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Human Molecular Genetics Advance Access originally published online on October 27, 2005
Human Molecular Genetics 2005 14(23):3723-3740; doi:10.1093/hmg/ddi403
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Consequences of mutations in the non-coding RMRP RNA in cartilage-hair hypoplasia

Pia Hermanns1,5, Alison A. Bertuch3, Terry K. Bertin1, Brian Dawson1,2, Mark E. Schmitt4, Chad Shaw1, Bernhard Zabel5 and Brendan Lee1,2,*

1Department of Molecular and Human Genetics, 2Howard Hughes Medical Institute and 3Department of Pediatrics, Hematology/Oncology Section, Baylor College of Medicine, Houston, TX 77030, USA, 4Department of Biochemistry and Molecular Biology, Upstate Medical University, Syracuse, NY 13210, USA and 5Children's Hospital, University of Mainz, Mainz D-55101, Germany

* To whom correspondence should be addressed at: Department of Molecular and Human Genetics, Howard Hughes Medical Institute, Baylor College of Medicine, One Baylor Plaza Room 635E, Mail Stop 225, Houston, TX 77030, USA. Tel: +1 7137988835; Fax: +1 7137985168; Email: blee{at}bcm.tmc.edu

Received August 10, 2005; Accepted October 19, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Cartilage-hair hypoplasia (CHH), also known as metaphyseal chondrodysplasia McKusick type (OMIM no. 250250 [OMIM] ), is an autosomal recessive, multi-systemic disease characterized by disproportionate short stature, fine and sparse hair, deficient cellular immunity and a predisposition to malignancy. It is caused by mutations in RMRP, the RNA component of the ribonucleoprotein complex RNase MRP, and, thus, CHH represents one of few Mendelian disorders caused by mutations in a nuclear encoded, non-coding RNA. While studies in yeast indicate that RMRP contributes to diverse cellular functions, the pathogenesis of the human condition is unknown. Studies of our CHH patient cohort revealed mutations in both the promoter and the transcribed region of RMRP. While mutations in the promoter abolished transcription in vitro, RMRP RNA levels in patients with transcribed mutations were also decreased suggesting an unstable RNA. RMRP mutations introduced into the yeast ortholog, NME1, exhibited normal mitochondrial function, chromosomal segregation and cell cycle progression, while a CHH fibroblast cell line exhibited normal mitochondrial content. However, the most commonly found mutation in CHH patients, 70A>G, caused an alteration in ribosomal processing by altering the ratio of the short versus the long form of the 5.8S rRNA in yeast. Transcriptional profiling of CHH patient RNAs showed upregulation of several cytokines and cell cycle regulatory genes, one of which has been implicated in chondrocyte hypertrophy. These data suggest that alteration of ribosomal processing in CHH is associated with altered cytokine signalling and cell cycle progression in terminally differentiating cells in the lymphocytic and chondrocytic cell lineages.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Cartilage-hair hypoplasia (CHH), also known as metaphyseal chondrodysplasia McKusick type (OMIM no. 250250 [OMIM] ), was first described in the Amish by Victor McKusick (1Go). It is an autosomal recessive disorder characterized by skeletal involvement with short stature, together with variable features like blond fine sparse hair, and defective cellular immunity affecting T-cell mediated responses (2Go). Patients may have severe combined immunodeficiency requiring bone marrow transplantation or they may be asymptomatic (3Go,4Go). Gastrointestinal dysfunction (5Go) such as malabsorption or Hirschsprung's disease is frequently observed (6Go). A predisposition to certain cancers, primarily lymphomas, has been reported as well (2Go,7Go). Shortening of tubular bones is evident at birth, with the metaphyses widened, scalloped and irregularly sclerotic. These metaphyseal changes are usually more severe in the knee region than in the proximal femora helping to distinguish CHH from other metaphyseal chondrodysplasias (8Go). The incidence of CHH in the Amish is 1.5 in 1000 births; and in Finland, it is 1 in 18 000–23 000 live births. CHH was mapped to 9p13 by linkage analysis (9Go) and Ridanpaa et al. (10Go) found causative mutations in the RMRP gene (OMIM no. 157660 [OMIM] ) in most of the CHH cases studied. The worldwide emerging mutation spectrum in CHH includes the major 70A>G transition mutation with an ancient founder origin established in Finland (11Go–14Go).

RMRP is the RNA component of the RNase MRP (ribonuclease mitochondrial RNA processing) complex. It is an untranslated, intronless gene transcribed by the DNA dependent RNA polymerase III (RNA PolIII). The human RMRP transcript is 267 bases long and the promoter region contains several putative promoter elements, a TATA box, a proximal sequence element (PSE), a SP1 and oct-1 binding elements. At the 3' end is a RNA PolIII stop signal with a run of five deoxythymidines. It is encoded in the nucleus (15Go) but the complex is localized primarily in the nucleolus and to a lesser extent in the mitochondria (16Go,17Go). The sequence of the RMRP transcript is highly conserved among a variety of different species, including human, mouse, rat, cow, Xenopus, yeast, Arabidopsis and tobacco (18Go). The length of the transcript varies among different species. Secondary structure models for RMRP reveal a complex structure, the core of which is required for assembly and function of the ribonucleoprotein complex (19Go,20Go). So far, in humans, ten proteins have been identified as part of this ribonucleoprotein complex (21Go).

In Saccharomyces cerevisiae, the RMRP ortholog NME1 (for nuclear mitochondrial endonuclease 1) is an essential gene required for viability (22Go). Extensive characterization of the gene has revealed that deletion of the least conserved portion (nt 186–211 forming the P8 hairpin) has no phenotypic effect (23Go). Studies in yeast have attributed multiple functions to this ribonucleoprotein complex. It is involved in mitochondrial DNA replication by cleaving the RNA that primes mitochondrial DNA replication (15Go,24Go). Some nme1 mutants exhibit a delay in the progression of the cell cycle at the end of mitosis in association with morphological changes. These mutants arrest in the late cycle of mitosis as large budded cells with dumbbell-shaped nuclei and extended spindles (25Go). One reason for the cell cycle delay in these mutants might be the increased level of CLB2 (B-type cyclin) mRNA. Normally, the RNase MRP complex cleaves the 5' UTR of CLB2 mRNA. That in turn causes a rapid degradation of CLB2 mRNA and efficient cell cycle progression (26Go). RNase MRP also plays a role in processing of ribosomal RNAs (27Go,28Go). In yeast, it cleaves pre-ribosomal RNA at the A3-site and promotes the production of the short form of the 5.8S rRNA. In addition to these multiple cellular roles, the functional analysis of the RNase MRP endoribonuclease is further complicated by the fact that eight proteins of the complex are shared with a related ribonucleoprotein, called RNase P. RNase P is also a endoribonuclease but it is mainly involved in tRNA precursors maturation (29Go). In yeast, two RNase MRP specific proteins have been identified, Snm1 (30Go) and most recently Rmp1 (31Go).

While mutations in RNAs can cause disease, this has been primarily restricted to mitochondrial disorders. An exception is the human telomerase RNA (hTR). Mutations affecting this transcript are responsible for the rare autosomal dominant form of dyskeratosis congenita (CD) (32Go). CD is a progressive bone marrow failure syndrome that is characterized by abnormal skin pigmentation, nail dystrophy and mucosal leukoplakia.

As RMRP is not translated into a protein, it is not obvious how mutations may affect its putative catalytic action as part of a larger ribonucleoprotein complex or in as yet uncharacterized functions. To answer this question, we have analyzed the expression pattern of RMRP during development and the effect of RMRP promoter mutations on RNA PolIII transcription. Furthermore, we analyzed the effects of base pair substitutions found in CHH patients that were localized in conserved regions of the RNA using S. cerevisiae as a model organism. Finally, we have correlated the findings of these experiments in humans by transcriptional profiling of CHH patient RNAs.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
RMRP is ubiquitously expressed during development
RMRP is ubiquitously expressed in Xenopus laevis throughout development. It is more highly expressed in those tissues containing large numbers of mitochondria (33Go). To determine whether the expression pattern of RMRP correlated with the distribution of affected tissues in CHH patients, we determined the temporal spatial pattern of RMRP expression during mouse development. We found that Rmrp is ubiquitously expressed at E10.5, E11.5, E12.5, E13.5, E14.5 and E17.5 during mouse embryogenesis by RNA in situ hybridization. Interestingly, Rmrp is more strongly expressed in the hypertrophic chondrocytes and pericondrium than in the zone of proliferating chondrocytes, correlating with the primary feature in human CHH as a metaphyseal dysplasia (Fig. 1A) (data not shown). RMRP is also ubiquitously expressed in all human adult tissues tested, though less so in skin and pancreas when compared with the GAPDH expression pattern that served as a RNA loading control (Fig. 1B).



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  		Figure 1. Expression pattern of the Rmrp gene. (A) In situ hybridization of an E15.5 mouse embryo. (B) Adult human multiple tissue northern blot. Rmrp is ubiquitously expressed in human and mouse. H, hypertrophic chondrocytes.

 
RMRP promoter duplications result in reduced expression
In vitro transcription studies have shown that an 84 bp 5' flanking region of the human RMRP promoter sequence is sufficient for RMRP transcription. When this 84 bp fragment and a 737 bp upstream sequence were injected into frog oocytes, the 737 bp promoter showed a stronger transcription efficiency than the 84 bp promoter in vivo, whereas no difference was observed in vitro (34Go).

To elucidate the effect of the RMRP promoter duplication found in CHH patients on RMRP transcription, the human RMRP promoter was further characterized in transfection studies in vitro. In RNA PolIII promoters, the TATA box is usually located at a fixed distance downstream of the PSE element (35Go–38Go). Because, promoters of RNA PolIII transcribed genes are usually very short, e.g. the human U6 promoter is 265 bp long (39Go) and the mouse U6 promoter 355 bp long (40Go), we studied two putative RMRP promoter sequences 352 and 841 bp in length (Fig. 2B). The putative 352 bp minimal promoter contains the TATA signal, PSE, oct-1 and the SP1 binding elements (Fig. 2A). To evaluate the strength of RMRP promoter variants, we inserted a short hairpin RNA (shRNA) directed against luciferase (shRNAluc) under the control of the RMRP promoter (RMRPshRNAluc). Hence, promoter strength can be correlated with the degree of downregulation of luciferase expression in cells co-transfected with RMRPshRNAluc and a luciferase expression plasmid. The U6 promoter driving the shRNAluc serves as a positive control for this assay (U6luc), whereas the U6 promoter (U6) or shLuc (luc) alone serve as negative controls for this assay.





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  		Figure 2. Human RMRP gene and promoter structure. (A) The RMRP transcript consists of 267 bp. The putative promoter region contains a TATA box, proximal sequence element (PSE), an Oct-1 binding site and a SP1 binding site. (B) The U6 promoter expressing the shRNA targeted against the luciferase gene serves as positive control. Two different lengths of the RMRP promoter were amplified: 352 and 841 bp. The U6 promoter without the shRNA and the shRNA without any promoter serve as negative controls and indicate the baseline luciferase activity without inhibition. The 352 bp RMRP promoter showed greater down-regulation of the luciferase activity than both the U6 and the 841 bp RMRP promoter. (C) The promoter activity of the mutant RMRP promoter is significantly decreased when compared with the RMRP wt promoter. The decrease in promoter activity results in an increase of luciferase activity.

 
As shown in Figure 2B, both RMRP promoters constructs tested resulted in the down-regulation of luciferase gene expression when compared with U6 promoter (U6) or the shRNAluc (luc) alone. The degree of down-regulation for each was comparable to that resulting from U6 promoter-driven shRNAluc expression. The 352 bp promoter seems to be stronger than the 841 bp promoter (P<0.002). This result suggests that the 352 bp sequence upstream of the RMRP transcription start site is sufficient to drive RNA expression and also might be stronger than the previously described 737 bp promoter.

We next investigated the impact of the promoter duplications identified in CHH patients on RMRP expression in this assay. The mutant promoters of CHH patient nos 4, 12 and 16 were amplified with the same primer pair as the wild-type (wt) 352 bp RMRP promoter. CHH no. 4 had a -11_-25dup, CHH no.12 a -6_-25dup, and CHH no. 16 a -15_-24dup, respectively. As shown in Figure 2C, the activities of the mutant RMRP promoters were reduced relative to the wt (–352 bp) promoter and were associated with lower shRNA expression and higher luciferase activities. Interestingly, the promoter activity was not completely abolished, as the measured luciferase activity was still diminished relative to the U6 promoter and shRNAluc alone controls. This suggests that the promoter duplications found in CHH patients are hypomorphic alleles that lead to decreased but not abolished RMRP transcription in vitro at least as defined in this assay.

Lobo and Hernandez (37Go) reported that the specificity of the RNA PolIII promoter can be converted to a RNA polymerase II promoter (RNA PolII) and vice versa by alterations in the distance between the TATA box and the transcription start site or by generating a TATA box (37Go). To test whether the promoter duplications found in the CHH cohort might convert the RNA PolIII-specific promoter to a RNA PolII-specific promoter, the same promoters as mentioned earlier were cloned upstream of the RNA PolII transcribed luciferase reporter gene. Transfection of these constructs into cos7 and HeLa cells showed that they had no activity, suggesting that CHH promoter duplications do not convert the RNA PolIII-specific promoter into a RNA PolII-specific promoter (data not shown).

RMRP expression level in CHH patients
To determine the effects of promoter duplications as well as single base pair substitution mutations on RMRP transcripts in vivo, we performed real-time RT–PCR to analyze the expression level of RMRP in three CHH patients with previously described mutations and six unaffected controls. Leukocytes from CHH patient nos 2 (compound heterozygous for 89C>G; 124C>T), 8 (homozygous for 70A>G) and 16 (with a promoter duplication -23_-14dup; and a 180G>A transition) as well as from sex and ethnicity matched controls were obtained. In addition, we collected samples from CHH no. 2 almost a year later to perform a longitudinal comparison. The RNA expression levels were normalized using the constitutively expressed gene HPRT as a reference (Fig. 3). Interestingly, RMRP RNA level is decreased in all three patients tested irrespective of the nature of mutation detected in these patients. These data suggest that mutations even in the transcribed region may affect transcription efficiency and/or RNA stability in vivo.



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  		Figure 3. RMRP RNA expression level in two CHH patients. Quantitative real-time RT–PCR was performed on total RNA isolated from CHH patient leukocytes. From patient CHH no. 2, two samples were obtained (October 2003 and July 2004). CHH no. 2 is compound heterozygous for 89C>G and 124C>T. CHH no. 8 is homozygous for the 70A>G base pair substitution. CHH no. 16 is compound heterozygous for the promoter duplication -14_-23dup and the base pair substitution 180G>A. The RMRP RNA level in leukocytes is approximately 6- to 7-fold down-regulated in both patients when compared with six control samples.

 
The cognate 70A>G RMRP mutation in the yeast ortholog NME1 alters rRNA processing
To study the functional consequences of RMRP mutations, we introduced mutations we found in CHH patients that were located at evolutionarily conserved positions from yeast to human into the yeast ortholog NME1 (Fig. 4A). This included the most frequently found mutation 70A>G (yeast84A>G), as well as 124C>T (yeast168C>T), 180G>A (yeast216G>A) and 262G>C (yeast331G>T) into the NME1 gene (Fig. 3A and B). In addition, we introduced a 2 bp deletion mutant (yeast267_268{Delta}AT) as a positive control, as previous random mutagenesis studies showed that many deletion mutants exhibited altered phenotypes (23Go). As the yeast NME1 gene is 72 bp longer than RMRP, the human mutation nomenclature was used to designate mutant strains. Figure 5 shows that the result of viability testing of haploid yeast strains bearing the mutant alleles. Each of the nme1 point mutant alleles was able to sustain growth in the absence of wild-type NME1, and, therefore, these mutations did not abolish the essential function of NME1 in yeast. In contrast, a strain bearing the 2 bp deletion had severely diminished growth in the absence of wild-type NME1, indicating its essential function was impaired. This supports the hypothesis that RMRP might be essential in humans and null alleles might be incompatible with life. Until now, no deletions of the entire RMRP gene have been reported in humans.



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  		Figure 4. RMRP sequence and gene structure. (A) RMRP is highly conserved among a variety of different species. Mutations that have been introduced into the yeast ortholog NME1 are highlighted [modified from Schmitt et al. (1992)]. (B) The positions of the human mutations in this complex are highlighted in a putative structure of the RNase MRP complex [modified from Welting et al. (24Go)].

 


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  		Figure 5. Viability tests of point mutations introduced into the NME1 gene. The experiment was performed in duplicate. To control for cell numbers the yeast strains were grown on –leu-ura medium to maintain the wt NME1 plasmid (left). The wt NME1 plasmid is shuffled out by growth on –leu/5FOA (right). The introduced nme1 point mutations grew equally well when compared with the wt control. The 2 bp-del mutant was lethal, which is indicated by a lack of growth on –leu 5-FOA.

 
A well-described function of NME1 is the processing of the pre-rRNA (27Go,28Go). To determine whether the human mutations could affect this function, we examined the steady state levels of the 5.8S rRNAs in yeast strains bearing the mutant nme1 allele (Fig. 6). The 5S rRNA and the tRNAs served as loading controls. As previously reported, there was a higher concentration of the 5.8SS rRNA (short form of the 5.8S rRNA) than the 5.8 SL rRNA (long form of the 5.8S rRNA) in wild-type cells: this ratio is usually 10:1. However, two of the five nme1 mutant strains exhibit an alteration in this ratio. The nme170A>G mutant strain had a ratio of approximately 2 : 3 of the short versus the long form of the 5.8S rRNA, whereas the 2 bp deletion mutant lacked the short form of the 5.8S rRNA completely. This latter mutant showed only minimal growth on non-fermentable carbon sources, indicating that most of its function is abolished. These data suggest that the human mutations including the common 70A>G variant can alter ribosomal RNA processing and that this is a highly conserved function for RMRP from yeast to humans. This is also the first example of a yeast mutant completely lacking the 5.8SS rRNA that still remains viable.



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  		Figure 6. Pre-ribosomal processing of mutant nme1 strains. Two micrograms of total RNA isolated from diploid yeast strains was separated on a 6% PAA, 7 M urea gel. nme170A>G, nme12 bp-del showed a defect in pre-ribosomal processing resulting in an alteration of the ratio 5.8SL:5.8SS rRNA, which is usually 1 : 10. The ratio of the nme170A>G was changed to 2:3, whereas the nme12 bp-del lacked the short form of the 5.8S rRNA completely.

 
Because RMRP has been shown to play a role in mitochondrial DNA replication (15Go), cell cycle progression at the end of mitosis (25Go), and chromosomal segregation during mitosis (41Go), we examined mitochondrial function, cell cycle progression and chromosome segregation in the nme1 mutant strains. However, each of these was indistinguishable from the wild-type control (data not shown).

Transcriptional profiling of human CHH leukocytes
To gain insight into the pathogenesis of human CHH caused by mutations in the RMRP gene, we performed transcriptional profiling of human leukocytes using the Affymterix Human Genome U133 chip set. For this study, two CHH patients were available; the first patient was compound heterozygous for an 89C>G transversion and a 124C>T transition, and the second patient was homozygous for the most frequently found 70A>G transition. The transcription profile was compared to two controls matched for ethnicity and sex. The data were normalized using GCRMA from the statistical analysis software, Bioconductor. The genes that were differentially expressed in the two patients relative to the normal controls are listed in Tables 1 and 2, respectively.


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  		Table 1. List of genes that are up regulated in CHH patients
 

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  		Table 2. Genes that are down regulated in CHH patients
 
Ninety-nine genes are commonly 2-fold or more up-regulated in both patients when compared with both controls. Forty-seven (47.5%) of these genes play a role in the immune system. Sixteen (16.2%) play a role in cell cycle regulation either via cell growth or apoptosis. Sixteen of the ninety-nine genes are involved in signal transduction, five of the sixteen genes belong to the family of G-coupled receptors. Five of them are transcription factors (Table 1).

Thirty-eight genes were 2-fold or more down-regulated in both patients when compared with unaffected controls. Seventeen genes (44.7%) play a role in the immune system. Seven genes are involved in cell cycle regulation and apoptosis. Nine genes (23.7%) are involved in signal transduction. Six genes (15.8%) have enzymatic activities. Four genes (10.5%) have an unknown function. Interestingly, no transcription factors were 2-fold or more down-regulated in this experiment.

PF4V1, IL8, CCR3 and STAT1 up-regulation was confirmed by real-time quantitative RT–PCR amplification (Fig. 7). There was not unexpectedly inter-patient variability, especially in PF4V1. The up-regulation of G0S2, AMF4, LRAP and HLA-DRB1 as well as the down-regulation of INDO has also been confirmed via quantitative RT–PCR of the patients (data not shown). Moreover, a longitudinal comparison of the same patient that was compound heterozygous for 89C>G and 124C>T showed up-regulation of seventy-two of the ninety-nine genes again when using the Affymetrix Human Genome 2.0 plus chip a year later in an independent assay. Also 24 genes previously found to be down-regulated were confirmed in this experiment.



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  		Figure 7. Verification of microarray data via quantitative real-time-RT–PCR. HPRT was used as a control housekeeping gene to normalize for expression level. The up-regulation of the genes tested correlates with the microarray analysis.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
To link the CHH pathogenesis with RMRP gene structure and transcript function in cellular and subcellular processes, we have generated genomic and functional data from in vitro and in vivo studies.

In eukaryotes, the RNA PolIII transcribes structural or catalytic RNAs that are usually shorter than 400 bp. There are three different types of RNA PolIII promoters. The type 1 promoter contains an intragenic internal control region consisting of an A box, an intermediate element and a C box. This is exemplified by the 5S rRNA gene (42Go). The type 2 promoter is also intragenic and consists of an A box and a B box and is exemplified by typical tRNAs (43Go–46Go). The type 3 RNA PolIII promoter is external. The core promoter consists of a PSE, element with a TATA box that is located at a fixed distance downstream of the PSE element (35Go,38Go). The PSE element on its own is sufficient for snRNA transcription by RNA PolIII (37Go,47Go).

The RMRP gene is transcribed by RNA PolIII and sequence elements of a type 3 promoter are present (48Go). The core sequence elements such as the PSE element and a TATA box can be found upstream of the transcription initiation site of the RMRP gene. In addition, transcription factor binding sites like a SP1 binding element and an octamer (recruits the transcription factor Oct-1) sequence could serve as distal sequence elements (DSE) to enhance the transcription of RMRP similar to the DSE element of the human U6 snRNA gene (49Go). Our in vitro studies show that 352 bp upstream of the transcription start site of the RMRP gene represents a region sufficient for activating transcription. The promoter duplications identified in CHH patients decrease the transcription activity in vitro, but they do not abolish transcription completely. This is consistent with our real-time RT–PCR data of patient leukocytes and correlates well with the yeast studies in that the transcribed human mutations cannot completely abolish the essential function required for viability. Hence, CHH mutant alleles are likely hypomorphic, as null alleles are likely incompatible with life.

Because RMRP is highly conserved among a variety of species, we studied the functional consequences of CHH mutations occurring at conserved positions between humans and yeast. Because RMRP may play a role in priming of mitochondrial DNA replication, loss of RNase MRP function might result in mitochondrial depletion.

However, our yeast studies and studies on human CHH fibroblasts do not support a significant defect in mitochondrial function or DNA content. It may be that other RNases compensate for RNase MRP in mitochondria (50Go) or mutations that affect the mitochondrial function of RNase MRP may be lethal.

Mutations in protein components of RNase MRP result in cell cycle delay in yeast. These mutants have an exit-from-mitosis defect (25Go). This defect is caused by an increase in Clb2 (B-type cyclin) levels caused by an increase in CLB2 mRNA stability. Normally, CLB2 mRNA levels decrease rapidly when the cell completes mitosis. RNase MRP cleaves the 5'-UTR of CLB2 causing rapid degradation of the mRNA by the Xrn1 nuclease (5'->3' exoribonuclease), but not by exosomes that are usually responsible for mRNA degradation (26Go). In this study, no cell cycle phenotype could be observed in the nme1mutant strains. There was no detectable change in the cell cycle progression under normal growth conditions or after stress induction through {gamma}-irradiation. Interestingly, we also did not see significant differences in human CLB2 mRNA in patient versus control white blood cells.

In yeast, RNase MRP plays a role in pre-ribosomal RNA processing. It cleaves at the A3-site that in turn leads to the production of the 5.8SS rRNA. Two of the five nme1mutant strains show an alteration in the normal ratio of the 5.8SL rRNA versus 5.8SS rRNA. Interestingly, the CHH 70A>G variant is just outside of the P4 domain. This position is highly conserved among a variety of species. In yeast, it can form a 9 bp duplex with the 5.8S rRNA (51Go). This mutation might, therefore, affect the maturation of the 5.8S rRNA that is reflected by the change of the ratio of the long versus short form of the 5.8S rRNA. The 70A>G might cause a subtle alteration in the local structure of the active site, and hence, lead to less efficient cleavage of the 5.8S rRNA. Similarly, the 2 bp deletion occurs in a stretch of nucleotides parallel to the P4 domain. This may have an even more severe affect on active site binding and processing of 5.8S rRNA. The reason for a lack of effect of the other point mutations is unclear, though they exist in predicted stem loop structures distal to the P4 domain. One possibility is that divergent functions may have arisen in mammals that require these distal structures that are non-essential in yeast. Alternatively, the yeast RMRP catalytic function may be less sensitive to these substitutions. In fact, while these substitutions affect the local stem loop structure in the yeast RNA, they do not appear to affect the P4 domain structure locally.

To gain insight into how such a defect might be translated in the human situation, we analyzed the transcriptional profile of CHH leukocytes. Interestingly, we saw up-regulation of cytokine family members and cell cycle regulatory genes, which is consistent with the in vitro studies performed on T-cells isolated from CHH patients (4Go). Our longitudinal analysis in one patient suggests that these changes persist over time and reflect the underlying RMRP genotype rather than environmental factors at the time of sampling. Some of the up-regulated genes may reflect an underlying common pathogenic mechanism in chondrocytes and lymphocytes. For example, up-regulation of IL8 and GRO-{alpha} is observed in osteoarthritic cartilage and is correlated with activation of the p38/MAPK pathway. This activation is also known to promote hypertrophic chondrocyte differentiation and apoptosis, and has been described as one of the cellular alterations in osteoarthritis (52Go). Altered hypertrophic chondrocyte differentiation would correlate with the primary clinical finding of metaphyseal chondrodysplasia. In addition, transglutaminase 2 is stimulated. TG2 among other transglutaminases modulates differentiation and calcification of chondrocytes and is a mediator of tissue repair (53Go).

The expression level of IL8 and IL6 are even higher in rheumatoid arthritis cartilage (54Go). In addition, it has been shown that IL8 is increased in several tumors (55Go,56Go). Therefore, IL8 up-regulation may be an important biomarker of both skeletal and hematopoietic disease, as CHH patients have not only metaphyseal changes but also an increased risk for developing lymphomas. Another candidate gene in the pathogenesis of CHH is the putative lymphocyte G0/G1 switch gene 2 (G0S2). In vitro studies of T-cells of CHH patients have shown a defect in the cell cycle transition of the G0 to G1 phase (57Go). Up-regulation of G0S2 leads to an arrest of cells in the G0 phase (58Go), which is a more permanent phase of end-differentiated cells. Increased STAT1, STAT5 and p21Cip1 protein levels are observed in the pre- and hypertrophic chondrocyte zone in achondroplasia and thanaphoric dysplasia. Moreover, expression level was correlated to the severity of the disease (59Go).

The studies in yeast suggest that there might be yet other functions of RNase MRP that it may have adopted during evolution. Studies in yeast suggest that it may act as a site-specific ribonuclease to cleave CLB2 mRNA (26Go). However, we did not observe alterations in CLB2 expression levels in the microarray studies performed here. The DNA sequence might be conserved among different species but not all functions might be. Not surprisingly, not all human or yeast protein subunits that bind RMRP and NME1, respectively, have clear orthologs in different species (31Go,60Go). These data suggest that while some functions are conserved between yeast and humans, others might have diverged. RMRP mutations can affect ribosomal processing in yeast. The human mutations are likely hypomorphic in that they do not abolish essential functions. Moreover, up-regulation of specific genes including IL8 and G0S2 may help explain the common effects on T-cell proliferation, malignancy risk and altered chondrocyte hypertrophy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
RMRP expression pattern
In situ hybridization
WT mouse embryos were collected at specified time points and fixed in 4% paraformaldehyde at 4°C for 12 h. The paraffin-embedding, sectioning and in situ hybridization were carried out as described by Albrecht et al. (61Go). The Rmrp gene was amplified using total RNA isolated from an E15.5 embryo as a template in the RT–PCR reaction. A NotI linker was added to the primers PH-21-Xho and PH-22-Pst, and the PCR product was subcloned into the NotI site of pBluescript SK– (Stratagene).

Northern blot
To elucidate the expression pattern of the human RMRP gene, a human multiple tissue northern blot (RNWAY Laboratories Inc., Korea) was hybridized with a RMRP probe amplified with the primers RMRP5 and RMRP6 at an annealing temperature of 61°C. Primer sequences are available in Supplementary Material. The PCR reaction was supplemented with DMSO to a final concentration of 2%. The PCR product was labeled with random hexamers (Invitrogen). 106 CPM probe was added per milliliter of Church-buffer and hybridized over night at 68°C. The blot was then rinsed twice with 5xSSC, 0.1% SDS at 68°C for 20 min followed by two washes with 2xSSC, 0.1% SDS also at 68°C, then twice with 1xSSC, 0.1% SDS and lastly once with 0.5xSSC, 0.1% SDS. A radiographic film was exposed over night at room temperature.

Transfections
The RMRP promoter was amplified from human genomic DNA with the primers RMRP8-EcoRI and RMRP14-ClaI. The second promoter included an additional 500 bp of upstream sequence, was 841 bp in length, and was amplified with the primers RMRP8-EcoRI and RMRP9-ClaI. Primer sequences are available in Supplementary Material. The annealing temperature for the PCR reaction was 61°C for both primer pairs. The PCR products were subcloned into the EcoRI and ClaI sites of the vector pSilencer U6 (Ambion) containing an RNAi oligo targeted against the luciferase reporter gene of the vector pGL3 Control (Promega). The U6 promoter of this vector was removed with a KpnI and ApaI digest. The remaining vector was then religated. The sequence for the RNAi oligo was provided by Steve Elledge at Baylor College of Medicine in Houston, TX, USA. The shRNA was cloned into the EcoRI and HindIII sites of the vector. Cos7 cells were plated at 0.6x106 cells/well in six-well plates just before transfection. Transfections were done in triplicates using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. Luciferase and beta-galactosidase activities were assayed 48 h after transfection as described by Zhou et al. (62Go). The transfections were repeated twice in independent assays.

RNA isolation of whole blood and quantitative real-time PCR
Blood from CHH patients was collected in ACD, Solution A yellow top BD VacutainersTM (BD, Franklin Lakes, NJ, USA) after informed consent was obtained. Total RNA from whole blood was isolated using the Versagene RNA isolation kit from Gentra according to the manufacture's protocol. Four micrograms of the total RNA was incubated for 90 min in an RT-reaction mix that consisted of 5 µM random hexamers (Invitrogen), 1.8 mM dNTPs (Roche), 40 U RNase Inhibitor (Invitrogen), first strand synthesis buffer (Invitrogen), 20 mM DTT (Sigma) and 40 U Reverse Transcriptase (Invitrogen).

The LightCycler® 1.1 Instrument and reagents from Roche Applied Science (Indianapolis, IN, USA) were used for the quantitative PCR and the recommendations of the manufacturer were followed exactly. Twenty microliter reactions are prepared with dNTP concentrations of 1200 mM, MgCl2 4 mM buffer and thermostable enzyme and Sybr green as part of the proprietary mastermix. cDNA samples were assayed by fluorometry on a BMG Fluostar plate reader using Ribo-green assay (Molecular Probes). Template concentration was ascertained over multiple dilutions and normalized to 40 ng/µl of which a 1:8 dilution was used for Lightcycler amplification. Two microliters of the 1:8 dilution (12 ng) was added to each 20 µl reaction and fluorescence monitored for 45 cycles. Crossing points were determined by a second derivative algorithm intrinsic to the Lightcycler software. Primer and primer sequence information are available in Supplementary Material.

Yeast strains and studies
The genetic background for the yeast strains used in Figure 5 is YPH275: MATa/MAT{alpha}, ura3-52, lys2-80, ade2-101, trp1-{Delta}1, his3-{Delta}200, leu2-{Delta}1, CF [TRP1, SUP11, CEN4] (Source: Peter Hieter). YPH1: MATa/MAT{alpha}, nme1-{Delta}::kanR/NME1ura3-52/ura3-52, lys2-80/lys2-80, ade2-101/ade2/101, trp1-{Delta}1/trp1-{Delta}1, his3-{Delta}200/his3-{Delta}200, leu2-{Delta}1/leu2-{Delta}1, CF pPH1[NME1 URA3CEN] was generated using the PCR-based strategy described by Wach et al.(63Go). YPH1 was transformed with pPH1 (pNME1 URA3 Cen), which contains NME1 and 259 and 205 bp of upstream and downstream sequence subcloned into pYClac33 to generate YPH2. YPH2 was sporulated to generate YPH3: MATa nme1-{Delta}::kanR/NME1ura3-52, lys2-80, ade2-101, trp1-{Delta}1, his3-{Delta}200, leu2-{Delta}1, CF [TRP1, SUP11, CEN4] pPH1[NME1 URA3 CEN]. Transformation of mutant nme1 plasmids was done as described by Gietz et al. (64Go).

Viability testing was performed by growing the yeast strains in –leu-ura media to late log phase at 30°C. Ten-fold serial dilution series were plated on –leu-ura and –leu 5-FOA (Fluoroorotic acid) plates and incubated for 3 and 6 days at 30°C, respectively.

For the experiment shown in Figure 7, TLG119: Mat a/alpha ade2-1/ade2-1 ura3-52/ura3-52 leu2-3,112/leu2-3,112 LYS2/lys2-1 his3/his3 cce1::HIS3/cce1::HIS3 trp1{Delta}-1/trp1{Delta}-1 nme1{Delta}2::TRP1/nme1{Delta}2::TRP1 pMES127[URA3 NME1 CEN] was transformed with mutant nme1 plasmids and pMES127 evicted by growth on media containing 5-FOA. Strains were grown to saturation and total RNA was isolated as described by Schmitt et al. (65Go).

NME1 PCR mutagenesis
The different nme1 point mutations were introduced into pPH2, which contains NME1 and flanking sequences as described earlier subcloned into pRS415, via a two-step PCR described by Higuchi et al. (1988). The flanking primers for each mutation were NME1-3 and NME1-5. The mutant primer were 70A->G, 124C->T, 180G->A and the 2 bp deletion. Primer sequences are available in Supplementary Material.

Microarray
The total RNA from leukocytes from two CHH patients and two matched controls was isolated using the RNAeasy Midi Prep kit from QIAGEN. The quality and the concentration of the RNA were determined using an Agilent 2100 Bioanalyzer. The HG-U133A and HG-U133B oligonucleotide arrays from Affymetrix were selected for the microarray analysis. Technical replicates were performed on samples from both patient and control subjects.

Samples were labeled using the standard Affymetrix T7 oligo(dT) primer protocol. Total RNA was reverse transcribed to produce double-stranded cDNA. The cDNA product was used as a template for the in vitro transcription reaction, producing biotin-labeled cRNA. The labeled cRNA was quantified using the NanoDrop® ND-1000 instrument. 15.0 µg of the labeled cRNA was fragmented and re-checked for concentration. A hybridization cocktail containing Affymetrix spike-in controls and fragmented labeled cRNA was loaded onto a GeneChip® array. The array was hybridized overnight at 45°C with rotation at 60 r.p.m. then washed and stained with a strepavidin, R-phycoerythrin conjugate stain. Signal amplification was done using biotinylated antistreptavidin. The stained array was scanned on the Affymetrix GeneChip® Scanner 3000. The images were analyzed and quality control metrics recorded using Affymetrix GCOS software version 1.1.2.

The signals of the chips were normalized using GCRMA (Robust Multi-array Analysis) from the statistical analysis software package, Bioconductor (http://128.32.135.2/users/bolstad/ComputeRMAFAQ/ComputeRMAFAQ.html). We averaged technical replicates and then computed a two sample T-test for the patient and control data using the technical averages for each biological replicate as the input. We used the empirical Bayes methods in the R package limma to generate moderated T-statistics and P-values for differential expression between control and CHH individuals for each Affymetrix probe set. The empirical Bayes method for computing T-statistics improves power by considering each gene's variance as a sample from a population of gene variances. An annotated gene or gene ontology list was generated using Affymetrix's web site at http://www.affymetrix.com/index.affx. The list of up- and down-regulated genes was created with Limma at http://bioinf.wehi.edu.au/l.imma/ and filtered in Excel. As cut-off, a 2-fold or higher up-regulation and down-regulation, respectively, was used as the logic to generate the gene lists.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENT
 
We would like to thank Professor Andrea Superti-Furga of the Centre for Pediatrics and Adolescent Medicine, Freiburg University Hospital (Freiburg, Germany) for providing us with a CHH patient fibroblast cell line, Dr Lee-Jun C. Wong of the Molecular Diagnostic Laboratory in the Department of Molecular and Human Genetics at Georgetown University Medical Center (Washington, DC, USA) for testing mitochondrial depletion in and Professor Hans-Anton Lehr of the Department of Pathology of the Johannes Gutenberg-University Clinics of Mainz (Mainz, Germany) for the EM analysis of this CHH patient fibroblast cell line. This work was supported by grants from the NIEHS P01 ES11253 (B.L.), NICHD P01 HD22657 (B.L. subproject from D. Rimoin), the Baylor MRDDRC NIH HD024064 and NIGMS GM063798 (M.E.S).

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

  1. McKusick, V.A., Eldridge, R., Hostetler, J.A., Ruangwit, U. and Egeland, J.A. (1965) Dwarfism in the Amish. Ii. Cartilage-hair hypoplasia. Bull. Johns. Hopkins. Hosp., 116, 285–326.[Web of Science][Medline]

  2. Makitie, O., Sulisalo, T., de la Chapelle, A. and Kaitila, I. (1995) Cartilage-hair hypoplasia. J. Med. Genet., 32, 39–43.[Free Full Text]

  3. Castigli, E., Irani, A.M., Geha, R.S. and Chatila, T. (1995) Defective expression of early activation genes in cartilage-hair hypoplasia (CHH) with severe combined immunodeficiency (SCID). Clin. Exp. Immunol., 102, 6–10.[Web of Science][Medline]

  4. Makitie, O., Kaitila, I. and Savilahti, E. (1998) Susceptibility to infections and in vitro immune functions in cartilage-hair hypoplasia. Eur. J. Pediatr., 157, 816–820.[CrossRef][Web of Science][Medline]

  5. Makitie, O. and Kaitila, I. (1993) Cartilage-hair hypoplasia—clinical manifestations in 108 Finnish patients. Eur. J. Pediatr., 152, 211–217.[CrossRef][Web of Science][Medline]

  6. Makitie, O., Kaitila, I. and Rintala, R. (2001) Hirschsprung disease associated with severe cartilage-hair hypoplasia. J. Pediatr., 138, 929–931.[CrossRef][Web of Science][Medline]

  7. Makitie, O., Pukkala, E., Teppo, L. and Kaitila, I. (1999) Increased incidence of cancer in patients with cartilage-hair hypoplasia. J. Pediatr., 134, 315–318.[CrossRef][Web of Science][Medline]

  8. Spranger, J.W., Brill, P.W. and Poznanski, A. (2002) In Bone Dysplasias. An Atlas of Genetic Disorders of Skeletal Development. 2nd edn. Oxford University Press, Oxford.

  9. Sulisalo, T., van der Burgt, I., Rimoin, D.L., Bonaventure, J., Sillence, D., Campbell, J.B., Chitayat, D., Scott, C.I., de la Chapelle, A., Sistonen, P. et al. (1995) Genetic homogeneity of cartilage-hair hypoplasia. Hum. Genet., 95, 157–160.[Web of Science][Medline]

  10. Ridanpaa, M., van Eenennaam, H., Pelin, K., Chadwick, R., Johnson, C., Yuan, B., vanVenrooij, W., Pruijn, G., Salmela, R., Rockas, S. et al. (2001) Mutations in the RNA component of RNase MRP cause a pleiotropic human disease, cartilage-hair hypoplasia. Cell, 104, 195–203.[CrossRef][Web of Science][Medline]

  11. Ridanpaa, M., Sistonen, P., Rockas, S., Rimoin, D.L., Makitie, O. and Kaitila, I. (2002) Worldwide mutation spectrum in cartilage-hair hypoplasia: ancient founder origin of the major70A->G mutation of the untranslated RMRP. Eur. J. Hum. Genet., 10, 439–447.[CrossRef][Web of Science][Medline]

  12. Bonafe, L., Schmitt, K., Eich, G., Giedion, A. and Superti-Furga, A. (2002) RMRP gene sequence analysis confirms a cartilage-hair hypoplasia variant with only skeletal manifestations and reveals a high density of single-nucleotide polymorphisms. Clin. Genet., 61, 146–151.[CrossRef][Web of Science][Medline]

  13. Kuijpers, T.W., Ridanpaa, M., Peters, M., de Boer, I., Vossen, J.M., Pals, S.T., Kaitila, I. and Hennekam, R.C. (2003) Short-limbed dwarfism with bowing, combined immune deficiency, and late onset aplastic anaemia caused by novel mutations in the RMPR gene. J. Med. Genet., 40, 761–766.[Free Full Text]

  14. Nakashima, E., Mabuchi, A., Kashimada, K., Onishi, T., Zhang, J., Ohashi, H., Nishimura, G. and Ikegawa, S. (2003) RMRP mutations in Japanese patients with cartilage-hair hypoplasia. Am. J. Med. Genet. A, 123, 253–256.[Medline]

  15. Chang, D.D. and Clayton, D.A. (1987) A mammalian mitochondrial RNA processing activity contains nucleus-encoded RNA. Science, 235, 1178–1184.[Abstract/Free Full Text]

  16. Reimer, G., Raska, I., Scheer, U. and Tan, E.M. (1988) Immunolocalization of 7-2-ribonucleoprotein in the granular component of the nucleolus. Exp. Cell. Res., 176, 117–128.[CrossRef][Web of Science][Medline]

  17. Li, K., Smagula, C.S., Parsons, W.J., Richardson, J.A., Gonzalez, M., Hagler, H.K. and Williams, R.S. (1994) Subcellular partitioning of MRP RNA assessed by ultrastructural and biochemical analysis. J. Cell. Biol., 124, 871–882.[Abstract/Free Full Text]

  18. Schmitt, M.E., Bennett, J.L., Dairaghi, D.J. and Clayton, D.A. (1993) Secondary structure of RNase MRP RNA as predicted by phylogenetic comparison. FASEB J., 7, 208–213.[Abstract]

  19. Walker, S.C. and Avis, J.M. (2004) A conserved element in the yeast RNase MRP RNA subunit can participate in a long-range base-pairing interaction. J. Mol. Biol., 341, 375–388.[CrossRef][Web of Science][Medline]

  20. Lindahl, L., Fretz, S., Epps, N. and Zengel, J.M. (2000) Functional equivalence of hairpins in the RNA subunits of RNase MRP and RNase P in Saccharomyces cerevisiae. RNA, 6, 653–658.[Abstract]

  21. Welting, T.J., van Venrooij, W.J. and Pruijn, G.J. (2004) Mutual interactions between subunits of the human RNase MRP ribonucleoprotein complex. Nucleic Acids Res., 32, 2138–2146.[Abstract/Free Full Text]

  22. Schmitt, M.E. and Clayton, D.A. (1992) Yeast site-specific ribonucleoprotein endoribonuclease MRP contains an RNA component homologous to mammalian RNase MRP RNA and essential for cell viability. Genes Dev., 6, 1975–1985.[Abstract/Free Full Text]

  23. Shadel, G.S., Buckenmeyer, G.A., Clayton, D.A. and Schmitt, M.E. (2000) Mutational analysis of the RNA component of Saccharomyces cerevisiae RNase MRP reveals distinct nuclear phenotypes. Gene, 245, 175–184.[CrossRef][Web of Science][Medline]

  24. Lee, D.Y. and Clayton, D.A. (1997) RNase mitochondrial RNA processing correctly cleaves a novel R loop at the mitochondrial DNA leading-strand origin of replication. Genes Dev., 11, 582–592.[Abstract/Free Full Text]

  25. Cai, T., Aulds, J., Gill, T., Cerio, M. and Schmitt, M.E. (2002) The Saccharomyces cerevisiae RNase mitochondrial RNA processing is critical for cell cycle progression at the end of mitosis. Genetics, 161, 1029–1042.[Abstract/Free Full Text]

  26. Gill, T., Cai, T., Aulds, J., Wierzbicki, S. and Schmitt, M.E. (2004) RNase MRP cleaves the CLB2 mRNA to promote cell cycle progression: novel method of mRNA degradation. Mol. Cell. Biol., 24, 945–953.[Abstract/Free Full Text]

  27. Chu, S., Archer, R.H., Zengel, J.M. and Lindahl, L. (1994) The RNA of RNase MRP is required for normal processing of ribosomal RNA. Proc. Natl Acad. Sci. USA, 91, 659–663.[Abstract/Free Full Text]

  28. Lygerou, Z., Allmang, C., Tollervey, D. and Seraphin, B. (1996) Accurate processing of a eukaryotic precursor ribosomal RNA by ribonuclease MRP in vitro. Science, 272, 268–270.[Abstract]

  29. Reich, C., Olsen, G.J., Pace, B. and Pace, N.R. (1988) Role of the protein moiety of ribonuclease P, a ribonucleoprotein enzyme. Science, 239, 178–181.[Abstract/Free Full Text]

  30. Schmitt, M.E. and Clayton, D.A. (1994) Characterization of a unique protein component of yeast RNase MRP: an RNA-binding protein with a zinc-cluster domain. Genes Dev., 8, 2617–2628.[Abstract/Free Full Text]

  31. Salinas, K., Wierzbicki, S., Zhou, L. and Schmitt, M.E. (2005) Characterization and purification of Saccharomyces cerevisiae RNase MRP reveals a new unique protein component. J. Biol. Chem., 280, 11352–11360.[Abstract/Free Full Text]

  32. Vulliamy, T., Marrone, A., Goldman, F., Dearlove, A., Bessler, M., Mason, P.J. and Dokal, I. (2001) The RNA component of telomerase is mutated in autosomal dominant dyskeratosis congenita. Nature, 413, 432–435.[CrossRef][Medline]

  33. Bennett, J.L. and Clayton, D.A. (1990) Efficient site-specific cleavage by RNase MRP requires interaction with two evolutionarily conserved mitochondrial RNA sequences. Mol. Cell. Biol., 10, 2191–2201.[Abstract/Free Full Text]

  34. Yuan, Y. and Reddy, R. (1991) 5' flanking sequences of human MRP/7-2 RNA gene are required and sufficient for the transcription by RNA polymerase III. Biochim. Biophys. Acta, 1089, 33–39.[Medline]

  35. Hernandez, N. and Lucito, R. (1988) Elements required for transcription initiation of the human U2 snRNA gene coincide with elements required for snRNA 3' end formation. EMBO J., 7, 3125–3134.[Web of Science][Medline]

  36. Jensen, R.C., Wang, Y., Hardin, S.B. and Stumph, W.E. (1998) The proximal sequence element (PSE) plays a major role in establishing the RNA polymerase specificity of DrosophilaU-snRNA genes. Nucleic Acids Res., 26, 616–622.[Abstract/Free Full Text]

  37. Lobo, S.M. and Hernandez, N. (1989) A 7 bp mutation converts a human RNA polymerase II snRNA promoter into an RNA polymerase III promoter. Cell, 58, 55–67.[CrossRef][Web of Science][Medline]

  38. Mattaj, I.W., Dathan, N.A., Parry, H.D., Carbon, P. and Krol, A. (1988) Changing the RNA polymerase specificity of U snRNA gene promoters. Cell, 55, 435–442.[CrossRef][Web of Science][Medline]

  39. Chen, Y.C., Song, C. and Luo, C.Q. (2003) Short hairpin RNAs induced RNA interference in human cells. Chinese J. Cancer, 22, 566–570.

  40. Sui, G., Soohoo, C., Affarel, B., Gay, F., Shi, Y. and Forrester, W.C. (2002) A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc. Natl Acad. Sci. USA, 99, 5515–5520.[Abstract/Free Full Text]

  41. Cai, T., Reilly, T.R., Cerio, M. and Schmitt, M.E. (1999) Mutagenesis of SNM1, which encodes a protein component of the yeast RNase MRP, reveals a role for this ribonucleoprotein endoribonuclease in plasmid segregation. Mol. Cell. Biol., 19, 7857–7869.[Abstract/Free Full Text]

  42. Bogenhagen, D.F. (1985) The intragenic control region of the Xenopus 5 S RNA gene contains two factor A binding domains that must be aligned properly for efficient transcription initiation. J. Biol. Chem., 260, 6466–6471.[Abstract/Free Full Text]

  43. Allison, D.S., Goh, S.H. and Hall, B.D. (1983) The promoter sequence of a yeast tRNAtyr gene. Cell, 34, 655–664.[CrossRef][Web of Science][Medline]

  44. Galli, G., Hofstetter, H. and Birnstiel, M.L. (1981) Two conserved sequence blocks within eukaryotic tRNA genes are major promoter elements. Nature, 294, 626–631.[CrossRef][Medline]

  45. Hofstetter, H., Kressman, A. and Birnstiel, M.L. (1981) A split promoter for a eucaryotic tRNA gene. Cell, 24, 573–585.[CrossRef][Web of Science][Medline]

  46. Sharp, S., DeFranco, D., Dingermann, T., Farrell, P. and Soll, D. (1981) Internal control regions for transcription of eukaryotic tRNA genes. Proc. Natl Acad. Sci. USA, 78, 6657–6661.[Abstract/Free Full Text]

  47. Kunkel, G.R. and Pederson, T. (1989) Transcription of a human U6 small nuclear RNA gene in vivo withstands deletion of intragenic sequences but not of an upstream TATATA box. Nucleic Acids Res., 17, 7371–7379.[Abstract/Free Full Text]

  48. Topper, J.N. and Clayton, D.A. (1990) Characterization of human MRP/Th RNA and its nuclear gene: full length MRP/Th RNA is an active endoribonuclease when assembled as an RNP. Nucleic Acids Res., 18, 793–799.[Abstract/Free Full Text]

  49. Schramm, L. and Hernandez, N. (2002) Recruitment of RNA polymerase III to its target promoters. Genes Dev., 16, 2593–2620.[Free Full Text]

  50. Cerritelli, S.M. and Crouch, R.J. (1998) Cloning, expression, and mapping of ribonucleases H of human and mouse related to bacterial RNase HI. Genomics, 53, 300–307.[CrossRef][Web of Science][Medline]

  51. Clayton, D.A. (1994) A nuclear function for RNase MRP. Proc. Natl Acad. Sci. USA, 91, 4615–4617.[Free Full Text]

  52. Kuhn, K., Shikhman, A.R. and Lotz, M. (2003) Role of nitric oxide, reactive oxygen species, and p38 MAP kinase in the regulation of human chondrocyte apoptosis. J. Cell Physiol., 197, 379–387.[CrossRef][Web of Science][Medline]

  53. Merz, D., Liu, R., Johnson, K. and Terkeltaub, R. (2003) IL-8/CXCL8 and growth-related oncogene alpha/CXCL1 induce chondrocyte hypertrophic differentiation. J. Immunol., 171, 4406–4415.[Abstract/Free Full Text]

  54. Kaneko, S., Satoh, T., Chiba, J., Ju, C., Inoue, K. and Kagawa, J. (2000) Interleukin-6 and interleukin-8 levels in serum and synovial fluid of patients with osteoarthritis. Cytokines Cell. Mol. Ther., 6, 71–79.[CrossRef][Web of Science][Medline]

  55. Orditura, M., De Vita, F., Catalano, G., Infusino, S., Lieto, E., Martinelli, E., Morgillo, F., Castellano, P., Pignatelli, C. and Galizia, G. (2002) Elevated serum levels of interleukin-8 in advanced non-small cell lung cancer patients: relationship with prognosis. J. Interferon Cytokine Res., 22, 1129–1135.[CrossRef][Web of Science][Medline]

  56. Ryningen, A., Wergeland, L., Glenjen, N., Gjertsen, B.T. and Bruserud, O. (2005) In vitro crosstalk between fibroblasts and native human acute myelogenous leukemia (AML) blasts via local cytokine networks results in increased proliferation and decreased apoptosis of AML cells as well as increased levels of proangiogenic Interleukin 8. Leuk. Res., 29, 185–196.[CrossRef][Web of Science][Medline]

  57. Kooijman, R., van der Burgt, C.J., Weemaes, C.M., Haraldsson, A., Scholtens, E.J. and Zegers, B.J. (1997) T cell subsets and T cell function in cartilage-hair hypoplasia. Scand. J. Immunol., 46, 209–215.[CrossRef][Web of Science][Medline]

  58. Cristillo, A.D., Heximer, S.P., Russell, L. and Forsdyke, D.R. (1997) Cyclosporin A inhibits early mRNA expression of G0/G1 switch gene 2 (G0S2) in cultured human blood mononuclear cells. DNA Cell Biol., 16, 1449–1458.[Web of Science][Medline]

  59. Legeai-Mallet, L., Benoist-Lasselin, C., Munnich, A. and Bonaventure, J. (2004) Overexpression of FGFR3, Stat1, Stat5 and p21Cip1 correlates with phenotypic severity and defective chondrocyte differentiation in FGFR3-related chondrodysplasias. Bone, 34, 26–36.[Medline]

  60. Aulds, J., Cai, T. and Schmittt, M.E. (1992) RNase MRP from Yeast to Humans, Cell Cycle Control and Cartilage-Hair Hypoplasia. Research Signpost, Kerala, India.

  61. Albrecht, U., Eichele, G., Helms, J.A. and Lu, H.-C. (1997) Visualization of Gene Expression Patterns by In Situ Hybridization. CRC Press, Inc.

  62. Zhou, G., Lefebvre, V., Zhang, Z., Eberspaecher, H. and de Crombrugghe, B. (1998) Three high mobility group-like sequences within a 48-base pair enhancer of the Col2a1 gene are required for cartilage-specific expression in vivo. J. Biol. Chem., 273, 14989–14997.[Abstract/Free Full Text]

  63. Wach, A., Brachat, A., Pohlmann, R. and Philippsen, P. (1994) New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast, 10, 1793–1808.[CrossRef][Web of Science][Medline]

  64. Gietz, R.D., Schiestl, R.H., Willems, A.R. and Woods, R.A. (1995) Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast, 11, 355–360.[CrossRef][Web of Science][Medline]

  65. Schmitt, M.E., Brown, T.A. and Trumpower, B.L. (1990) A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res., 18, 3091–3092.[Free Full Text]


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