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PubMed2006

GENCODE: producing a reference annotation for ENCODE.

BACKGROUND: The GENCODE consortium was formed to identify and map all protein-coding genes within the ENCODE regions. This was achieved by a combination of initial manual annotation by the HAVANA team, experimental validation by the GENCODE consortium and a refinement of the annotation based on these experimental results. RESULTS: The GENCODE gene features are divided into eight different categories of which only the first two (known and novel coding sequence) are confidently predicted to be protein-coding genes. 5' rapid amplification of cDNA ends (RACE) and RT-PCR were used to experimentally verify the initial annotation. Of the 420 coding loci tested, 229 RACE products have been sequenced. They supported 5' extensions of 30 loci and new splice variants in 50 loci. In addition, 46 loci without evidence for a coding sequence were validated, consisting of 31 novel and 15 putative transcripts. We assessed the comprehensiveness of the GENCODE annotation by attempting to validate all the predicted exon boundaries outside the GENCODE annotation. Out of 1,215 tested in a subset of the ENCODE regions, 14 novel exon pairs were validated, only two of them in intergenic regions. CONCLUSION: In total, 487 loci, of which 434 are coding, have been annotated as part of the GENCODE reference set available from the UCSC browser. Comparison of GENCODE annotation with RefSeq and ENSEMBL show only 40% of GENCODE exons are contained within the two sets, which is a reflection of the high number of alternative splice forms with unique exons annotated. Over 50% of coding loci have been experimentally verified by 5' RACE for EGASP and the GENCODE collaboration is continuing to refine its annotation of 1% human genome with the aid of experimental validation.

Chromosome Mapping↗
PubMed2004

Differential expression of splice variants of chemokine CCL27 mRNA in lens, cornea, and retina of the normal mouse eye.

PURPOSE: Constitutive expression of RNA sequences complementary to the chemokine CCL27 mRNA has been found in the normal mouse eye. This study examines the nature and location of these endogenous RNAs in ocular tissues. METHODS: Conventional RT-PCR, 5' RACE, and dideoxy DNA sequencing were used to examine the sequences of CCL27 related RNAs in the eye. Expression levels of specific RNAs were measured by real time PCR. Tissue distribution of RNA transcripts was determined by RT-PCR using RNA from microdissected tissues and by in situ hybridization with radiolabeled riboprobes. RESULTS: We detect 5 distinct splice variants derived from transcription of the CCL27 gene locus. The most abundant form codes for a non-secreted protein, PESKY, and is expressed in lens, cornea, and retina. Another variant corresponds to the mRNA of the secreted chemokine and is synthesized in the cornea, but not in retina or lens. The remaining splice variants are novel and may be eye specific, but have only short open reading frames (<50 amino acids). CCL27 transcripts are most abundantly expressed in the retina, as judged by in situ hybridization. CONCLUSIONS: PESKY and other CCL27 splice variants of unknown function are widely expressed in ocular tissues. Analysis of CCL27 transcripts from lens, retina, and cornea indicates that mRNA for the secreted chemokine, CCL27, is endogenously expressed only in the cornea and may play a role in ocular immune responses involving CD4 lymphocytes in this tissue.

Alternative Splicing↗
PubMed1996

Effects of an exon 5 variant of the estrogen receptor in MCF-7 breast cancer cells.

The estrogen receptor gene gives rise to variant mRNAs, generated by alternative mRNA splicing, as well as the full-length mRNA containing eight coding exons. It has been postulated that one of these, the exon 5 variant, may be important in the development of hormone-independent and anti-estrogen-resistant breast cancer since it has the potential to encode a truncated receptor that retains the N-terminal activation domain AF-1, but lacks the hormone-binding domain. We have expressed the variant using an inducible promoter in estrogen receptor-positive MCF-7 cells and analyzed the effect of the variant protein on gene expression and cell growth. Inducible expression was validated using a specific antiserum that recognized a novel epitope on the exon 5 variant. The variant was able to stimulate transcription of a reporter gene in transiently transfected chicken embryo fibroblasts in the absence of hormone but showed weak constitutive activity when it was stably expressed in MCF-7 cells. The variant had no effect on the expression of the estrogen target genes, pS2, and the progesterone receptor. Finally, we analyzed whether the proliferation of MCF-7 cells was altered by the expression of the exon 5 variant and found that the stimulatory effects of estrogen and growth inhibitory effects of tamoxifen and ICI 182780 were unchanged. We therefore conclude that expression of the variant alone is not sufficient to give rise to hormone independence or tamoxifen resistance of breast cancers.

Animals↗
PubMed2001

Mutations of the BRCA1 and BRCA2 genes in patients with bilateral breast cancer.

Mutations of the BRCA1 or BRCA2 genes have been shown to strongly predispose towards the development of contralateral breast cancer in patients from large multi-case families. In order to test the hypothesis that BRCA1 and BRCA2 mutations are more frequent in patients with bilateral breast cancer, we have investigated a hospital-based series of 75 consecutive patients with bilateral breast cancer and a comparison group of 75 patients with unilateral breast cancer, pairwise matched by age and family history, for mutations in the BRCA1 and BRCA2 genes. Five frameshift deletions (517delGT in BRCA1; 4772delA, 5946delCT, 6174delT and 8138del5 in BRCA2) were identified in patients with bilateral disease. No further mutations, apart from polymorphisms and 3 rare unclassified variants, were found after scanning the whole BRCA1 and BRCA2 coding sequence. Three pathogenic BRCA1 mutations (Cys61Gly, 3814del5, 5382insC) were identified in the group of patients with unilateral breast cancer. The frequencies of common BRCA1 and BRCA2 missense variants were not different between the 2 groups. In summary, we did not find a significantly increased prevalence of BRCA1 and BRCA2 mutations in a hospital-based cohort of German patients with bilateral breast cancer. We conclude that bilaterality of breast cancer on its own is not strongly associated with BRCA1 and BRCA2 mutations when adjusted for age and family history. The high frequency of bilateral disease in multi-case breast cancer families may be due to a familial aggregation of additional susceptibility factors modifying the penetrance of BRCA1 and BRCA2 mutations.

Adult↗
PubMed2005

Automated splicing mutation analysis by information theory.

Information theory-based software tools have been useful in interpreting noncoding sequence variation within functional sequence elements such as splice sites. Individual information analysis detects activated cryptic splice sites and associated splicing regulatory sites and is capable of distinguishing null from partially functional alleles. We present a server (https://splice.cmh.edu) designed to analyze splicing mutations in binding sites in either human genes, genome-mapped mRNAs, user-defined sequences, or dbSNP entries. Standard HUGO-approved gene symbols and HGVS-approved systematic mutation nomenclature (or dbSNP format) are entered via a web portal. After verifying the accuracy of input variant(s), the surrounding interval is retrieved from the human genome or user-supplied reference sequence. The server then computes the information contents (Ri) of all potential constitutive and/or regulatory splice sites in both the reference and variant sequences. Changes in information content are color-coded, tabulated, and visualized as sequence walkers, which display the binding sites with the reference sequence. The software was validated by analyzing approximately 1,300 mutations from Human Mutation as well as eight mapped SNPs from dbSNP designated as splice site variants. All of the splicing mutations and variants affected splice site strength or activated cryptic splice sites. The server also detected several missense mutations that were unexpectedly predicted to have concomitant effects on splicing or appeared to activate cryptic splicing.

Automation↗
PubMed1993

Evidence from mutation spectra that the UV hypermutability of xeroderma pigmentosum variant cells reflects abnormal, error-prone replication on a template containing photoproducts.

Xeroderma pigmentosum (XP) variant patients are genetically predisposed to sunlight-induced skin cancer. Fibroblasts derived from these patients are extremely sensitive to the mutagenic effect of UV radiation and are abnormally slow in replicating DNA containing UV-induced photoproducts. However, unlike cells from the majority of XP patients, XP variant cells have a normal or nearly normal rate of nucleotide excision repair of such damage. To determine whether their UV hypermutability reflected a slower rate of excision of photoproducts specifically during early S phase when the target gene for mutations, i.e., the hypoxanthine (guanine) phosphoribosyltransferase gene (HPRT), is replicated, we synchronized diploid populations of normal and XP variant fibroblasts, irradiated them in early S phase, and compared the rate of loss of cyclobutane pyrimidine dimers and 6-4 pyrimidine-pyrimidones from DNA during S phase. There was no difference. Both removed 94% of the 6-4 pyrimidine-pyrimidones within 8 h and 40% of the dimers within 11 h. There was also no difference between the two cell lines in the rate of repair during G1 phase. To determine whether the hypermutability resulted from abnormal error-prone replication of DNA containing photoproducts, we determined the spectra of mutations induced in the coding region of the HPRT gene of XP variant cells irradiated in early S and G1 phases and compared with those found in normal cells. The majority of the mutations in both types of cells were base substitutions, but the two types of cells differed significantly from each other in the kinds of substitutions, but the two types differed significantly from each other in the kinds of substitutions observed either in mutants from S phase (P < 0.01) or from G1 phase (P = 0.03). In the variant cells, the substitutions were mainly transversions (58% in S, 73% in G1). In the normal cells irradiated in S, the majority of the substitutions were G.C --> A.T, and most involved CC photoproducts in the transcribed strand. In the variant cells irradiated in S, substitutions involving cytosine in the transcribed strand were G.C --> T.A transversions exclusively. G.C --> A.T transitions made up a much smaller fraction of the substitutions than in normal cells (P < 0.02), and all of them involved photoproducts located in the nontranscribed strand. The data strongly suggest that XP variant cells are much less likely than normal cells to incorporate either dAMP or dGMP opposite the pyrimidines involved in photoproducts. This would account for their significantly higher frequency of mutants and might explain their abnormal delay in replicating a UV-damaged template.

Base Sequence↗
PubMed2006

Association of the F352V variant of the Klotho gene with bone mineral density.

Klotho gene codes for a protein with glucuronidase activity and is thought to influence bone and vascular homeostasis. We studied the relationship of a common T/G polymorphism, resulting in a phenylalanine (F) to valine (V) substitution at aminoacid position 352, with bone mineral density (BMD) and osteoporotic fractures. The study group comprised 914 Spanish women, including 438 control subjects, 190 patients with osteoporosis, 198 with hip fractures, and 88 patients with severe osteoarthritis. BMD was measured by DEXA in 540 women from the control and osteoporosis groups. Allele frequencies were 86% and 14%, for the F and V alleles, respectively. In comparison with the most common FF genotype, postmenopausal women with FV/VV genotypes had higher hip BMD (femoral neck: 0.673 +/- 0.011 vs. 0.644 +/- 0.006 g/cm(2); P = 0.02; total hip: 0.807 +/- 0.014 vs. 0.774 +/- 0.008 g/cm(2); P = 0.03). Klotho alleles explained about 1.5% of BMD variance, but were not associated to the risk of osteoporotic spine or hip fractures. The Klotho genotype was not associated to BMD in premenopausal women. In conclusion, the F352V Klotho polymorphism is associated with BMD in postmenopausal women, suggesting that Klotho gene variants influence skeletal aging.

Adult↗
PubMed1995

Common genetic variants of lipoprotein lipase that relate to lipid transport in patients with premature coronary artery disease.

Two common coding sequence mutations of lipoprotein lipase (serine447-ter, producing a carboxy terminal truncation; and asp9-asn variants) were studied in 329 Caucasian subjects, of whom 243 had angiographic features of premature atheroscelerosis (220 with coronary artery disease; 23 with coronary and peripheral artery disease). As expected, the mean levels of cholesterol, triglycerides, LDL-cholesterol, ApoB and Lp(a) were significantly higher in the arterial disease group than in the controls. HDL levels were lower in the patient group. With regard to the common mutations, plasma triglycerides and VLDL-triglycerides were lower in subjects possessing the Serine447-Ter mutation (p = 0.06 and < 0.05, respectively). When the lipid distributions were analysed by tertiles, the Ser447-Ter mutation was significantly less frequent in the highest tertiles for triglycerides (p < 0.02), and VLDL (p < 0.04). The Asp9-Asn substitution was significantly more frequent in the lowest tertiles for ApoAI (p = 0.05). Case-control analyses of genotypic distributions between the two groups with or without arterial disease did not show any significant differences. The possible functional effects of these common mutants of lipoprotein lipase are discussed.

Asparagine↗
PubMed2004

Multiple novel nonsynonymous CYP2B6 gene polymorphisms in Caucasians: demonstration of phenotypic null alleles.

The human microsomal cytochrome P450, CYP2B6, is involved in the biotransformation of several clinically important drugs. By complete sequence analysis of the human CYP2B6 gene coding regions in selected Caucasian DNA samples, we identified the five novel missense mutations 62A>T (Q21L in exon 1), 136A>G (M46V in exon 1), 12820G>A (G99E in exon 2), 13076G>A (R140Q in exon 3), and 21388T>A (I391N in exon 8). The recently described but functionally uncharacterized variant 13072A>G (K139E) was also observed. Haplotype analysis indicated the presence of at least six novel alleles that code for the protein variants CYP2B6.10 (Q21L, R22C), CYP2B6.11 (M46V), CYP2B6.12 (G99E), CYP2B6.13 (K139E, Q172H, K262R), CYP2B6.14 (R140Q), and CYP2B6.15 (I391N). Heterologous expression in COS-1 cells revealed comparable levels of CYP2B6 apoprotein and bupropion hydroxylase activity for CYP2B6.1 (wild type) and CYP2B6.10, whereas all other variants exhibited reduced expression and/or function. The three amino acid changes M46V, G99E, and I391N resulted in almost unmeasurable (M46V) or undetectable (G99E and I391N) enzyme activity, despite the presence of residual protein. The K139E change led to completely abolished protein expression; as a consequence, no function was detected. Expression in insect cells by recombinant baculoviruses confirmed these results and demonstrated the virtual absence of incorporated heme in these protein variants. The collective allele frequency of the four very low or null activity variants M46V, G99E, K139E, and I391N was 2.6% in a Caucasian study population. These data provide further insight into the genetic variability of CYP2B6 and demonstrate the existence of phenotypic null alleles in this gene.

Alleles↗
PubMed1994

Mutations in the R-type pyruvate kinase gene and altered enzyme kinetic properties in patients with hemolytic anemia due to pyruvate kinase deficiency.

The biochemical properties of erythrocyte pyruvate kinase (PK) together with mutations found in the coding sequence of the R-PK gene in five patients with severe hemolytic anemia due to PK deficiency are described. The enzyme variants were designated PK 'Mosul' (homozygote), PK 'Bukarest', PK 'Hamburg', PK 'Köln', and PK 'Essen' (compound heterozygote). PK 'Mosul' showed normal positive cooperative substrate binding, PK 'Bukarest' exhibited non-cooperative behavior, and PK 'Hamburg' and PK 'Köln' displayed mixed cooperativity, whereas PK 'Essen' was negative cooperative. PK 'Mosul' was found to be homozygous for the mutation 1151 ACG to ATG, resulting in an amino acid substitution 384 Thr to Met. In one allele of PK 'Bukarest' a single nucleotide substitution GAG-TAG was found at nucleotide 721, causing a change of 241 Glu to a chain termination codon (PK 'Bukarest'). Additionally, in the second allele of this patient a point mutation at position 1594 (CGG-TGG) occurs, changing 532 Arg to Trp (PK 'Bukarest'). Direct sequencing showed the heterozygosity of the patient's mother (PK 'Bukarest'/normal) at position 721 and of the patient's father (PK 'Bukarest'/normal) at position 1594. A point mutation at position 1529 (CGA-CAA), causing an amino acid substitution 510 Arg-Gln, was identified in PK 'Hamburg' and PK 'Köln'. The second mutation in these variants was not detected. In PK 'Essen' no mutation in the coding sequence was found at all. Screening for the mutation at position 1529 in further compound heterozygote patients and in normal subjects of Western European origin showed that this exchange is a common mutation responsible for PK deficiency in this population.

Adult↗
PubMed2003

Variants in CHEK2 other than 1100delC do not make a major contribution to breast cancer susceptibility.

We recently reported that a sequence variant in the cell-cycle-checkpoint kinase CHEK2 (CHEK2 1100delC) is a low-penetrance breast cancer-susceptibility allele in noncarriers of BRCA1 or BRCA2 mutations. To investigate whether other CHEK2 variants confer susceptibility to breast cancer, we screened the full CHEK2 coding sequence in BRCA1/2-negative breast cancer cases from 89 pedigrees with three or more cases of breast cancer. We identified one novel germline variant, R117G, in two separate families. To evaluate the possible association of R117G and two germline variants reported elsewhere, R145W and I157T with breast cancer, we screened 737 BRCA1/2-negative familial breast cancer cases from 605 families, 459 BRCA1/2-positive cases from 335 families, and 723 controls from the United Kingdom, the Netherlands, and North America. All three variants were rare in all groups, and none occurred at significantly elevated frequency in familial breast cancer cases compared with controls. These results indicate that 1100delC may be the only CHEK2 allele that makes an appreciable contribution to breast cancer susceptibility.

Base Sequence↗
PubMed2004

A novel missense mutation in the galactosyltransferase-I (B4GALT7) gene in a family exhibiting facioskeletal anomalies and Ehlers-Danlos syndrome resembling the progeroid type.

The Ehlers-Danlos syndrome (EDS) is a heterogeneous group of heritable connective tissue disorders characterized by skin hyperextensibility, joint hypermobility, and tissue fragility. Several genes have been implicated to result in EDS phenotypes. The progeroid type of EDS is characterized by wrinkled, loose skin on the face, curly fine hair, scanty eyebrows and eyelashes, in addition to the classical features of EDS. Here we describe two similarly affected individuals in two sibships of a large consanguineous family from Qatar. DNA samples from affected and unaffected members of the family were analyzed for homozygosity of polymorphic markers associated with genes that have been implicated in EDS. Among 28 markers analyzed, homozygosity was only observed for D5S469 and D5S2111, which were markers for galactosyltransferase-I (B4GALT7) located on chromosome 5q35.2, where the previously reported progeroid-like variant of EDS has been mapped. Exons harboring the coding regions and exon-intron junctions of B4GALT7 were amplified by PCR and examined for mutations. A homozygous misssense C to T substitution at nucleotide 808 in the coding region was discovered in both affected individuals. The carrier parents were heterozygous for this mutation, which was not found among 76 DNA samples from control individuals of the same ethnicity. Segregation of this novel mutation in the family further confirmed the allelic variant and its recessive mode of inheritance in this type of EDS. The C to T substitution results in an arginine to cysteine change at amino acid residue 270 that is located in the catalytically active extracellular C-terminal domain. This change could result in abnormal protein folding and/or aberrant interactions of mutated galactosyltransferase-I with other extracellular matrix proteins leading to the development of a progeroid-like phenotype in affected individuals.

Adult↗
PubMed2001

A novel apolipoprotein E5 variant with a 24-bp insertion causing hyperlipidemia.

A tandem 24-bp insertion in the apolipoprotein E (apo E) gene was detected in a patient with elevated triglyceride, apolipoprotein (apo) CII, and apo CIII levels. This novel variant, apo E5ss, showed in position apo E5 by isoelectric focusing and was of larger molecular weight than apo E3 during two-dimensional gel electrophoresis. Polymerase chain reaction-single strand conformation polymorphism analysis using the primer pairs that cover all the coding regions was useful for rapid detection of the variant of the apo E allele. Apo E5ss may have a 24-bp insertion caused by slipped mispairing, resulting in a tandem duplication of amino acid residues 135-142 [APOE, 24-BP INS, DUP CODONS 135-142]. The proband was the only person with apo E5ss among the 806 Japanese males that we examined. We inspected six other reported apo E5 variants in the literature.

Amino Acid Sequence↗
PubMed2005

Genetic variants of the human dipeptide transporter PEPT1.

We tested whether genetic polymorphisms affect activity of the dipeptide transporter PEPT1, which mediates bioavailability of peptidomimetic drugs. All 23 exons and adjoining intronic sections of PEPT1 (SLC15A1) were sequenced in 247 individuals of various ethnic origins (Coriell collection). Of 38 single nucleotide polymorphisms (SNPs), 21 occurred in intronic and noncoding regions and 17 in exonic coding region, of which nine were nonsynonymous. Eight nonsynonymous variants were cloned into expression vectors and functionally characterized after transient transfection into Cos7 and Chinese hamster ovary cells. None of the variants had altered transport activity for various ligands, supporting previous results, except for the new, low-frequency PEPT1-F28Y. This variant displayed significantly reduced cephalexin uptake attributable to increased K(m). Altered pH dependence of substrate transport suggested a role for F28Y in H(+)-driven translocation. Haplotype analysis revealed significant differences among ethnic populations. To search for cis-acting polymorphisms affecting transcription and mRNA processing, we measured allelic PEPT1 mRNA expression in human intestinal biopsy samples using a frequent-marker SNP in exon 17. Of 24 heterozygous samples, significant differences in allelic mRNA levels of 20 to 30% were observed in seven tissues. However, the small difference suggests that cis-acting regulatory factors have only limited effects on transporter activity. We also measured the relative formation of a splice variant (PEPT1-RF). PEPT1-RF mRNA levels ranged from 2 to 44% of total PEPT1-related mRNA, with potential consequences for drug absorption. Together with previous results, this study reveals a relatively low level of genetic variability in polymorphisms affecting both protein function and gene regulation.

Animals↗
PubMed2006

A state-specific polarizable continuum model time dependent density functional theory method for excited state calculations in solution.

An effective state specific (SS) model for the inclusion of solvent effects in time dependent density functional theory (TD-DFT) computations of excited electronic states has been developed and coded in the framework of the so-called polarizable continuum model (PCM). Different relaxation time regimes can be treated thus giving access to a number of different spectroscopic properties together with solvent relaxation energies of paramount relevance in electron transfer processes. SS and conventional linear response (LR) models have been compared for two benchmark systems (coumarin 153 and formaldehyde in different solvents) and in the limiting simple case of a dipolar solute embedded in a spherical cavity. The results point out the complementarity of LR and SS approaches and the advantages of the latter model especially for polar solvents. The favorable scaling properties of PCM-TD-DFT models in both SS and LR variants and their availability in effective quantum mechanical codes pave the route for the computation of reliable spectroscopic properties of large molecules of technological and/or biological interest in their natural environments.

Biophysics↗
PubMed2005

Translation and assembly of CABYR coding region B in fibrous sheath and restriction of calcium binding to coding region A.

CABYR is a highly polymorphic, sperm flagellar calcium-binding protein that is tyrosine as well as serine/threonine phosphorylated during capacitation. Six alternative splice variants of human CABYR (I-VI) have previously been identified, involving two coding regions, CR-A and CR-B, separated by an intervening stop codon. It is presently unknown if proteins encoded by the predicted coding region B of CABYR are translated during spermiogenesis, where they localize, or which CABYR isoforms bind calcium. Immunofluorescent and electron microscopic studies using polyclonal antibodies generated to the recombinant c-terminal 198 aa CABYR-B localized the isoforms containing CABYR-B to the ribs and longitudinal columns of the fibrous sheath in the principal piece of the flagellum. Antisera to recombinant CABYR-A and CABYR-B proteins recognized distinct populations of CABYR isoforms encoded by either CR-A alone and/or CR-B as well as a common population of CABYR isoforms. Only the recombinant CABYR-A and not the CABYR-B bound calcium in vitro, which is consistent with the hypothesis that CABYR-A is the only form that binds calcium in sperm. These observations confirmed that, despite the presence of the stop codon in CR-A, splice variants containing CR-B are expressed during spermiogenesis and assemble into the fibrous sheath of the principal piece; however, calcium binding occurs only to those CABYR isoforms containing CABYR-A.

Alternative Splicing↗
PubMed1985

Some effects of plasmids coding for antibiotic resistance on the virulence of Staphylococcus aureus.

The relative virulence of pairs of staphylococci differing in resistance plasmid content has been studied using the neonatal mouse weight gain test. Both clinical and laboratory strains were used which had undergone genetic manipulation, either curing for loss of plasmids or transduction for gain of plasmids. A difference in virulence was detected between two variants of S. aureus NCTC 8325 possessing different plasmids coding for penicillinase. However in most cases any form of genetic manipulation seemed to reduce the virulence of the staphylococcus. In the case of NCTC 9789 (PS 80) which was originally an epidemic strain, curing of a plasmid coding for cadmium resistance resulted in reduced virulence but original virulence could not be restored by transduction of the plasmid into the cured derivative.

Animals↗
PubMed2003

Excision of the first intron from the gonadotropin-releasing hormone (GnRH) transcript serves as a key regulatory step for GnRH biosynthesis.

The mammalian gonadotropin-releasing hormone (GnRH) gene consists of four short exons (denoted as 1, 2, 3, and 4) and three intervening introns (A, B, and C). Recently, we demonstrated that excision of the first intron (intron A) from the GnRH transcript is regulated in a tissue- and developmental stage-specific fashion and is severely attenuated in hypogonadal (hpg) mouse because of its lack of exonic splicing enhancers (ESE) 3 and 4. In the present study, we examined the influence of intron A on translational efficiency, thereby establishing a post-transcriptional control over GnRH biosynthesis. First, we verified that an intron A-retained GnRH transcript is a splicing variant but not a splicing intermediate. Intron A-retained transcripts can be transported to the cytoplasm in contrast to intron B-containing transcripts, which are restricted to the nucleus. This result implicates the intron A-retained GnRH transcript as a splicing variant; it has a long 5'-untranslated region, as the GnRH prohormone open reading frame (ORF) begins on exon 2. We investigated whether an intron A-retained GnRH transcript can properly initiate translation at the appropriate start codon and found that intron A completely blocks the translation initiation of its downstream reporter ORF both in vivo and in vitro. The inhibition of translation initiation appears to be due to the presence of a tandem repeat of ATG sequences within intron A. Constructs bearing mutations of ATGs to AAGs restored translation initiation at the downstream start codon; the extent of this restoration correlated with the number of mutated ATGs. Besides the failure in the translation initiation of GnRH-coding region in the intron A-containing variant, the present study also suggests that the interference between mature GnRH mRNA and intron A-retained splicing variant could occur to lower the efficiency of GnRH biosynthesis in the GT1-1-immortalized GnRH-producing cell line. Therefore, our results indicate that the precise and efficient excision of intron A and the joining of adjacent exons may be a critical regulatory step for the post-transcriptional regulation of GnRH biosynthesis.

Alternative Splicing↗