribution of K(M) and V(Max) to the processing efficiency of selected variants. In one substitution (C3A), mis-folding correlates with processing defects. In another (C69A), a disruption of structure appears to be transmitted laterally to both ends of the acceptor stem. Poor processing of C69A by RNase P is due entirely to a reduction in V(Max), but for 3'-tRNase, it is due to an increase in K(M).

The 3' end CCA of mature tRNA is an antideterminant for eukaryotic 3'-tRNase.

Cytoplasmic tRNAs undergo posttranscriptional 5' and 3' end processing in the eukaryotic nucleus, and CCA (which forms the mature 3' end of all tRNAs) must be added by tRNA nucleotidyl transferase before tRNA can be aminoacylated and utilized in translation. Eukaryotic 3'-tRNase can endonucleolytically remove a 3' end trailer by cleaving on the 3' side of the discriminator base (the unpaired nucleotide 3' of the last base pair of the acceptor stem). This reaction proceeds despite a wide range in length and sequence of the 3' end trailer, except that mature tRNA containing the 3' terminal CCA is not a substrate for mouse 3'-tRNase (Nashimoto, 1997, Nucleic Acids Res 25:1148-1154). Herein, we extend this result with Drosophila and pig 3'-tRNase, using Drosophila melanogaster tRNAHis as substrate. Mature tRNA is thus prevented from recycling through 3' end processing. We also tested a series of tRNAs ending at the discriminator base (-), with one C added (+C), two Cs added (+CC), and CCA added (+CCA) as 3'-tRNase inhibitors. Inhibition was competitive with both Drosophila and pig 3'-tRNase. The product of the 3'-tRNase reaction (-) is a good 3'-tRNase inhibitor, with a KI approximately two times KM for the normal 3'-tRNase substrate. KI increases with each nucleotide added beyond the discriminator base, until when tRNA+CCA is used as inhibitor, KI is approximately forty times the substrate KM. The 3'-tRNase can thus remain free to process precursors with 3' end trailers because it is barely inhibited by tRNA+CCA, ensuring that tRNA can progress to aminoacylation. The active site of 3'-tRNase may have evolved to make an especially poor fit with tRNA+CCA.

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 KI approximately 7 times greater than the substrate KM of approximately 200 nM and is a better inhibitor of 3'-tRNase, with a KI approximately two times the KM of approximately 80 nM. We generated matrices of substitutions at positions G18/U55 and G19/C56 (two contiguous universally conserved D/T loop base pairs) in Drosophila 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 in KM with relatively little change in Vmax, 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.

Sequence and structure requirements for Drosophila tRNA 5'- and 3'-end processing.

Eukaryotic tRNAs are processed at their 5'- and 3'- ends by endonucleases RNase P and 3'-tRNase, respectively. We have prepared substrates for both enzymes, separated the activities from a Drosophila extract, and designed variant tRNAs to assess the effects of sequence and structure on processing. Mutations affect these reactions in similar ways; thus, RNase P and 3'-tRNase probably require similar substrate structures to maintain the catalytic fit. RNase P is more sensitive to substrate substitutions than 3'-tRNase. In three of the four stems, one substitution prevents both processing reactions while the opposite one has less effect; anticodon stem substitutions hardly affect processing, and double substitution intended to restore base pairing also restore processing to the wild type rate. Structure probing suggests that tRNA misfolding sometimes coincides with reduced processing. In other cases, processing inhibition probably results from specific unfavorable stem appositions leading to local helix deformation. A single T loop substitution disrupts the tertiary D-T loop interaction and reduces processing. We have thus begun mapping tRNA processing determinants on the global, local, and tertiary structure levels.

Point mutations distal to the processing site affect Drosophila pre-5 S RNA processing. Long range cooperation and a breathing model.

Drosophila pre-5 S RNA, which consists of five conserved stem-loop domains and a 15-nucleotide 3' tail, is 3'-end processed to 120 nucleotide mature 5 S RNA before ribosome assembly. Large deletions in stems II and III, all of stems IV and V, and loop C prohibit Drosophila 5 S RNA processing; deletion of stem IV and half of V does not (Preiser, P. R., and Levinger, L. (1991a) J. Biol. Chem. 266, 7509-7516). Several point mutations in stem I reduce, while certain neighboring sequence changes stimulate, processing (Levinger, L., Vasisht, V., Greene, V., and Arjun, I. (1992) J. Biol. Chem. 267, 23683-23687). Herein we extend this 5 S RNA fine structure analysis to regions farther from the processing site. Most point mutations in loop B, stem III, and loop C severely inhibit processing. One loop C substitution stimulates processing; when combined with stimulatory sequence changes in stem I and loop A, these dispersed mutations improve processing manyfold, perhaps by stabilizing a required conformation or strengthening a protein-binding site. Central stem II sequence changes inhibit processing; several adjacent sequence substitutions which weaken base pairing improve processing. Combining these results with earlier work from stem I and loop A, we hypothesize that slight reduction in base pairing may improve groove access of polypeptide chains to essential contact positions.

Poly(U)-binding protein inhibits Drosophila pre-5 S RNA 3'-exonuclease digestion.

A approximately 50-kDa protein binds specifically to the 3' terminus of 135-nucleotide Drosophila pre-5 S RNA. Unlabeled poly(U) competes out protein binding and stimulates the activity of a 3'-exonuclease, which eventually degrades the substrate to 120 nucleotides, the size of mature 5 S RNA. In its RNA binding and UV cross-linking properties, the endogenous poly(U)-binding protein resembles human La, an autoantigen that binds the U > 3 3' ends of vertebrate RNA polymerase III primary transcripts. This protein appears to inhibit a 3' exonuclease and could protect 5 S RNA for faithful processing and transport.

The effects of stem I and loop A on the processing of 5 S rRNA from Drosophila melanogaster.

The 135-nucleotide Drosophila melanogaster 5 S RNA precursor is processed by removal of 15 nucleotides from its 3' end before incorporation into the large ribosomal subunit. Mature 5 S RNA consists of five helical stem-loops; stem IV and part of V are dispensable, whereas stem III and the 1/118 G-C base pair closest to the processing site at nucleotide 120 are required for processing (Preiser, P., and Levinger, L. (1991) J. Biol. Chem. 266, 7509-7516; Preiser, P., and Levinger, L. (1991) J. Biol. Chem. 266, 23602-23605). We have investigated the effects of stem I and loop A transversions, transitions, selected additions and deletions on 5 S RNA processing. Stem I single substitutions generally prevent processing, whereas compensatory double substitutions restore a range of processing rates. Proximal to the processing site, stem I double substitutions inhibit processing. In the distal portion of stem I and loop A, the processing effect of paired sequence changes varies widely in an irregular pattern. The 7/112 GU pair and nucleotide 13A least tolerate sequence changes; several mutations clustered close to the stem I-loop A boundary stimulate processing. We interpret these results in terms of the RNA helix path and possible RNA-protein contacts.

Drosophila 5 S RNA processing requires the 1-118 base pair and additional sequence proximal to the processing site.

Using an in vitro processing system, we have identified a required sequence surrounding the Drosophila melanogaster 5 S RNA processing site at nucleotide 120. Mutations in this region vary the processing rate from complete inhibition to a level equal to or greater than wild type. Analysis of mutants at +1 and in the region 118-122 separates the inhibitory effect into two parts. 1) Nucleotide 118 C, the base-paired nucleotide in helix I proximal to the processing site, plays an essential role. Changing it to a purine inhibits processing. The +1-118 base pair must be intact, but this alone is not sufficient for processing, since compensatory changes at +1 do not restore down-processing mutants at 118 to the wild type level. 2) The processing site has to be pyrimidine rich; multiple contiguous purines inhibit processing. On the other hand, multiple pyrimidines can largely negate the inhibitory effect of a mutation at position 118. Thus a base-paired C at 118 followed by a stretch of pyrimidines is the processing signal, which may be recognized by the processing enzyme and/or a required accessory factor.

In vitro processing of Drosophila melanogaster 5 S ribosomal RNA. 3' end effects and requirement for internal domains of mature 5 S RNA.

5 S RNA processing in Drosophila melanogaster, the removal of 15 nucleotides from the 3' end of the 135-nucleotide (nt) primary transcript, may play an important role in the regulation of 5 S RNA transport and ribosome assembly. We have uncoupled processing from transcription using gel purified primary transcripts processed in vitro by a cellular S100 extract. The RNA was generated by a homologous transcription system or by a T7 RNA polymerase reaction using a constructed 5 S RNA gene linked to a T7 promoter. In vitro D. melanogaster 5 S RNA processing is heat- and EDTA-sensitive, suggesting a requirement for protein, and produces a 3' end characteristic of mature 5 S RNA. Processing of substrate RNAs with altered 3' ends shows that the 3'-U5 tail (nt 131-135) inhibits the reaction. 30 nt, including all of domain IV and most of domain V, are dispensible for processing, whereas deletions including the base of stem V and all or part of stem III severely inhibit it. Several possible mechanisms are discussed.

Visualization of RNA binding proteins by sequential gel shift and ultraviolet cross-linking.

RNA binding proteins partially constitute the ribonucleoprotein or protein machinery for RNA processing (splicing, polyadenylation and 3' end formation), transport, and storage. We have devised a novel method for the detection of RNA binding proteins in vitro. The template for transcription is a cloned Drosophila melanogaster 5S rRNA gene. The method is a two-dimensional gel analysis involving: in vitro transcription of 32P-labeled 5S rRNA using a cellular S-100; resolution of labeled RNA protein complexes from unbound RNA on a first-dimension mobility shift gel; cross-linking of RNA to protein in gel by ultraviolet irradiation; degradation of the RNA by RNase A and T1; and analysis of 32P-protein patterns on a second-dimension discontinuous SDS gel by autoradiography. The pattern of proteins associated with 32P-5S rRNA is obtained by covalent transfer of 32P-nucleotides from RNA to the proteins with which the RNA was bound. This method could be useful in the analysis of RNA maturation and processing pathways.

Human protein binding to DNA sequences surrounding the human T-cell lymphotropic virus type-I long terminal repeat polyadenylation site.

The long terminal repeats (LTRs) of RNA tumor viruses, including human T-cell lymphotropic virus type I (HTLV-I), contain the control elements for expression of viral genes. Sequence-specific LTR-DNA-binding proteins could regulate viral functions. To search for such proteins we have used an in vitro non-denaturing polyacrylamide gel assay, with restriction fragments of the HTLV-I LTR and nuclear protein extracts from several HTLV-I-infected cell lines and an uninfected T-cell line, H9. Four DNA-binding activities were observed, including non-specific DNA-binding activity and at least two activities (forms I and II) which bind specifically to a HinfI restriction fragment from nucleotides +181 to +334 relative to the transcription start site. DNA-binding activities I and II were partially resolved by ion-exchange chromatography and mapped by protection experiments to two 10-20-bp blocks surrounding the polyadenylation site at +221. Of the cell lines tested, form II was abundantly found in C10/MJ, and forms I and IV were also found in C91/PL, C81-66-45, MT2 and H9 cells.

A unique nucleoprotein structure associated with the Drosophila melanogaster 18-28 S rDNA nontranscribed spacer.

We have detected unique nucleoprotein particles specific for the 18-28 S rDNA nontranscribed spacer of Drosophila melanogaster. The particles migrate between di- and trinucleosomes on nucleoprotein gels, and are between mono- and dinucleosomal in DNA length. These migration properties suggest that the nontranscribed spacer particles could have a protein component larger than a histone core. The variant nucleoprotein structures map primarily within the nontranscribed spacer 235 base pair internal subrepeat, which is AT-rich and possesses a 50 base pair sequence homologous to the RNA polymerase I binding site.

Group meetings for parents and spouses of bone marrow transplant patients.

To reduce the stress and social isolation of being the parent or spouse of a patient hospitalized for bone marrow transplantation (BMT), a support group for family members was established. Sharing the common experience of the BMT procedure was valuable to family members despite differences in patient's age or disease, relationship to the patient, or socio-economic status. Recommendations are made to include similar groups in planning to meet the psychosocial needs of family members at other centers where patients are treated with high-risk, high intensity procedures.

D1 protein of Drosophila melanogaster. Purification and AT-DNA binding properties.

D1 protein of Drosophila melanogaster is a sequence-specific DNA-binding protein which recognizes AT-rich DNA sequences. AT-rich DNA sequences in eukaryotic organisms are distributed in two characteristic ways: flanking transcriptional units and in constitutive heterochromatin. D1 could play a role in regulation of gene expression and in geographical localization of DNA sequences within the nucleus. D1 has been partially purified by ion exchange chromatography. DNA-binding activity was investigated by nucleoprotein gel electrophoresis, using end-labeled restriction fragments varying in AT sequence content. D1 binds most tightly to the satellite sequence -AATAT-, with intermediate strength to the complex satellite (359-base pair repeat) and another AT-rich (68% AT) mixed sequence DNA, and least to the simple satellite sequence -AAGAG-.

Nucleosomal structure of two Drosophila melanogaster simple satellites.

Nucleosomes have been fractionated on nondenaturing polyacrylamide gels, and nucleosome subtypes containing the Drosophila melanogaster specific protein D1 and ubiquitinated core histone H2A were identified by solubility in 0.1 M NaCl before nucleoprotein gel electrophoresis. Nucleosomes which contain DNA complementary to the 1.672 density simple satellite (sequence-AATAT-) bind protein D1, as demonstrated by two-dimensional hybridization mapping. This hybridization pattern allows the identification of D1 dinucleosomes, which, like D1 mononucleosomes, are reduced in mobility on the first dimension (nucleoprotein) gel by the addition of D1, an AT sequence-specific DNA-binding protein. The 1.705 density simple satellite (sequence-AAGAG-) is also found in nucleosomes, in a radically different subset from those of the -AATAT- DNA sequence. -AAGAG- nucleosomes do not contain D1 protein, but appear to be enriched in ubiquitinated core histone H2A. One-dimensional hybridization patterns suggest that -AAGAG- nucleosomal DNA is rapidly trimmed to a shorter DNA length than either bulk or -AATAT- nucleosomes.