Pseudo-messenger RNA: Phantoms of the transcriptome

The mammalian transcriptome harbours shadowy entities that resist classification and analysis. In analogy with pseudogenes, we define pseudo-messenger RNA to be RNA molecules that resemble protein- coding mRNA, but cannot encode full-length proteins owing to disruptions of the reading frame. Using a rigorous computational pipeline, which rules out sequencing errors, we identify 10,679 pseudo - messenger RNAs ( approximately half of which are transposonassociated) among the 102,801 FANTOM3 mouse cDNAs: just over 10% of the FANTOM3 transcriptome. These comprise not only transcribed pseudogenes, but also disrupted splice variants of otherwise protein- coding genes. Some may encode truncated proteins, only a minority of which appear subject to nonsense- mediated decay. The presence of an excess of transcripts whose only disruptions are opal stop codons suggests that there are more selenoproteins than currently estimated. We also describe compensatory frameshifts, where a segment of the gene has changed frame but remains translatable. In summary, we survey a large class of non- standard but potentially functional transcripts that are likely to encode genetic information and effect biological processes in novel ways. Many of these transcripts do not correspond cleanly to any identifiable object in the genome, implying fundamental limits to the goal of annotating all functional elements at the genome sequence level.

The prion protein gene: Identifying regulatory signals using marsupial sequence

The function of the prion protein gene (PRNP) and its normal product PrPC is elusive. We used comparative genomics as a strategy to understand the normal function of PRNP. As the reliability of comparisons increases with the number of species and increased evolutionary distance, we isolated and sequenced a 66.5 kb BAC containing the PRNP gene from a distantly related mammal, the model Australian marsupial Macropus eugenii (tammar wallaby). Marsupials are separated from eutherians such as human and mouse by roughly 180 million years of independent evolution. We found that tammar PRNP, like human PRNP, has two exons. Prion proteins encoded by the tammar wallaby and a distantly related marsupial, Monodelphis domestica (Brazilian opossum) PRNP contain proximal PrP repeats with a distinct, marsupial-specific composition and a variable number. Comparisons of tammar wallaby PRNP with PRNPs from human, mouse, bovine and ovine allowed us to identify non-coding gene regions conserved across the marsupial-eutherian evolutionary distance, which are candidates for regulatory regions. In the PRNP 3' UTR we found a conserved signal for nuclear-specific polyadenylation and the putative cytoplasmic polyadenylation element (CPE), indicating that post-transcriptional control of PRNP mRNA activity is important. Phylogenetic footprinting revealed conserved potential binding sites for the MZF-1 transcription factor in both upstream promoter and intron/intron 1, and for the MEF2, MyTI, Oct-1 and NFAT transcription factors in the intron(s). The presence of a conserved NFAT-binding site and CPE indicates involvement of PrPC in signal transduction and synaptic plasticity. (c) 2004 Elsevier B.V. All rights reserved.

Opsins: Evolution in waiting

Complete vertebrate genome sequencing has revealed a remarkable stability and uniformity in the protein-coding gene set, which at first glance might suggest that gene duplication events are relatively rare. This may be a red herring, or at least a red cichlid, as the Lake Malawi cichlid fishes show rapid and extensive duplication and diversification of their retinal cone photoreceptor opsin genes.

Characterisation of three ACC synthase gene family members during post-harvest-induced senescence in broccoli (Brassica oleracea L. var. italica)

We investigated the gene expression profiles of different members of the 1-aminocyclopropane-1-carboxilic acid (ACC) synthase (EC 4.4.1.14) gene family in broccoli (Brassica oleracea L. var. italica) during the post-harvest-induced senescence process. Using RT-PCR, three different cDNAs coding for ACC synthase (BROCACS1, BROCACS2 and BROCACS3) were amplified from floret tissue at the start of the senescence process. The three genes share relatively little homology, but have highly homologous sequences in Arabidopsis thaliana, and could be functionally related to these counterparts. Southern analyses suggest that BROCACS1 and BROCACS3 are present as single copy genes, while there are probably two copies of BROCACS2. All three genes showed different expression patterns: BROCACS1 is likely to be either wound - or mechanical stress-induced showing high transcript levels after harvesting, but no detectable expression afterwards. BROCACS2 shows steady expression throughout senescence, increasing at the latest stages, and BROCACS3 is almost undetectable until the final stages. Our results suggest that BROCACS1 could be required to initiate the senescence process, while BROCACS2 would be the main ACC synthase gene involved throughout the post-harvest-induced senescence. BROCACS3's expression pattern indicates that it is not directly involved in the initial stages of senescence, but in the final remobilization of cellular resources.

No disease-associated mutations found in the coding sequence of the canine polycystic kidney disease gene 1 in Bull Terriers with polycystic kidney disease

The aim of this study was to identify possible disease-associated mutations in the canine homologue of the polycystic kidney disease gene 1 (PKD1) in Bull Terriers with autosomal dominant polycystic kidney disease. Messenger RNA was obtained from the blood or renal tissue of five Bull Terriers with the disease and four close relatives without the disease. Reverse transcription, PCR and 3' rapid amplification of cDNA ends were used to amplify the coding and 3' untranslated regions of this transcript. Comparison of PKD1 sequence between the affected and unaffected Bull Terriers, revealed six polymorphisms, but no disease-associated mutations.

Challenging the dogma: the hidden layer of non-protein-coding RNAs in complex organisms

The central dogma of biology holds that genetic information normally flows from DNA to RNA to protein. As a consequence it has been generally assumed that genes generally code for proteins, and that proteins fulfil not only most structural and catalytic but also most regulatory functions, in all cells, from microbes to mammals. However, the latter may not be the case in complex organisms. A number of startling observations about the extent of non-protein-coding RNA (ncRNA) transcription in the higher eukaryotes and the range of genetic and epigenetic phenomena that are RNA-directed suggests that the traditional view of the structure of genetic regulatory systems in animals and plants may be incorrect. ncRNA dominates the genomic output of the higher organisms and has been shown to control chromosome architecture, mRNA turnover and the developmental timing of protein expression, and may also regulate transcription and alternative splicing. This paper re-examines the available evidence and suggests a new framework for considering and understanding the genomic programming of biological complexity, autopoletic development and phenotypic variation. BioEssays 25:930-939,2003. (C) 2003 Wiley Periodicals, Inc.

An investigation of the 5-HT2C receptor gene as a migraine candidate gene

Migraine is a common complex disorder, currently classified into two main subtypes, migraine with aura (MA) and migraine without aura (MO). The strong preponderance of females to males suggests an X-linked genetic component. Recent studies have identified an X chromosomal susceptibility region (Xq24-q28) in two typical migraine pedigrees. This region harbours a potential candidate gene for the disorder, the serotonin receptor 2C (5-HT2C) gene. This study involved a linkage and association approach to investigate two single nucleotide variants in the 5-HT2C gene. In addition, exonic coding regions of the 5-HT2C gene were also sequenced for mutations in X-linked migraine pedigrees. Results of this study did not detect any linkage or association, and no disease causing mutations were identified. Hence, results for this study do not support a significant role of the 5-HT2C gene in migraine predisposition. (C) 2003 Wiley-Liss, Inc.

Sequence diversity in the coat protein coding region of the genome RNA of Johnsongrass mosaic virus in Australia

Sequence diversity in the coat protein coding region of Australian strains of Johnsongrass mosaic virus (JGMV) was investigated. Field isolates were sampled during a seven year period from Johnsongrass, sorghum and corn across the northern grain growing region. The 23 isolates were found to have greater than 94% nucleotide and amino acid sequence identity. The Australian isolates and two strains from the U.S.A. had about 90% nucleotide sequence identity and were between 19 and 30% different in the N-terminus of the coat protein. Two amino acid residues were found in the core region of the coat protein in isolates obtained from sorghum having the Krish gene for JGMV resistance that differed from those found in isolates from other hosts which did not have this single dominant resistance gene. These amino acid changes may have been responsible for overcoming the resistance conferred by the Krish gene for JGMV resistance in sorghum. The identification of these variable regions was essential for the development of durable pathogen-derived resistance to JGMV in sorghum.

Post-transcriptional regulation of the cystic fibrosis gene in cardiac development and hypertrophy

Eukaryotic gene expression, reflected in the amount of steady-state mRNA, is regulated at the post-transcriptional level. The 5'-untranslated regions (5'-UTRs) of some transcripts contain cis-acting elements, including upstream open reading frames (uORFs), that have been identified as being fundamental in modulating translation efficiency and mRNA stability. Previously, we demonstrated that uORFs present in the 5'-UTR of cystic fibrosis transmembrane conductance regular (CFTR) transcripts expressed in the heart were able to modulate translation efficiency of the main CFTR ORF. Here, we show that the same 5'-UTR elements are associated with the differential stability of the 5'-UTR compared to the main coding region of CFTR transcripts. Furthermore, these post-transcriptional mechanisms are important factors governing regulated CFTR expression in the heart, in response to developmental and pathophysiological stimuli. (C) 2004 Elsevier Inc. All rights reserved.

Association between chronic fatigue syndrome and the corticosteroid-binding globulin gene ALA SER224 polymorphism

Chronic fatigue syndrome (CFS) is characterized by idiopathic fatigue of greater than 6 months' duration with postexertional exacerbation and many other symptoms. A trend toward relative hypocortisolism is described in CFS. Twin and family studies indicate a substantial genetic etiologic component to CFS. Recently, severe corticosteroid-binding globulin (CBG) gene mutations have been associated with CFS in isolated kindreds. Human leukocyte elastase, an enzyme important in CBG catabolism at inflammatory sites, is reported to be elevated in CFS. We hypothesized that CBG gene polymorphisms may act as a genetic risk factor for CFS. A total of 248 patients with CFS defined by Centers for Disease Control criteria, and 248 controls were recruited. Sequencing and restriction enzyme testing of the CBG gene coding region allowed detection of severe CBG gene mutations and a common exon 3 polymorphism (c.825G --> T, Ala-Ser(224)). Plasma CBG levels were measured in 125 CFS patients and 198 controls by radioimmunoassay. Total and free (calculated and measured) cortisol levels were ascertained in single samples between 8-10 a.m. The age of onset (mid 30s) and gender ratio (2.2:1, female:male) of the patients were similar to those reported in U.S. epidemiologic studies. A trend toward a preponderance of serine(224) homozygosity among the CFS patients was noted, compared with controls (chi(2) = 5.31, P = 0.07). Immunoreactive-CBG (IR-CBG) levels were higher in Serine/Alanine (Ser/Ala) than Ala/Ala subjects and higher again in Ser/Ser subjects, this effect was strongest in controls; Ser/Ser: 46.1 +/- 1.8 (n = 31, P = 0.03) vs. Ser/Ala: 42.4 +/- 1.0 (n = 56, P = 0.05) vs. Ala/Ala: 40.8 +/- 1.7 mug/mL (n = 21). Despite higher CBG levels, there was a nonsignificant trend toward lower total and free plasma cortisol in serine allele positive patients, total cortisol: Ser/Ser: 13.3 +/- 1.4 (n = 34) vs. Ser/Ala: 14.0 +/- 0.7 (n = 66) vs. Ala/Ala: 15.4 +/- 1.0 (n = 23). Homozygosity for the serine allele of the CBG gene may predispose to CFS, perhaps due to an effect on hypothalamic-pituitary-adrenal axis function related to altered CBG-cortisol transport function or immune-cortisol interactions.

Normal HPRT coding region in a male with gout due to HPRT deficiency (Brief Communication)

A deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT; EC 2.4.2.8) is associated with a spectrum of disease that ranges from gouty arthritis (OMIM 300323) to the more severe Lesch-Nyhan syndrome (OMIM 300322). To date, all cases of HPRT deficiency have shown a mutation within the HPRT cDNA. In the present study of an individual with gout due to HPRT deficiency, we found a normal HPRT cDNA sequence. This is the first study to provide an example of HPRT deficiency which appears to be due to a defect in the regulation of the gene. © 2005 Elsevier Inc. All rights reserved.

Gene codon composition determines differentiation-dependent expression of a viral capsid gene in keratinocytes in vitro and in vivo

By establishing mouse primary keratinocytes (KCs) in culture, we were able, for the first time, to express papillomavirus major capsid (L1) proteins by transient transfection of authentic or codon-modified L1 gene expression plasmids. We demonstrate in vitro and in vivo that gene codon composition is in part responsible for differentiation-dependent expression of L1 protein in KCs. L1 mRNA was present in similar amounts in differentiated and undifferentiated KCs transfected with authentic or codon-modified L1 genes and had a similar half-life, demonstrating that L1 protein production is posttranscriptionally regulated. We demonstrate further that KCs substantially change their tRNA profiles upon differentiation. Aminoacyl-tRNAs from differentiated KCs but not undifferentiated KCs enhanced the translation of authentic L1 mRNA, suggesting that differentiation-associated change to tRNA profiles enhances L1 expression in differentiated KCs. Thus, our data reveal a novel mechanism for regulation of gene expression utilized by a virus to direct viral capsid protein expression to the site of virion assembly in mature KCs. Analysis of two structural proteins of KCs, involucrin and keratin 14, suggests that translation of their mRNAs is also regulated, in association with KC differentiation in vitro, by a similar mechanism

Identification of two evolutionarily conserved and functional regulatory elements in intron 2 of the human BRCA1 gene

Cross-species comparative genomics is a powerful strategy for identifying functional regulatory elements within noncoding DNA. In this paper, comparative analysis of human and mouse intronic sequences in the breast cancer susceptibility gene (BRCA1) revealed two evolutionarily conserved noncoding sequences (CNS) in intron 2, 5 kb downstream of the core BRCA1 promoter. The functionality of these elements was examined using homologous-recombination-based mutagenesis of reporter gene-tagged cosmids incorporating these regions and flanking sequences from the BRCA1 locus. This showed that CNS-1 and CNS-2 have differential transcriptional regulatory activity in epithelial cell lines. Mutation of CNS-1 significantly reduced reporter gene expression to 30% of control levels. Conversely mutation of CNS-2 increased expression to 200% of control levels. Regulation is at the level of transcription and shows promoter specificity. Both elements also specifically bind nuclear proteins in vitro. These studies demonstrate that the combination of comparative genomics and functional analysis is a successful strategy to identify novel regulatory elements and provide the first direct evidence that conserved noncoding sequences in BRCA1 regulate gene expression. (c) 2005 Elsevier Inc. All rights reserved.