Osteogenesis imperfecta type IV. Detection of a point mutation in one alpha 1(I) collagen allele (COL1A1) by RNA/RNA hybrid analysis

We have identified a point mutation in one alpha 1(I) collagen allele (COL1A1) of a child with the type IV osteogenesis imperfecta phenotype. When compared to parental and control samples, skin fibroblasts of the proband synthesized two populations of type I collagen molecules. One population was normal; the other was delayed in secretion and electrophoretic migration due to post-translational overmodification. Two-dimensional gel electrophoresis of the CNBr peptides demonstrated a gradient of overmodification beginning near the carboxyl-terminal CB peptides. This predicts that the mutation delaying helix formation is near the carboxyl-terminal end of one of the component chains of type I collagen. The mRNA of the patient was probed with overlapping antisense riboprobes to type I collagen cDNA. Cleavage of a mismatch in RNA/RNA hybrids of RNase A allowed the location of the mutation to a 225-base pair region of alpha 1(I) cDNA. The mismatch was not present in RNA/RNA hybrids from either parent. This region of both alpha 1(I) alleles of the patient was isolated by screening a lambda ZAP cDNA library. Sequence determination of both alleles demonstrated a single nucleotide change, G----A, resulting in the substitution of a serine for a glycine at amino acid residue 832. This point mutation occurs in the coding region for alpha 1(I) CB6 and is concordant with the protein data. The finding of a glycine substitution in an alpha 1(I) chain of a patient with the milder type IV osteogenesis imperfecta phenotype requires modification of current molecular models for types II and IV osteogenesis imperfecta.     

Detection of mutations in human type I collagen mRNA in osteogenesis imperfecta by indirect RNase protection

A method for detecting a wide variety of mutations within type I collagen has been developed and evaluated on a series of patients with osteogenesis imperfecta. RNA, extracted from the nuclear and cytoplasmic compartment of cultured fibroblasts from affected individuals, is hybridized with antisense single-stranded cDNA to the alpha 1(I) mRNA. The hybrid is digested with RNase A and T1 under varying degrees of stringency. The resistant RNA bands are separated by electrophoresis in agarose, transferred to nitrocellulose, and hybridized with antisense cRNA colinear with the protecting probe. This approach is capable of detecting previously defined mutations such as 252-base pair deletion and a 1-base pair mismatch within the alpha 1(I) mRNA. The method appears to be particularly useful in detecting abnormalities of RNA processing that behave as an insert or deletion within the mature mRNA. The procedure should be generally applicable for the identification and localization of any mutation within an entire gene if the gene is expressed as an RNA and a complete cDNA for the mRNA is available.     

A 9-base pair deletion in COL1A1 in a lethal variant of osteogenesis imperfecta

A proband with lethal osteogenesis imperfecta has been investigated for the causative defect at the levels of collagen protein, mRNA, and DNA. Analysis of type I collagen synthesized by the proband's fibroblasts showed excessive post-translational modification of alpha 1(I) chains along the entire length of the helix. Oververmodification of alpha chains could be prevented by incubation of the cells at 30 rather than 37 degrees C, and the thermal stability of the triple helix, as determined by protease digestion, was normal. RNase A cleavage of RNA:RNA hybrids formed between the proband's mRNA and antisense RNA derived from normal pro-alpha 1(I) chain cDNA clones was used to locate an abnormality to exon 43 of the proband's pro-alpha 1(I) collagen gene (COL1A1). The nucleotide sequence of the corresponding gene region showed, in one allele, the deletion of 9 base pairs, not present in either parent, within a repeating sequence of exon 43. The mutation causes the loss of one of three consecutive Gly-Ala-Pro triplets at positions 868-876, but does not otherwise disrupt the Gly-X-Y sequence. Procollagen processing in fibroblast cultures and susceptibility of the mutant collagen I to cleavage with vertebrate collagenase were normal, indicating that the slippage of collagen chains by one Gly-X-Y triplet does not abolish amino-propeptidase and collagenase cleavage sites. How the mutation produces the lethal osteogenesis imperfecta phenotype is not entirely clear; the data suggest that the interaction of alpha chains immediately prior to helix formation may be affected.     

A critical survey of the structure-function of the antisense oligo/RNA heteroduplex as substrate for RNase H

The aim of this review is to draw a correlation between the structure of the DNA/RNA hybrid and its properties as a substrate for the RNase H, as well as to point the crucial structural requirements for the modified AONs to preserve their RNase H potency. The review is divided into the following parts: (1) mechanistic considerations, (2) target RNA folding-AON folding-RNase H assistance in AON/RNA hybrid formation, (3) carbohydrate modifications, (4) backbone modifications, (5) base modifications, (6) conjugated AONs, (7) importance of the tethered chromophore in AON for the AON/RNA hybrid interactions with the RNase H. The structural changes in the AON/RNA hybrid duplexes brought by different modifications of the sugar, backbone or base in the antisense strand, and the effect of these changes on the RNase H recognition of the modified substrates have been addressed. Only those AON modifications and the corresponding AON/RNA hybrids, which have been structurally characterized by spectroscopic means and functionally analyzed by their ability to elicit RNase H potency in comparison with the native counterpart have been presented here.     

Structural basis of cleavage by RNase H of hybrids of arabinonucleic acids and RNA

The origins of the substrate specificity of Escherichia coli RNase H1 (termed RNase H here), an enzyme that hydrolyzes the RNA strand of DNA-RNA hybrids, are not understood at present. Although the enzyme binds double-stranded RNA, no cleavage occurs with such duplexes [Lima, W. F., and Crooke, S. T. (1997) Biochemistry 36, 390]. Therefore, the hybrid substrates may not adopt a canonical A-form geometry. Furthermore, RNase H is exquisitely sensitive to chemical modification of the DNA strands in hybrid duplexes. This is particularly relevant to the RNase H-dependent pathway of antisense action. Thus, only very few of the modifications currently being evaluated as antisense therapeutics are tolerated by the enzyme, among them phosphorothioate DNA (PS-DNA). Recently, hybrids of RNA and arabinonucleic acid (ANA) as well as the 2'F-ANA analogue were shown to be substrates of RNase H [Damha, M. J., et al. (1998) J. Am. Chem. Soc. 120, 12976]. Using X-ray crystallography, we demonstrate here that ANA analogues, such as 2'F-ANA [Berger, I., et al. (1998) Nucleic Acids Res. 26, 2473] and [3.3.0]bicyclo-ANA (bc-ANA), may not be able to adopt sugar puckers that are compatible with pure A- or a B-form duplex geometries, but rather prefer the intermediate O4'-endo conformation. On the basis of the observed conformations of these ANA analogues in a DNA dodecamer duplex, we have modeled a duplex of an all-C3'-endo RNA strand and an all-O4'-endo 2'F-ANA strand. This duplex exhibits a minor groove width that is intermediate between that of A-form RNA and B-form DNA, a feature that may be exploited by the enzyme in differentiating between RNA duplexes and DNA-RNA hybrids. Therefore, the combination of the established structural and functional properties of ANA analogues helps settle existing controversies concerning the discrimination of substrates by RNase H. Knowlegde of the structure of an analogue that exhibits enhanced RNA affinity while not interfering with RNase H activity may prove helpful in the design of future antisense modifications.     

Sequence of normal canine COL1A1 cDNA and identification of a heterozygous alpha1(I) collagen Gly208Ala mutation in a severe case of canine osteogenesis imperfecta

The sequence of canine COL1A1 cDNA was determined from four overlapping COL1A1 RT-PCR products generated from canine fibroblast RNA. In the translated region, nucleotide identity between canine and human COL1A1 cDNA was 93.2%, although the canine sequence lacked nucleotides 204 to 215 in the region coding for the N-propeptide. Amino acid identity was 97.7%. Total RNA and type I collagen were collected from cultured skin fibroblasts of a 12-week-old male golden retriever with pathologic fractures suggestive of osteogenesis imperfecta (OI) and dentinogenesis imperfecta. Sequential, overlapping approximately 1,000-bp fragments of COL1A1 and COL1A2 cDNA were each amplified by RT-PCR using primers containing 5' T7 polymerase sites. These PCR products were transcribed with T7 RNA polymerase, hybridized into RNA duplexes, and cleaved at mismatch sites with RNase. The proband had an unique cleavage pattern for the fragment of COL1A1 mRNA spanning nucleotides 709 to 1,531. Sequence analysis identified a G to C point mutation for nucleotide 1,276, predicting a codon change from glycine (GGA) to alanine (GCA) for amino acid 208. This change disrupts the normal Gly-X-Y pattern of the collagen triple helix. Restriction enzyme digestion of the RT-PCR product was consistent with a heterozygous COL1A1 mutation. Type I collagen was labeled with 3H-proline, salt precipitated, and analyzed by SDS-PAGE. Pepsin digested alpha chains were over-hydroxylated, and procollagen processing was delayed. Thus, canine and human OI appear homologous in terms of clinical presentation, etiology, and pathogenesis.     

Substitution of serine for alpha 1(I)-glycine 844 in a severe variant of osteogenesis imperfecta minimally destabilizes the triple helix of type I procollagen. The effects of glycine substitutions on thermal stability are either position of amino acid specific

Recent reports have demonstrated that a series of probands with severe osteogenesis imperfecta had single base mutations in one of the two structural genes for type I procollagen that substituted amino acids with bulkier side chains for glycine residues and decreased the melting temperature of the triple helix. Here we demonstrate that the type I procollagen synthesized by cultured fibroblasts from a proband with a severe form of osteogenesis imperfecta consisted of normal molecules and molecules over-modified by post-translational reactions. The thermal stability of the intact type I collagen was normal as assayed by protease digestion under conditions in which a decrease in thermal stability was previously observed with eight other substitutions for glycine in the alpha 1(I) chain. In contrast, the thermal stability of the one-quarter length B fragment generated by digestion with vertebrate collagenase was decreased by 2-3 degrees C under the same conditions. Nucleotide sequencing of cDNAs and genomic DNA established that the proband had a substitution of A for G in one allele of the pro alpha 1(I) gene that converted the codon for alpha 1-glycine 844 to a codon for serine. The results also established that the alpha 1-serine 844 was the only mutation that could account for the decrease in thermal stability of the collagenase B fragment. There are at least two possible explanations for the failure of the alpha 1-serine 844 substitution to decrease the thermal stability of the collagen molecule whereas eight similar mutations decreased the melting temperature. One possibility is that the effects of glycine substitutions are position specific because not all glycine residues make equivalent contributions to cooperative blocks of the triple helix that unfold in the predenaturation range of temperatures. A second possible explanation is that substitutions of glycine by serine have much less effect on the stability of protein than the substitutions by arginine, cysteine, and aspartate previously studied.     

A single base mutation in type I procollagen (COL1A1) that converts glycine alpha 1-541 to aspartate in a lethal variant of osteogenesis imperfecta: detection of the mutation with a carbodiimide reaction of DNA heteroduplexes and direct sequencing of products of the PCR.

Skin fibroblasts from a proband with a lethal variant of osteogenesis imperfecta synthesized both apparently normal type I procollagen and a type I procollagen that had slow electrophoretic mobility because of posttranslational overmodifications. The thermal unfolding of the collagen molecules as assayed by protease digestion was about 2 degrees C lower than normal. It is surprising, however, that collagenase A and B fragments showed an essentially normal melting profile. Assay of cDNA heteroduplexes with a new technique involving carbodiimide modification indicated a mutation at about the codon for amino acid 550 of the alpha 1(I) chain. Subsequent amplification of the cDNA by the PCR and nucleotide sequencing revealed a single-base mutation that substituted an aspartate codon for glycine at position alpha 1-541 in the COL1A1 gene. The results here confirm previous indications that the effects of glycine substitutions in type I procollagen are highly position specific. They also demonstrate that a recently described technique for detecting single-base differences by carbodiimide modification of DNA heteroduplexes can be effectively employed to locate mutations in large genes.     

细菌中RNase HI介导RNA降解的研究进展

在细菌细胞中,为了维持基因组稳定和正常的生命活动,RNase HI通常以降解RNA/DNA杂合链中RNA的方式来防止复制中引物的积累以及转录中R环的形成。RNase HI对底物的识别主要依赖于DNA与RNA结合槽,对底物的催化主要依赖于DEDD基序和位于活性位点附近柔性环中的一个组氨酸。以Mg²⁺为代表的金属离子在催化过程中发挥了至关重要的作用。杂交双链中ssDNA突出部分的类型决定了RNase HI的作用模式：在没有突出或在ssDNA的5′端存在突出部分的情况下,RNase HI作为一种非序列特异性核酸内切酶随机地降解RNA;当ssDNA的3′端存在突出部分时,RNase HI依靠5′核酸外切酶活性对RNA进行连续切割。RNase HI、Rep、DinG和UvrD通过与单链DNA结合蛋白(single-stranded DNA-binding protein, SSB)的C端尾部的6个残基相互作用被招募到复制叉附近,并可能以协作的方式解决复制-转录冲突。RNaseHI的缺失或活性降低将引起DNA结构不稳定、基因突变、转录装置回溯和复制不协调等一系列有害后果。RN...     

Lethal perinatal osteogenesis imperfecta due to the substitution of arginine for glycine at residue 391 of the alpha 1(I) chain of type I collagen

A baby with the lethal perinatal form of osteogenesis imperfecta was shown to have a structural defect in the alpha 1(I) chain of type I procollagen. Normal and mutant alpha 1(I) CB8 cyanogen bromide peptides, from the helical part of the alpha 1(I) chains, were purified from bone. Amino acid sequencing of tryptic peptides derived from the mutant alpha 1(I) CB8 peptide showed that the glycine residue at position 391 of the alpha 1(I) chain had been replaced by an arginine residue. This substitution accounted for the more basic charged form of this peptide that was observed on two-dimensional electrophoresis of the collagen peptides obtained from the tissues. The substitution was associated with increased enzymatic hydroxylation of lysine residues in the alpha 1(I) CB8 and the adjoining CB3 peptides but not in the carboxyl-terminal CB6 and CB7 peptides. This finding suggested that the sequence abnormality had interfered with the propagation of the triple helix across the mutant region. The abnormal collagen was not incorporated into the more insoluble fraction of bone collagen. The baby appeared to be heterozygous for the sequence abnormality and as the parents did not show any evidence of the defect it is likely that the baby had a new mutation of one allele of the pro-alpha 1(I) gene. The amino acid substitution could result from a single nucleotide mutation in the codon GGC (glycine) to produce the codon CGC (arginine).     

Interferon-alpha and antisense K-ras RNA combination gene therapy against pancreatic cancer

Interferon alpha (IFN-alpha) is used worldwide for the treatment of a variety of cancers. For pancreatic cancer, recent clinical trials using IFN-alpha in combination with standard chemotherapeutic drugs showed some antitumor activity of the cytokine, but the effect was not significant enough to enlist pancreatic cancer as a clinically effective target of IFN-alpha. In general, an improved therapeutic effect and safety are expected for cytokine therapy when given in a gene therapy context, because the technology would allow increased local concentrations of this cytokine in the target sites. In this study, we first examined the antiproliferative effect of IFN-alpha gene transduction into pancreatic cancer cells. The expression of IFN-alpha effectively induced growth suppression and cell death in pancreatic cancer cells, an effect which appeared to be more prominent when compared with other types of cancers and normal cells. Another strategy we have been developing for pancreatic cancer targets its characteristic genetic aberration, K-ras point mutation, and we reported that the expression of antisense K-ras RNA significantly suppressed the growth of pancreatic cancer cells. When these two gene therapy strategies are combined, the expression of antisense K-ras RNA significantly enhanced IFN-alpha-induced cell death (1.3- to 3.5-fold), and suppressed subcutaneous growth of pancreatic cancer cells in mice. Because the 2',5'-oligoadenylate synthetase/RNase L pathway, which is regulated by IFN and induces apoptosis of cells, is activated by double-strand RNA, it is plausible that the double-strand RNA formed by antisense and endogenous K-ras RNA enhanced the antitumor activity of IFN-alpha. This study suggested that the combination of IFN-alpha and antisense K-ras RNA is a promising gene therapy strategy against pancreatic cancer.     

Antisense oligonucleotide inhibition of encephalomyocarditis virus RNA translation

We report the inhibition of encephalomyocarditis virus (EMCV) RNA translation in cell-free rabbit reticulocyte lysates by antisense oligonucleotides (13-17-base oligomers) complementary to (a) the viral 5' non-translated region, (b) the AUG start codon and (c) the coding sequence. Our results demonstrate that the extent of translation inhibition is dependent on the region where the complementary oligonucleotides bind. Non-complementary and 3'-non-translated-region-specific oligonucleotides had no effect on translation. A significant degree of translation inhibition was obtained with oligonucleotides complementary to the viral 5' non-translated region and AUG initiation codon. Digestion of the oligonucleotide:RNA hybrid by RNase H did not significantly increase translation inhibition in the case of 5'-non-translated-region-specific and initiator-AUG-specific oligonucleotides; in contrast, RNase H digestion was necessary for inhibition by the coding-region-specific oligonucleotide. We propose that (a) 5'-non-translated-region-specific oligonucleotides inhibit translation by affecting the 40S ribosome binding and/or passage to the AUG start codon, (b) AUG-specific oligonucleotides inhibit translation initiation by inhibiting the formation of an active 80S ribosome and (c) the coding-region-specific oligonucleotide does not prevent protein synthesis because the translating 80S ribosome can dislodge the oligonucleotide from the EMCV RNA template.     

Preparation of Stereoregulated Antisense Oligodeoxyribonucleoside Phoshorothioate and Interaction with its Complementary DNA And RNA

Abstract

Diastereoisomcric specificity of oligodeoxyribonucleoside phosphorothioate (OPT) in DNA/OPT and RNA/OPT hybrid formation was investigated. The difference in the configuration between RRRR and SSSS was reflected in the conformation and the stability of the DNA/OPT and RNA/OPT hybrids. Therefore, findings of this report rationalize the antisense effect by non-stereoregulated OPT and the difference of diastereoisomerism in susceptibility to RNase H.

     

Predicting the clinical lethality of osteogenesis imperfecta from collagen glycine mutations

Osteogenesis imperfecta (OI), or brittle bone disease, often results from missense mutation of one of the conserved glycine residues present in the repeating Gly-X-Y sequence characterizing the triple-helical region of type I collagen. A composite model was developed for predicting the clinical lethality resulting from glycine mutations in the alpha1 chain of type I collagen. The lethality of mutations in which bulky amino acids are substituted for glycine is predicted by their position relative to the N-terminal end of the triple helix. The effect of a Gly --> Ser mutation is modeled by the relative thermostability of the Gly-X-Y triplet on the carboxy side of the triplet containing the substitution. This model also predicts the lethality of Gly --> Ser and Gly --> Cys mutations in the alpha2 chain of type I collagen. The model was validated with an independent test set of six novel Gly --> Ser mutations. The hypothesis derived from the model of an asymmetric interaction between a Gly --> Ser mutation and its neighboring residues was tested experimentally using collagen-like peptides. Consistent with the prediction, a significant decrease in stability, calorimetric enthalpy, and folding time was observed for a peptide with a low-stability triplet C-terminal to the mutation compared to a similar peptide with the low-stability triplet on the N-terminal side. The computational and experimental results together relate the position-specific effects of Gly --> Ser mutations to the local structural stability of collagen and lend insight into the etiology of OI.     

Substitution of cysteine for glycine within the carboxyl-terminal telopeptide of the alpha 1 chain of type I collagen produces mild osteogenesis imperfecta

We have characterized a mutation that produces mild, dominantly inherited osteogenesis imperfecta. Half of the alpha 1 (I) chains of type I collagen synthesized by cells from an affected individual contain a cysteine residue in the 196-residue carboxyl-terminal cyanogen bromide peptide of the triple-helical domain (Steinmann, B., Nicholls, A., and Pope, F. M. (1986) J. Biol. Chem. 261, 8958-8964). Unexpectedly, sequence determined from a proteolytic fragment of the alpha 1 (I) chain derived from procollagen molecules synthesized in the presence of both [3H]proline and [35S]cysteine indicated that the cysteine is located at the third residue carboxyl-terminal to the triple-helical domain, normally a glycine. The nucleotide sequence of a fragment amplified from genomic DNA confirmed the location of the cysteine residue and showed that the mutation was a single nucleotide change in one COL1A1 allele. This represents a new class of mutations, point mutations outside the triple-helical domain of the chains of type I collagen, that produce the osteogenesis imperfecta phenotype.     

Molecular requirements for degradation of a modified sense RNA strand by Escherichia coli ribonuclease H1

The structural requirements for DNA/RNA hybrids to be suitable substrates for RNase H1 are well described; however the tolerance level of this enzyme towards modifications that do not alter the duplex conformation is not clearly understood, especially with respect to the sense RNA strand. In order to investigate the molecular requirements of Escherichia coli RNase H1 (termed RNase H1 here) with respect to the sense RNA strand, we synthesized a series of oligonucleotides containing 2'-deoxy-2'-fluoro-beta-D-ribose (2'F-RNA) as a substitute for the natural beta-D-ribose sugars found in RNA. Our results from a series of RNase H1 binding and cleavage studies indicated that 2'F-RNA/DNA hybrids are not substrates of RNase H1 and ultimately led to the conclusion that the 2'-hydroxyl moiety of the RNA strand in a DNA/RNA hybrid is required for both binding and hydrolysis by RNase H1. Through the synthesis of a series of chimeric sense oligonucleotides of mixed RNA and 2'F-RNA composition, the gap requirements of RNase H1 within the sense strand were examined. Results from these studies showed that RNase H1 requires at least five or six natural RNA residues within the sense RNA strand of a hybrid substrate for both binding and hydrolysis. The RNase H1-mediated degradation patterns of these hybrids agree with previous suggestions on the processivity of RNase H1, mainly that the binding site is located 5' to the catalytic site with respect to the sense strand. They also suggest, however, that the binding and catalytic domains of RNase H1 might be closer than has been previously suggested. In addition to the above, physicochemical studies have revealed the thermal stabilities and relative conformations of these modified heteroduplexes under physiological conditions. These findings offer further insights into the physical binding and catalytic properties of the RNase H1-substrate interaction, and have been incorporated into a general model summarizing the mechanism of action of this unique enzyme.     

Mutations in collagen genes: causes of rare and some common diseases in humans

More than 70 mutations in the two structural genes for type I procollagen (COL1A1 and COL1A2) have been found in probands with osteogenesis imperfecta, a heritable disease of children characterized by fragility of bone and other tissues rich in type I collagen. The mutations include deletions, insertions, RNA splicing mutations, and single-base substitutions that convert a codon for glycine to a codon for an amino acid with a bulkier side chain. With a few exceptions, the most severe phenotypes of the disease are explained largely by synthesis of structurally defective pro alpha chains of type I procollagen that either interfere with the folding of the triple helix or with self-assembly of collagen into fibrils. The results emphasize the extent to which the zipperlike folding of the collagen triple helix and the self-assembly of collagen fibrils depend on the principle of nucleated growth whereby a few subunits form a nucleus and the nucleus is then propagated to generate a large structure with a precisely defined architecture. The principle of nucleated growth is a highly efficient mechanism for the assembly of large structures, but biological systems that depend extensively on nucleated growth are highly vulnerable to mutations that cause synthesis of structurally abnormal but partially functional subunits. Recently, several mutations in three other collagen genes (COL2A1, COL3A1, and COL4A5) have been found in probands with genetic diseases involving tissues rich in these collagens. Most of the probands have rare genetic diseases but a few appear to have phenotypes that are difficult to distinguish from more common disorders such as osteoarthritis, osteoporosis, and aortic aneurysms. Therefore, the results suggest that mutations in procollagen genes may cause a wide spectrum of both rare and common human diseases.