Major mRNA Transcripts Research Articles

Dysregulation of RNA Splicing in Tauopathies

SUMMARYPathological aggregation of RNA binding proteins (RBPs) is associated with dysregulation of RNA splicing in PS19 P301S tau transgenic mice and in Alzheimer’s disease brain tissues. The dysregulated splicing particularly affects genes involved in synaptic transmission. The effects of neuroprotective TIA1 reduction on PS19 mice are also examined. TIA1 reduction reduces disease-linked alternative splicing events for the major synaptic mRNA transcripts examined, suggesting that normalization of RBP functions is associated with the neuroprotection. Use of the NetDecoder informatics algorithm identifies key upstream biological targets, including MYC and EGFR, underlying the transcriptional and splicing changes in the protected compared to tauopathy mice. Pharmacological inhibition of MYC and EGFR activity in neuronal cultures tau recapitulates the neuroprotective effects of TIA1 reduction. These results demonstrate that dysfunction of RBPs and RNA splicing processes are major elements of the pathophysiology of tauopathies, as well as potential therapeutic targets for tauopathies.

A multi-centre international quality control study comparing mRNA splicing assay protocols and reporting practices from the ENIGMA consortium

Classification of intronic and predicted missense changes in the breast cancer susceptibility genes BRCA1 and BRCA2 remains a significant challenge for management of patients carrying these variants. Defective mRNA splicing is established as a pathway to disease, and mRNA analysis of unclassified variants has been shown to assist in classification and genetic counselling. However the interpretation of splicing assay results can be difficult, particularly for those variants that give rise to aberrations in a background of naturally occurring isoforms. The ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles) consortium was set up to facilitate research and improve research methods used to classify rare variants in the BRCA1 and BRCA2 (and potentially other) breast cancer predisposition genes. ENIGMA has established a Splicing Working Group, with stated purpose to pool the expertise of different active research groups to conduct large-scale studies that improve the clinical classification of likely spliceogenic variants. An initial project of the Splicing Working Group is to assess the consistency of protocols and results obtained across the multiple participating laboratories from Australia, Europe, UK and the USA. A comparison of mRNA assay protocols in use across 21 labs has identified differences in source material for RNA assays (cultured and uncultured lymphocytes, lymphoblastoid cell lines (LCLs) or constructs), differential use of nonsense-mediated decay inhibitors, and numerous differences in mRNA extraction, DNase treatment and cDNA synthesis methods. A second phase of the project is now underway to determine the impact of the splicing assay methods routinely used by these laboratories on assay data and clinical interpretation of a panel of variants. LCLs were selected from the kConFab repository from carriers of a variant associated with single major aberrant mRNA transcript absent in controls (n=4); carriers of a variant associated with a complicated aberrant mRNA splicing profile involving multiple transcripts including naturally occurring isoforms (n=5); female cancer-free controls (n=11). LCLs have already been distributed to 15 of 20 participating sites, and mRNA assays are underway. Preliminary results indicate that major aberrations associated with several variants mirror results previously observed for mRNA from uncultured lymphocytes. In addition, there is evidence for notable differences in expression of some isoforms compared to results previously observed for RNA from uncultured lymphocytes . This collaborative effort will provide information to inform optimal standardised mRNA splicing assay methodology, and to improve guidelines for clinical interpretation of assay results.

Abstract 1085: RbBP6 isoform 3/DWNN is essential for arsenic trioxide-induced G2/M arrest in cancer cell lines

Abstract Retinoblastoma binding protein 6 (RbBP6) is a gene that encodes RbBP6 protein products (isoforms 1,2 and 3) that has previously been documented to bind retinoblastoma protein (pRb) and p53, which are key components of cell cycle regulation and localises on chromosome 16p12.2 in humans. These isoforms are derived from two major mRNA transcripts, a 1.1 kb (Isoform 3) and 6.1 kb (isoform 1and 2). These two tumour suppressors are important to the understanding the function of the RbBP6. Arsenic trioxide has been used to treat APLS and has been shown to arrest cells at G1 and G2. In this study, the involvement of the DWNN in cell cycle arrest caused by As2O3 was investigated. RNA interference was employed to investigate the involvement of the DWNN in cell cycle regulation. Immunocytochemistry was employed to determine the localization of the DWNN-containing proteins in human cancer cells as well as non-cancerous cells. Propidium iodide cell cycle analysis was used to quantify cell cycle arrest induced by As2O3. Western blotting and real-time PCR were used to determine the expression of the DWNN in both control and treated cells. RNA interference was employed to investigate the involvement of the DWNN in cell cycle regulation. Immunocytochemistry was employed to determine the localization of the DWNN-containing proteins in human cancer cells as well as non-cancerous cells. Propidium iodide cell cycle analysis was utilised to quantify cell cycle arrest induced by As2O3. Western blotting and real-time PCR were used to determine the expression of the DWNN in both control and treated cells. DWNN knockdown increased cell proliferation and did not have an effect on apoptosis. Treatment of human cells with low concentrations of As2O3 induced G2 cell cycle arrest and resulted in induced expression of DWNN. This induced cell cycle arrest was also associated with deglycosylation of the DWNN domain. This finding is in line with the localization of the DWNN proteins in human cancer cells, which showed high expression of the DWNN proteins in G2/M cell cycle phase. Expression analysis showed that the up-regulation of the DWNN domain resulted in p53 up-regulation. These results strongly suggest that the DWNN may be either required for G2 cell cycle arrest or may be required for p53 stabilization during this phase or may be needed for signalling for progression to the next phase of cell cycle. DWNN involvement in cell cycle may be in a p53-dependent manner. Staurosporine which induces G1 cell cycle arrest did not result in DWNN up-regulation and that suggest that DWNN may not be required for G1 cell cycle arrest or regulation. The effect of DWNN in pRb phosphorylation and G1 cell cycle arrest still needs more attention. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1085.

PU.1 Positively Regulates GATA-1 Expression in Mast Cells

Coexpression of PU.1 and GATA-1 is required for proper specification of the mast cell lineage; however, in the myeloid and erythroid lineages, PU.1 and GATA-1 are functionally antagonistic. In this study, we report a transcriptional network in which PU.1 positively regulates GATA-1 expression in mast cell development. We isolated a variant mRNA isoform of GATA-1 in murine mast cells that is significantly upregulated during mast cell differentiation. This isoform contains an alternatively spliced first exon (IB) that is distinct from the first exon (IE) incorporated in the major erythroid mRNA transcript. In contrast to erythroid and megakaryocyte cells, in mast cells we show that PU.1 and GATA-2 predominantly occupy potential cis-regulatory elements in the IB exon region in vivo. Using reporter assays, we identify an enhancer flanking the IB exon that is activated by PU.1. Furthermore, we observe that in PU.1(-/-) fetal liver cells, low levels of the IE GATA-1 isoform is expressed, but the variant IB isoform is absent. Reintroduction of PU.1 restores variant IB isoform and upregulates total GATA-1 protein expression, which is concurrent with mast cell differentiation. Our results are consistent with a transcriptional hierarchy in which PU.1, possibly in concert with GATA-2, activates GATA-1 expression in mast cells in a pathway distinct from that seen in the erythroid and megakaryocytic lineages.

The human ABCG1 gene: identification of LXR response elements that modulate expression in macrophages and liver

The ABC transporter ABCG1 (ATP binding cassette transporter G1), expressed in macrophages, liver, and other tissues, has been implicated in the efflux of cholesterol to high density lipoprotein. The ABCG1 gene is transcriptionally activated by cholesterol loading and activators of liver X receptors (LXRs) and retinoid X receptors (RXRs) through genomic sequences that have not been fully characterized. Here we show that ABCG1 mRNA is induced by LXR agonists in RAW264.7 macrophage cells, HepG2 hepatoma cells, and primary mouse hepatocytes. We identify two evolutionarily highly conserved LXR response elements (LXREs), LXRE-A and LXRE-B, located in the first and second introns of the human ABCG1 gene. Each element conferred robust LXR-agonist responsiveness to ABCG1 promoter-directed luciferase gene constructs in RAW264.7 and HepG2 cells. Overexpression of LXR/RXR activated the ABCG1 promoter in the presence of LXRE-A or LXRE-B sequences. In gel-shift assays, LXR/RXR heterodimers bound to wild-type but not to mutated LXRE-A and LXRE-B sequences. In chromatin immunoprecipitation assays, LXR and RXR were detected at LXRE-A and -B regions of DNA of human THP-1 macrophages. These studies clarify the mechanism of transcriptional upregulation of the ABCG1 gene by oxysterols in macrophages and liver, two key tissues where ABCG1 expression may affect cholesterol balance and atherogenesis.

MEK-ERK Signaling Controls Hdm2 Oncoprotein Expression by Regulating hdm2 mRNA Export to the Cytoplasm

The physical and functional interaction between the transcription factor p53 and its negative regulatory partner protein Hdm2 (Mdm2 in mouse) is a key point of convergence of multiple signaling pathways that regulates cell proliferation and survival. hdm2 mRNA transcription is induced by p53, forming the basis of an auto-regulatory feedback loop. Growth and survival factor-activated Ras-Raf-MEK-ERK signaling can also regulate Hdm2 expression independently of p53, contributing to the pro-survival effect of these factors. In murine fibroblasts, this occurs through the regulation of mdm2 mRNA transcription. Here we show that, in human breast cancer epithelial cells, MEK-dependent regulation of Hdm2 expression also occurs at a post-transcriptional level. Pharmacological blockade of MEK activity in T47D cells inhibits Hdm2 protein synthesis by 80-90%. This occurs in the absence of changes in the expression of the major hdm2-P1 mRNA transcript and only an approximately 40% reduction in hdm2-P2 transcript levels. The amounts of both transcripts that are associated with polyribosomes and are, hence, being actively translated are reduced by >80% by the MEK inhibitor, U0126. We show here that this is due to the inhibition of hdm2 mRNA export from the nucleus when MEK activity is inhibited. In MCF-7 breast cancer cells that express wild-type p53, Hdm2 is required to suppress p53-dependent transcription when MEK kinase is active. Regulation of the nuclear export of hdm2 mRNA provides, therefore, a mechanism whereby mitogen-stimulated cells avoid p53-dependent cell cycle arrest or apoptosis by maintaining the dynamic equilibrium of the Hdm2-p53 feedback loop.

Transcription Factor NFIC Undergoes N-Glycosylation during Early Mammary Gland Involution

Expression of a 74-kDa nuclear factor I (NFI) protein is triggered in early involution in the mouse mammary gland, and its expression correlates with enhanced occupation of a twin (NFI) binding element in the clusterin promoter, a gene whose transcription is induced at this time (Furlong, E. E., Keon, N. K., Thornton, F. D., Rein, T., and Martin, F. (1996) J. Biol. Chem. 271, 29688-29697). We now identify this 74-kDa NFI as an NFIC isoform based on its interaction in Western analysis with two NFIC-specific antibodies. A transition from the expression of a 49-kDa NFIC in lactation to the expression of the 74-kDa NFIC in early involution is demonstrated. We show that the 74-kDa NFIC binds specifically to concanavalin A (ConA) and that this binding can be reversed by the specific ConA ligands, methyl alpha-D-mannopyranoside and methyl alpha-D-glucopyranoside. In addition, its apparent molecular size was reduced to approximately 63 kDa by treatment with the peptide N-glycosidase. The 49-kDa lactation-associated NFIC did not bind ConA nor was it affected by peptide N-glycosidase. Tunicamycin, a specific inhibitor of N-glycosylation, blocked formation of the 74-kDa NFI in involuting mouse mammary gland in vivo when delivered from implanted Elvax depot pellets. Finally, the production of the ConA binding activity could be reiterated in "mammospheres" formed from primary mouse mammary epithelial cells associated with a laminin-rich extracellular matrix. Synthesis of the 74-kDa NFIC was also inhibited in this setting by tunicamycin. Thus, involution triggers the production of an NFIC isoform that is post-translationally modified by N-glycosylation. We further show, by using quantitative competitive reverse transcriptase-PCR, that there is increased expression of the major mouse mammary NFIC mRNA transcript, mNFIC2, in early involution, suggesting that the involution-associated change in NFIC expression also has a transcriptional contribution.

The genomic structure of the human SPEC1 gene reveals complex splicing and close promoter proximity to the AF1q translocation gene

SPECs are small Cdc42 signaling molecules. In mammals, two genes, SPEC1 and SPEC2, encode proteins of 79 and 84 amino acid residues, respectively. Here we report the expression and genomic organization of the human SPEC1 gene. Using Northern blot analysis, three major SPEC1 mRNA transcripts of 1.6, 3.3, and 6.3 kb were detected. Identification and sequencing of different sized SPEC1 cDNA clones revealed that the transcript size heterogeneity was due to alternative splicing in the 3′-untranslated region. In addition, a distinct SPEC1 splice variant from within the coding sequence, SPEC1-β, was identified and detected in a variety of human tissues. Analysis of the genomic organization of SPEC1 revealed that the coding sequence of the SPEC1 isoform was derived from exons 2, 3 and 4, while the SPEC1-β isoform was derived from exon 2 and a read-through event of intron 2. Examination of the 5′-end of the SPEC1 genomic sequence revealed that AF1q, a previously identified gene involved in translocations with the MLL (mixed-lineage leukemia) gene, was 631 bp away in a head-to-head orientation. This intergenic sequence containing the putative promoter region for both SPEC1 and AF1q genes did not contain a TATA box or CAAT box. Transfection experiments using an AF1q promoter luciferase reporter construct in a variety of cells including Cos1 cells, Jurkat T-cells, MCF-7 breast cancer cells, and NIH-3T3 fibroblasts showed no promoter activity. In contrast, a SPEC1 promoter luciferase reporter construct showed high levels of reporter activity in Cos1 and MCF-7 cells, low activity in NIH-3T3 fibroblasts and no activity in Jurkat T-cells. These promoter analyses suggest that although SPEC1 and AF1q genes share the same promoter region, they are not coordinately regulated.

TRH-R2 exhibits similar binding and acute signaling but distinct regulation and anatomic distribution compared with TRH-R1.

TRH (thyroliberin) is a tripeptide (pGlu-His-ProNH2) that signals via G protein-coupled receptors. Until recently, only a single receptor for TRH was known (TRH-R1), but two groups identified a second receptor, TRH-R2. We independently discovered TRH-R2. Using an extensive set of TRH analogs, we found no differences in TRH-R1 and TRH-R2 binding or in acute stimulation of signaling. TRH-R2 was more rapidly internalized upon binding TRH and exhibited a greater level of TRH-induced down-regulation than TRH-R1. During prolonged exposure to TRH, cells expressing TRH-R2 exhibited a lower level of gene induction than cells expressing TRH-R1. TRH-R2 receptor mRNA was present in very discrete nuclei and regions of rat brain. A major mRNA transcript for TRH-R2 was seen in the cerebral cortex, pons, thalamus, hypothalamus, and midbrain with faint bands found in the striatum and pituitary. The extensive distribution of TRH-R2 in the brain suggests that it mediates many of the known functions of TRH that are not transduced by TRH-R1. The variations in agonist-induced internalization and down-regulation/desensitization, and anatomic distribution of TRH-R2 compared with TRH-R1, suggest important functional differences between the two receptors.

Expression of the components of the insulin-like growth factor axis across the growth-plate

Linear bone growth occurs as the result of proliferation and differentiation of growth-plate chondrocytes. These two phases of chondrocyte growth are regulated separately, with insulin-like growth factor I (IGF-I) being the primary stimulator of proliferation. We studied the expression of the components of the growth hormone GH/IGF system to learn if this proliferative signal is altered as chondrocytes undergo differentiation. Growth-plate chondrocytes were isolated from fetal cows and fractionated on discontinuous Percoll gradients. Five populations were recovered, ranging from high density cells (proliferative chondrocytes) to low density cells (hypertrophic chondrocytes). Messenger RNAs (mRNAs) were analyzed by a reverse transcriptase/quantitative polymerase chain reaction (RT/qPCR) technique. Results showed that mRNA of IGF-I and IGF-II in proliferative chondrocytes was 32 and five fold more abundant, respectively, than in hypertrophic chondrocytes. Of the four major IGF-I mRNA transcripts, the class 1-Ea transcript was predominant. Messenger RNA levels for IGFBP-3, -4, and -5 were also reduced in hypertrophic chondrocytes. Levels of GH receptor, the type 1 IGF receptor, and IGF binding protein-2 (IGFBP-2) mRNAs were unchanged across the growth-plate. Since IGF-I and -II are potent stimulators of proliferation, the down-regulation of these genes may be necessary in order for hypertrophy to proceed.

Cloning and expression of Aplysia carboxypeptidase D, a candidate prohormone-processing enzyme.

Many peptide hormones in a variety of species are produced from larger precursors by limited proteolysis at basic amino acid-containing sites. The marine mollusc Aplysia has homologs of mammalian peptide-processing enzymes, including furin, prohormone convertase 1 (PC1), PC2, and carboxypeptidase E (CPE). A novel neuronal Aplysia enzyme was recently identified that was most closely related to carboxypeptidase D (CPD; Fan and Nagle, DNA Cell Biol. 15, 937-945, 1996), a second carboxypeptidase thought to be present in the secretory pathway and to contribute to peptide hormone processing. We have identified and cloned multiple overlapping bag-cell neuron cDNAs that encode two proteins that are members of the CPD family. Sequence analyses demonstrate that the longer CPD protein (1446 residues) contains an N-terminal signal peptide and four carboxypeptidase-like domains; the third and fourth domains are not predicted to form active enzymes, as several critical residues are absent. The shorter CPD protein is predicted to contain two active carboxypeptidase-like domains. Northern blot analysis identified a major Aplysia CPD mRNA (5.3 kb) and several smaller minor transcripts in central nervous system tissue. The CPD was purified from Aplysia ovotestis using a method previously developed for mammalian CPD. The purified Aplysia CPD binds antisera raised against regions of the protein encoded by the Aplysia cDNA clone, as well as an antiserum raised against duck CPD. The enzymatic properties of purified Aplysia CPD are generally similar to those of mammalian CPD. Aplysia CPD is a candidate prohormone-processing enzyme that may play a role in the processing of Aplysia prohormones in the secretory pathway.

Alternative transcription initiation sites generate two LCA1 Ca2+-ATPase mRNA transcripts in tomato roots.

The tomato LCA1 gene encodes a Ca2+-ATPase and gives rise to two major mRNA transcripts and two distinct protein products of different size in tomato roots. The basis of the transcript size difference was investigated to assess whether the mRNA transcripts encoded distinct protein products. Primer extension and S1 nuclease analysis identified two transcription initiation sites at -72 and -1392 from the start of translation. RNA gel blot analysis of poly(A)+ RNA isolated from phosphate-starved tomato roots using probes designed to domains of the 5'-untranslated region (UTR) or the full-length LCA1 cDNA identified mRNAs of 4.7 and 3.6 kb, corresponding to mRNA originating from transcription initiation sites -1392 and -72, respectively. Screening of a cDNA library derived from phosphate-starved tomato roots yielded three cDNA clones, LCA1A, LCA1B and LCA1C (3.6, 4.5 and 5.1 kb respectively). These cDNAs contain full-length LCA1 mRNA sequence derived from each transcription initiation site, with LCA1C additionally containing an intron of 0.6 kb. Sequence analysis indicated 100% identity between the three size classes of cDNA clones except for the differential 5'-UTR and the unspliced intron. Overall, the results indicate that the two major LCA1 mRNA transcripts are derived by differential transcription initiation and that two of the mRNAs may encode identical protein products, while a third mRNA may correspond to a non-functional truncated protein.

Analysis of CD39/ATP Diphosphohydrolase (ATPDase) Expression in Endothelial Cells, Platelets and Leukocytes

Purinergic signaling may influence hemostasis, inflammatory responses and apoptosis. Therefore, hydrolysis of extracellular ATP and ADP by the ATP diphosphohydrolase (ATPDase) could regulate these processes. We have previously demonstrated the identity between the vascular ATPDase and CD39. Here we show that levels of CD39 expression correlate with ATPDase activity in human endothelial cells (EC), platelets and selected monocyte, NK, and megakaryocyte cell lines. Western blotting revealed one to three isoforms of CD39/ATPDase: mobility variations of major protein resulted from post-translational modifications. Northern blotting and primer extension indicated two major mRNA transcripts and one transcription start point, respectively. In addition, mRNAs specific for purinergic P2 receptors were detected in all of the investigated cells, suggesting that the coexpressed CD39/ATPDase may regulate purinergic signaling. Thrombotic and inflammatory responses may be modulated by the expression of CD39/ATPDase.

Two Isoforms of a Human Intersectin (ITSN) Protein Are Produced by Brain-Specific Alternative Splicing in a Stop Codon

Using selected trapped exons with homology to specific protein domains, we identified a new full-length cDNA encoding a protein containing many motifs for protein–protein interactions. There are two major mRNA transcripts, a ubiquitously expressed mRNA of 5.3 kb and a brain-specific transcript of approximately 15 kb, encoding proteins of 1220 and 1721 amino acids, respectively. The stop codon of the ORF of the shorter transcript is split between adjacent exons. In brain tissues the last exon of the short transcript is skipped, and an alternative downstream exon, the first of several additional, is used to produce the 15-kb mRNA. The putative human protein is highly homologous toXenopusintersectin (81% identical) and toDrosophiladynamin-associated protein, Dap160 (31% identical) and was termed intersectin (ITSN). Both human proteins contain five SH3 (Src homology 3) domains, two EH (Eps15 homology) domains, and an α-helix-forming region. The brain-specific long transcript encodes for three additional domains: a GEF (guanine-nucleotide exchange factors), a PH (pleckstrin homology), and a C2 domain. TheDrosophilahomologue is associated with dynamin, a protein family involved in the endocytic pathway and/or synaptic vesicle recycling. The structure of the human ITSN protein is consistent with its involvement in membrane-associated molecular trafficking and signal transduction pathways. The human ITSN gene has been mapped to 21q22.1–q22.2 between markers D21S319 and D21S65, and its importance in Down syndrome and monogenic disorders is currently unknown.

Molecular cloning of a cDNA encoding the neuropeptides APGWamide and cerebral peptide 1: localization of APGWamide-like immunoreactivity in the central nervous system and male reproductive organs of Aplysia.

While much is known about the neural and endocrine mechanisms that control egg laying in the gastropod mollusk Aplysia, relatively little is known about the regulation of male reproductive activity in this simultaneous hermaphrodite. In the present study, we have cloned and sequenced a cDNA that encodes a precursor protein, the predicted posttranslational processing of which presumably generates nine copies of the neuropeptide Ala-Pro-Gly-Trp-NH2 (APGWamide), five connecting peptide sequences, and a C-terminal peptide. The sequence of one connecting peptide is identical to the previously characterized cerebral peptide 1. Northern blot analysis identified two major APGWamide mRNA transcripts (approximately 1.3 kb, approximately 2.4 kb), which were present in central nervous system ganglia, but were most abundant in the right cerebral and right pedal ganglia. Immunohistochemical studies using sexually mature Aplysia demonstrated that the vast majority of APGWamide-like immunoreactivity was localized in 30-40 neurons along the anterior and medial margins of the right cerebral ganglion and in a cluster of 15-20 neurons in the right pedal ganglion. A total of only about ten immunoreactive neurons were located in other ganglia. Immunohistochemistry also demonstrated that APGWamide was present in the reproductive organs that participate in the storage or transport of sperm, including the small hermaphroditic duct (site of sperm storage before mating), the white hemiduct (also known as the copulatory duct), and penial complex. As a group, these data suggest that APGWamide may play a role in regulating male reproductive function in Aplysia, as it does in other gastropods.

Expression and localization of prolactin receptor messenger ribonucleic acid in red deer ovary during the estrous cycle and pregnancy.

In this study, expression of the prolactin receptor (PRL-R) gene in the ovaries of cycling and pregnant red deer (Cervus elaphus) hinds was investigated. A 1.9-kilobase (kb) cDNA encoding the cervine long-form PRL-R was amplified by reverse transcriptase polymerase chain reaction from corpus luteum (CL) and liver poly(A)+ RNA. Northern hybridization revealed a major mRNA transcript of 3.5 kb in both tissues. PRL-R mRNA transcripts were localized by in situ hybridization in 15-micron frozen sections of red deer ovaries, collected during the estrous cycle and early pregnancy, with homologous 45-mer [35S]dATP-labeled sense and antisense oligonucleotide probes. Specific hybridization was assessed by measurement of autoradiograph optical density (OD) in CL, follicles and stroma. PRL-R mRNA expression was higher (p < 0.001) in the CL (OD = 22.2 +/- 3.77, n = 11 CL) than in follicles (OD = 2.8 +/- 0.10, n = 224 follicles) and was undetectable in the stroma (OD < 1, limit of detection). No differences in abundance of PRL-R mRNA were observed between follicles divided on the basis of size, health vs. atresia, or stage of estrous cycle or pregnancy, or between CL from pregnant and nonpregnant hinds. In the follicles, PRL-R mRNA was localized to the theca layer. These results suggest a direct role for PRL in red deer ovarian physiology during the estrous cycle and pregnancy.

Differential regulation of TGF-β2 by hormones in rat uterus and mammary gland

Previous work from this laboratory has shown that transforming growth factor β2 (TGF-β2) mRNA is abundant in the pregnant uterus. In the present study, we examined the synthesis and secretion of TGF-β1,2 and 3 in the rat uterus and mammary gland and show differential secretion and expression of TGF-β2 in a tissue specific manner. Elevated levels of TGF-β2 were detected in late pregnant maternal plasmas (> 100 pM), and in the milk (> 500 pM) during early lactation. High concentrations of TGF-β2 (> 200 pM) were also detected in uterine fluids collected from ovariectomized adult rats after high dose estrogen treatment. TGF-β2 mRNA levels were elevated in lobuloalveolar epithelial cells isolated from pregnant mammary gland. Three major transcripts of 3.5, 4.0, and 4.7 kb were seen, of which the 4.7 kb, dominates. Mammary glands of estrogen treated ovariectomized rats showed a similar pattern of TGF-β2 transcripts. In contrast, four major TGF-β2 mRNA transcripts of 5.7, 4.7, 4.0, and 3.5 kb, with the dominant species of 4.0 and 5.7 kb, were observed in uteri from the estrogen treated animals up to 48 h after the last estrogen injection. This suggests that TGF-β2 is regulated in a tissue specific manner. We conclude that the secretion of TGF-β2 is tightly regulated by hormones and that estrogen and prolactin are critical factors in the tissue-specific regulation of the local production of TGF-β2 in the mammary gland and female reproductive tract.

Characterisation of mouse CD37: cDNA and genomic cloning

The leukocyte surface antigen CD37 is a member of the transmembrane 4 superfamily (TM4SF) of glycoproteins which are predicted to span the lipid bilayer four times. The protein sequence and gene structure of mouse CD37 ( Cd37) have been deduced through the isolation of cDNA and genomic clones. The Cd37 gene produces a major mRNA transcript of 1.2 kb that is restricted to cells of lymphoid and myeloid origin. Mouse CD37 is a glycoprotein of 281 amino acids in length, encoded by eight exons that span approximately 5.2 kb. CD37 is highly conserved between species, the mouse sequence sharing amino acid identities of 98% and 79% with rat and human, respectively. Cd37 shows a striking similarity in genomic organisation to other members of the TM4SF, which is consistent with the theory that this superfamily has evolved by gene duplication and divergence from a common ancestral gene.

Hormonal regulation of steroidogenic acute regulatory (StAR) protein messenger ribonucleic acid expression in the rat ovary

Steroidogenic acute regulatory (StAR) protein is thought to mediate the rapid increase in steroid hormone biosynthesis in response to tropic hormones by facilitating cholesterol transport to the inner mitochondrial membrane where the P450 side-chain cleavage enzyme (P450scc) is located. Since cholesterol delivery is the regulated step in steroidogenesis and is dependent onde novo protein synthesis, StAR mRNA levels were examined in response to the tropic hormones, pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). The results of this investigation revealed that major StAR mRNA transcripts of 3.4 and 1.6 kb and a less abundant transcript of 1.2 kb were detected in the adrenal, ovary, and testis. Within the ovary, StAR mRNA levels were regulated by PMSG and hCG. The two major transcripts were increased in the immature rat ovary following PMSG administration and in the ovary, 8 d after ovulation, in response to stimulation by hCG. Serum progesterone levels were increased following hCG treatment in parallel with the enhanced expression of StAR. Following PMSG treatment, ovarian StAR transcripts at 3.4 and 1.6 kb were each increased twofold. In the ovary, 8 d following ovulation, basal ovarian StAR mRNA levels were elevated up to sixfold relative to the preovulatory StAR mRNA levels. Even with the enhanced basal level of StAR mRNA within the ovary 8 d postovulation, hCG administration still resulted in a 2.5- and 7-fold increase in the 3.4 and 1.6 kb (p<0.025) transcripts, respectively, and a 58% increase in serum progesterone. In contrast to the dramatic alterations in StAR mRNA expression following hormonal stimulation, P450scc mRNA levels remained unchanged in response to hCG stimulation. The levels of serum progesterone paralleled the change in ovarian StAR mRNA in all experiments. This study provides the first evidence that StAR mRNA expression in the rat ovary is mediated by gonadotropins, further supporting its important role in the regulation of steroid hormone biosynthesis.

Target specificity of neuronal RNA-binding protein, Mel-N1: direct binding to the 3' untranslated region of its own mRNA

We have identified cDNAs encoding Mel-N1, the mouse homologue of a human nervous system-specific RNA-binding protein, Hel-N1. Two major mRNA transcripts of Mel-N1 were detected predominantly in the adult mouse brain by Northern blot analysis. To gain insight into the RNA binding specificity of Mel-N1, we performed iterative in vitro RNA selection. The resulting in vitro selected RNAs were found to contain AU-rich sequences as well as a GAAA motif in the majority of clones. By means of in vitro binding assays we demonstrate that this GAAA sequence appears to significantly affect the Mel-N1 RNA-binding efficiency. Our studies further reveal that Mel-N1 can bind to its own 3' untranslated region (3'UTR) as well as to the c-fos 3'UTR, and is localized predominantly in the cytoplasmic region in cells, suggesting that posttranscriptional autoregulation of Mel-N1 gene expression occurs in vivo.