Untranslated Region Transcripts Research Articles

IMP1 3' UTR shortening enhances metastatic burden in colorectal cancer.

The RNA-binding protein insulin-like growth factor 2 mRNA binding protein 1 (IMP1) is overexpressed in colorectal cancer (CRC); however, evidence for a direct role for IMP1 in CRC metastasis is lacking. IMP1 is regulated by let-7 microRNA, which binds in the 3' untranslated region (UTR) of the transcript. The availability of binding sites is in part controlled by alternative polyadenylation, which determines 3' UTR length. Expression of the short 3' UTR transcript (lacking all microRNA sites) results in higher protein levels and is correlated with increased proliferation. We used in vitro and in vivo model systems to test the hypothesis that the short 3' UTR isoform of IMP1 promotes CRC metastasis. Herein we demonstrate that 3' UTR shortening increases IMP1 protein expression and that this in turn enhances the metastatic burden to the liver, whereas expression of the long isoform (full length 3' UTR) does not. Increased tumor burden results from elevated tumor surface area driven by cell proliferation and cell survival mechanisms. These processes are independent of classical apoptosis pathways. Moreover, we demonstrate the shifts toward the short isoform are associated with metastasis in patient populations where IMP1-long expression predominates. Overall, our work demonstrates that different IMP1 expression levels result in different functional outcomes in CRC metastasis and that targeting IMP1 may reduce tumor progression in some patients.

A comprehensive analysis of SNCA-related genetic risk in sporadic parkinson disease.

The goal of this study was to refine our understanding of disease risk attributable to common genetic variation in SNCA, a major locus in Parkinson disease, with potential implications for clinical trials targeting α-synuclein. We aimed to dissect the multiple independent association signals, stratify individuals by SNCA-specific risk profiles, and explore expression quantitative trait loci. We analyzed participant-level data from 12,503 patients and 12,502 controls, optimizing a risk model and assessing SNCA-specific risk scores and haplotypes as predictors of individual risk. We also explored hypotheses about functional mechanisms and correlated risk variants to gene expression in human brain and protein levels in cerebrospinal fluid. We report and replicate a novel, third independent association signal at genome-wide significance level downstream of SNCA (rs2870004, p = 3.0*10-8 , odds ratio [OR] = 0.88, 95% confidence interval [CI] = 0.84-0.92). SNCA risk score stratification showed a 2-fold difference in disease susceptibility between top and bottom quintiles (OR = 1.99, 95% CI = 1.78-2.23). Contrary to previous reports, we provide evidence supporting top variant rs356182 as functional in itself and associated with a specific SNCA 5' untranslated region transcript isoform in frontal cortex. The SNCA locus harbors a minimum of 3 independent association signals for Parkinson disease. We demonstrate a fine-grained stratification of α-synuclein-related genetic burden in individual patients of potential future clinical relevance. Further efforts to pinpoint the functional mechanisms are warranted, including studies of the likely causal top variant rs356182 and its role in regulating levels of specific SNCA mRNA transcript variants. Ann Neurol 2018;83:117-129.

Human cellular CYBA UTR sequences increase mRNA translation without affecting the half-life of recombinant RNA transcripts

Modified nucleotide chemistries that increase the half-life (T1/2) of transfected recombinant mRNA and the use of non-native 5′- and 3′-untranslated region (UTR) sequences that enhance protein translation are advancing the prospects of transcript therapy. To this end, a set of UTR sequences that are present in mRNAs with long cellular T1/2 were synthesized and cloned as five different recombinant sequence set combinations as upstream 5′-UTR and/or downstream 3′-UTR regions flanking a reporter gene. Initial screening in two different cell systems in vitro revealed that cytochrome b-245 alpha chain (CYBA) combinations performed the best among all other UTR combinations and were characterized in detail. The presence or absence of CYBA UTRs had no impact on the mRNA stability of transfected mRNAs, but appeared to enhance the productivity of transfected transcripts based on the measurement of mRNA and protein levels in cells. When CYBA UTRs were fused to human bone morphogenetic protein 2 (hBMP2) coding sequence, the recombinant mRNA transcripts upon transfection produced higher levels of protein as compared to control transcripts. Moreover, transfection of human adipose mesenchymal stem cells with recombinant hBMP2-CYBA UTR transcripts induced bone differentiation demonstrating the osteogenic and therapeutic potential for transcript therapy based on hybrid UTR designs.

Efficient inhibition of human immunodeficiency virus replication using novel modified microRNA-30a targeting 3'-untranslated region transcripts.

RNA interference (RNAi)-based gene therapy is currently considered to be a combinatorial anti-human immunodeficiency virus-1 (HIV-1) therapy. Although artificial polycistronic microRNAs (miRs) can reduce HIV-1 escape mutant variants, this approach may increase the risk of side effects. The present study aimed to optimize the efficiency of anti-HIV RNAi gene therapy in order to reduce the cell toxicity induced by multi-short hairpin RNA expression. An artificial miR-30a-3'-untranslated region (miR-3-UTR) obtained from a single RNA polymerase II was used to simultaneously target all viral transcripts. The results of the present study demonstrated that HIV-1 replication was significantly inhibited in the cells with the miR-3-UTR construct, suggesting that miR-3'-UTR may serve as a promising tool for RNAi-based gene therapy in the treatment of HIV-1.

Alternative polyadenylation diversifies post‐transcriptional regulation by selective RNA–protein interactions

Recent research has uncovered extensive variability in the boundaries of transcript isoforms, yet the functional consequences of this variation remain largely unexplored. Here, we systematically discriminate between the molecular phenotypes of overlapping coding and non-coding transcriptional events from each genic locus using a novel genome-wide, nucleotide-resolution technique to quantify the half-lives of 3' transcript isoforms in yeast. Our results reveal widespread differences in stability among isoforms for hundreds of genes in a single condition, and that variation of even a single nucleotide in the 3' untranslated region (UTR) can affect transcript stability. While previous instances of negative associations between 3' UTR length and transcript stability have been reported, here, we find that shorter isoforms are not necessarily more stable. We demonstrate the role of RNA-protein interactions in conditioning isoform-specific stability, showing that PUF3 binds and destabilizes specific polyadenylation isoforms. Our findings indicate that although the functional elements of a gene are encoded in DNA sequence, the selective incorporation of these elements into RNA through transcript boundary variation allows a single gene to have diverse functional consequences.

12-Lipoxygenase Products Reduce Insulin Secretion and β-Cell Viability in Human Islets

Context: Inflammation is increasingly recognized as an important contributing factor in diabetes mellitus. Lipoxygenases (LOs) produce active lipids that promote inflammatory damage by catalyzing the oxidation of linoleic and arachidonic acid, and LO is expressed in rodent and human islets. Little is known about the differential effect of the various hydroxyeicosatetraenoic acids (HETEs) that result from LO activity in human islets. Design: We compared the effect of stable compounds derived from LOs: 12(S)-HETE, 15HETE, 12HPETE, and 12RHETE. Interventions: At both 1 and 100 nm, insulin secretion was consistently reduced by 12(S)-HETE and 12HPETE. 12(S)-HETE also reduced viability activity by 32% at 1 nm. Insulin and reduced viability were partially reversed by treatment with lisofylline, a small-molecule antiinflammatory compound that protects mitochondrial function. 12(S)-HETE also increased cell death rate of human islets by 50% at 100 nm. To investigate mechanisms of 12-LO-mediated islet inhibition, we examined the p38-MAPK and JNK stress-activated pathways. Results: Treatment of 12(S)-HETE significantly increased phosphorylated p38-MAPK (pp38) protein activity in human islets. We further explored the in vivo role of 12-LO in regulating p38-MAPK using mouse models. Knockdown of 12-LO by injecting 12-LO siRNA into C57BL/6 mice decreased pp38 protein levels in mouse islets. The addition of proinflammatory cytokines increased pp38 levels in normal mouse islets but not in siRNA-treated islets. Conclusions: These data suggest that 12(S)-HETE reduces insulin secretion and increases cell death in human islets. The 12-LO pathway is present in human islets, and expression is up-regulated by inflammatory cytokines. Reduction of 12-LO activity could thus provide a new therapeutic approach to protect human -cells from inflammatory injury. Genotype and Tissue-Specific Effects on Alternative Splicing of the Transcription Factor 7-Like 2 Gene in Humans Ashis K. Mondal, Swapan K. Das, Giulia Baldini, Winston S. Chu, Neeraj K. Sharma, Oksana G. Hackney, Jianhua Zhao, Struan F. A. Grant, and Steven C. Elbein (J Clin Endocrinol Metab, 10.1210/jc.2009-2064) ABSTRACT Context: Noncoding single-nucleotide polymorphisms (SNPs) within the TCF7L2 gene are confirmed risk factors for type 2 diabetes, but the mechanism by which they increase risk is unknown. Objective: We hypothesized that associated SNPs alter TCF7L2 splicing and that splice forms have altered biological roles. Design: Splice forms and 5 and 3 untranslated regions were characterized in sc adipose, muscle, liver, HepG2 cells, pancreas, and islet. Isoform-specific transcript levels were quantified in sc adipose. Alternative splice forms were characterized in HepG2 liver cells under glucose and insulin conditions and in SGBS cells with differentiation. Major isoforms were characterized by transfection. Setting: The study was conducted at an ambulatory general clinical research center. Patients: Patients included 78 healthy, nondiabetic study subjects characterized for insulin sensitivity and secretion. Results: We identified 32 alternatively spliced transcripts and multiple-length 3 untranslated region transcripts in adipose, muscle, islet, and pancreas. Alternative exons 3a, 12, 13, and 13a were observed in all tissues, whereas exon 13b was islet specific. Transcripts retaining exons 13 and 13a but not total TCF7L2 transcripts were significantly correlated with both obesity measures (P 0.01) and rs7903146 genotype (P 0.026) in sc adipose. Insulin (5–10 nM) suppressed all TCF7L2 isoforms in SGBS cells but T R A N S L A T I O N A L H I G H L I G H T S F R O M J C E MContext: Noncoding single-nucleotide polymorphisms (SNPs) within the TCF7L2 gene are confirmed risk factors for type 2 diabetes, but the mechanism by which they increase risk is unknown. Objective: We hypothesized that associated SNPs alter TCF7L2 splicing and that splice forms have altered biological roles. Design: Splice forms and 5 and 3 untranslated regions were characterized in sc adipose, muscle, liver, HepG2 cells, pancreas, and islet. Isoform-specific transcript levels were quantified in sc adipose. Alternative splice forms were characterized in HepG2 liver cells under glucose and insulin conditions and in SGBS cells with differentiation. Major isoforms were characterized by transfection. Setting: The study was conducted at an ambulatory general clinical research center. Patients: Patients included 78 healthy, nondiabetic study subjects characterized for insulin sensitivity and secretion. Results: We identified 32 alternatively spliced transcripts and multiple-length 3 untranslated region transcripts in adipose, muscle, islet, and pancreas. Alternative exons 3a, 12, 13, and 13a were observed in all tissues, whereas exon 13b was islet specific. Transcripts retaining exons 13 and 13a but not total TCF7L2 transcripts were significantly correlated with both obesity measures (P 0.01) and rs7903146 genotype (P 0.026) in sc adipose. Insulin (5–10 nM) suppressed all TCF7L2 isoforms in SGBS cells but T R A N S L A T I O N A L H I G H L I G H T S F R O M J C E M

Genotype and Tissue-Specific Effects on Alternative Splicing of the Transcription Factor 7-Like 2 Gene in Humans

Context: Noncoding single-nucleotide polymorphisms (SNPs) within the TCF7L2 gene are confirmed risk factors for type 2 diabetes, but the mechanism by which they increase risk is unknown. Objective: We hypothesized that associated SNPs alter TCF7L2 splicing and that splice forms have altered biological roles. Design: Splice forms and 5′ and 3′ untranslated regions were characterized in sc adipose, muscle, liver, HepG2 cells, pancreas, and islet. Isoform-specific transcript levels were quantified in sc adipose. Alternative splice forms were characterized in HepG2 liver cells under glucose and insulin conditions and in SGBS cells with differentiation. Major isoforms were characterized by transfection. Setting: The study was conducted at an ambulatory general clinical research center. Patients: Patients included 78 healthy, nondiabetic study subjects characterized for insulin sensitivity and secretion. Results: We identified 32 alternatively spliced transcripts and multiple-length 3′ untranslated region transcripts in adipose, muscle, islet, and pancreas. Alternative exons 3a, 12, 13, and 13a were observed in all tissues, whereas exon 13b was islet specific. Transcripts retaining exons 13 and 13a but not total TCF7L2 transcripts were significantly correlated with both obesity measures (P < 0.01) and rs7903146 genotype (P < 0.026) in sc adipose. Insulin (5–10 nM) suppressed all TCF7L2 isoforms in SGBS cells but suppressed exon 13a-containing isoforms most significantly (P < 0.001). The isoform distribution differed throughout SGBS cell differentiation. Isoforms with predicted early stop codons yielded stable proteins of the predicted size, bound β-catenin, and targeted correctly to the nucleus. Conclusions: Intronic TCF7L2 variants may regulate alternative transcript isoforms, which in turn may have distinct physiologic roles. a a a Page 1 of 16 Genotype and Tissue-Specific Effects on Alternative Splicing of the Transcription Factor ... 6/19/2014 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2841530/?report=printable Type 2 diabetes (T2D) is among the common, complex diseases for which genetic predisposition is well accepted (1). Grant et al. (2) reported the association of single-nucleotide polymorphisms (SNPs) in the transcription factor 7-like 2 gene (TCF7L2), and this association has been convincingly replicated as the strongest genetic risk factor for T2D (3). Nonetheless, the SNPs implicated are intronic (4) and are not associated with total TCF7L2 transcripts in adipose and muscle (5). Physiological studies of TCF7L2 risk variants have most consistently demonstrated reduced insulin secretion in large epidemiology studies (6,7,8), but other findings suggest a broader role consistent with widespread gene expression (5,9,10,11). TCF7L2 plays a key role in the Wnt signaling pathway (12) and may be important in the expansion and maintenance of β-cell mass (9). However, both increased and decreased TCF7L2 expression in β-cells has been associated with altered insulin secretion and apoptosis in human islets (11,13). Other studies suggested reduced glucagon-like peptide (GLP)-1 induced insulin secretion among carriers (14) or altered proinsulin processing (15). GLP-1 and GLP-1 agonists may act through TCF7L2 and the Wnt pathway to expand and maintain β-cell mass (12). Whereas recent data strongly support a role for TCF7L2 in the β-cell, the means by which intronic variants alter β-cell function or mass remain unclear. Wnt signaling is known to be important in other tissues, including adipose (16), and the proinflammatory cytokine TNFα may inhibit adipogenesis by way of β-catenin/TCF7L2 signaling (17). Studies from our laboratory and others supported a role for TCF7L2 SNPs in altered insulin sensitivity (10), but we and others (5) failed to find a genotype effect on total adipose TCF7L2 transcript levels (5,18,19). Reports of differences in TCF7L2 expression between visceral and sc adipose are inconsistent (18,20,21). The T2D-associated TCF7L2 variants are located in a linkage disequilibrium block that encompasses introns 3 and 4. The T allele of SNP rs7903146 showed reduced binding to protein factors in several cell lines (22), but the relevance of this finding to T2D risk is uncertain. Previous studies suggested multiple TCF7L2 splice forms in tumor tissue (23) and while this work was in preparation, tissue-specific splicing was confirmed in pancreatic islets, lymphocytes, skeletal muscle, and adipose tissue in nondiabetic human subjects (24,25). We hypothesized that TCF7L2 susceptibility variants act by altering the levels of specific transcript isoforms and that splice forms differ in biological function. We characterized the transcript isoforms and untranslated regions in human tissues relevant to diabetes and quantified isoform-specific transcript levels by genotype in nondiabetic subjects. To examine the physiological role of transcript isoforms, we examined the isoform distribution in response to glucose and insulin and during adipocyte cell differentiation. Finally, we determined whether transcript isoforms yielded stable, nuclear targeted proteins that properly bound β-catenin. Materials and Methods

Genotype and Tissue-Specific Effects on Alternative Splicing of the Transcription Factor 7-Like 2 Gene in Humans

Noncoding single-nucleotide polymorphisms (SNPs) within the TCF7L2 gene are confirmed risk factors for type 2 diabetes, but the mechanism by which they increase risk is unknown. We hypothesized that associated SNPs alter TCF7L2 splicing and that splice forms have altered biological roles. Splice forms and 5' and 3' untranslated regions were characterized in sc adipose, muscle, liver, HepG2 cells, pancreas, and islet. Isoform-specific transcript levels were quantified in sc adipose. Alternative splice forms were characterized in HepG2 liver cells under glucose and insulin conditions and in SGBS cells with differentiation. Major isoforms were characterized by transfection. The study was conducted at an ambulatory general clinical research center. PATIENTS included 78 healthy, nondiabetic study subjects characterized for insulin sensitivity and secretion. We identified 32 alternatively spliced transcripts and multiple-length 3' untranslated region transcripts in adipose, muscle, islet, and pancreas. Alternative exons 3a, 12, 13, and 13a were observed in all tissues, whereas exon 13b was islet specific. Transcripts retaining exons 13 and 13a but not total TCF7L2 transcripts were significantly correlated with both obesity measures (P < 0.01) and rs7903146 genotype (P < 0.026) in sc adipose. Insulin (5-10 nm) suppressed all TCF7L2 isoforms in SGBS cells but suppressed exon 13a-containing isoforms most significantly (P < 0.001). The isoform distribution differed throughout SGBS cell differentiation. Isoforms with predicted early stop codons yielded stable proteins of the predicted size, bound beta-catenin, and targeted correctly to the nucleus. Intronic TCF7L2 variants may regulate alternative transcript isoforms, which in turn may have distinct physiologic roles.

RNA Mapping of Mutant Myotonic Dystrophy Protein Kinase 3'-Untranslated Region Transcripts

Myotonic dystrophy type 1 (DM1), which is a dominantly inherited neurodegenerative disorder, results from a CTG trinucleotide repeat expansion in the 3’-untranslated region (3’-UTR) of the myotonic dystrophy protein kinase (DMPK) gene. Retention of mutant DMPK (mDMPK) transcripts in the nuclei of affected cells has been known to be the main cause of pathogenesis of the disease. Thus, reducing the RNA toxicity through elimination of the mutant RNA has been suggested as one therapeutic strategy against DM1. In this study, we suggested RNA replacement with a trans-splicing ribozyme as an alternate genetic therapeutic approach for amelioration of DM1. To this end, we identified the regions of mDMPK 3’-UTR RNA that were accessible to ribozymes by using an RNA mapping strategy based on a transsplicing ribozyme library. We found that particularly accessible sites were present not only upstream but also downstream of the expanded repeat sequence. Repair or replacement of the mDMPK transcript with the specific ribozyme will be useful for DM1 treatment through reduction of toxic mutant transcripts and simultaneously restore wild-type DMPK or release nucleus-entrapped mDMPK transcripts to the cytoplasm.

Regulation of Plasminogen Activator Inhibitor-1 Expression by Tumor Suppressor Protein p53

H1299 lung carcinoma cells lacking p53 (p53-/-) express minimal amounts of plasminogen activator inhibitor-1 (PAI-1) protein as well as mRNA. p53(-/-) cells express highly unstable PAI-1 mRNA. Transfection of p53 in p53(-/-) cells enhanced PAI-1 expression and stabilized PAI-1 mRNA. On the contrary, inhibition of p53 expression by RNA silencing in non-malignant human lung epithelial (Beas2B) cells decreased basal as well as urokinase-type plasminogen activator-induced PAI-1 expression because of accelerated degradation of PAI-1 mRNA. Purified p53 protein specifically binds to the PAI-1 mRNA 3'-un-translated region (UTR), and endogenous PAI-1 mRNA forms an immune complex with p53. Treatment of purified p53 protein with anti-p53 antibody abolished p53 binding to the 3'-UTR of PAI-1 mRNA. The p53 binding region maps to a 70-nucleotide PAI-1 mRNA 3'-UTR sequence, and insertion of the p53-binding sequence into beta-globin mRNA destabilized the chimeric transcript. Deletion experiments indicate that the carboxyl-terminal region (amino acid residues 296-393) of p53 protein interacts with PAI-1 mRNA. These observations demonstrate a novel role for p53 as an mRNA-binding protein that regulates increased PAI-1 expression and stabilization of PAI-1 mRNA in human lung epithelial and carcinoma cells.

Translational regulation of lipoprotein lipase in adipocytes: depletion of cellular protein kinase Cα activates binding of the C subunit of protein kinase A to the 3′-untranslated region of the lipoprotein lipase mRNA

Adipose LPL (lipoprotein lipase) plays an important role in regulating plasma triacylglycerols and lipid metabolism. We have previously demonstrated that PKCalpha (protein kinase Calpha) depletion inhibits LPL translation in 3T3-F442A adipocytes. Using in vitro translation experiments, the minimum essential region on the 3'UTR (3'-untranslated region) of LPL mRNA required for the inhibition of translation was identified as the proximal 39 nt. These results were confirmed by RNase protection analysis using cytoplasmic proteins isolated from the adipocytes treated with PKCalpha antisense oligomers and the LPL 3'UTR transcript (LPL 3'UTR nt: 1512-1640). The protein components involved in this RNA-binding interaction from PKCalpha depletion were passed through an affinity column containing a sequence of the LPL 3'UTR and, after Western blotting, the RNA-binding proteins were identified as the catalytic and the regulatory subunits of PKA (protein kinase A), Calpha and RIIbeta, and AKAP (A-kinase-anchoring protein) 121. This RNA inhibitory complex consisted of the same RNA-binding proteins that have been identified previously as mediators of LPL translational inhibition by PKA activation, suggesting that PKCalpha depletion inhibits LPL translation through PKA activation. In additional experiments, PKC depletion by prolonged PMA treatment or PKCalpha antisense oligomers resulted in an increase in PKA activity in 3T3-F442A adipocytes, comparable with PKA activation with adrenaline (epinephrine) treatment. These results demonstrate that LPL translational inhibition occurs through an RNA-binding complex involving PKA subunits and AKAP121, and this complex can be activated either through traditional PKA activation methods or through the depletion of PKCalpha.

Expression of Pyruvate Carboxylase mRNA Variants in Liver of Dairy Cattle at Calving

BackgroundBovine liver expresses six pyruvate carboxylase (PC) transcript variants, bPC5′A, bPC5′B, bPC5′C, bPC5′D, bPC5′E, and bPC5′F, which only differ at the 5′ untranslated region (UTR) and contain a common coding region. The objective of this experiment was to determine the profile and abundance of PC transcripts in bovine liver at calving.Methodology/Principal FindingsA ribonuclease protection assay (RPA) protocol was developed to simplify analysis of these variants and investigate the changes in abundance of each 5′ UTR transcript relative to total PC mRNA. Liver biopsy samples collected from seven cows on +1 d relative to calving were analyzed by RPA to determine the profile in PC 5′ UTR variants. Results show that all six bovine PC 5′ UTR variants are detected at calving. Data indicate that bovine PC 5′ UTR variant A is the most abundant, variants C and E are least abundant and expression of variants B, D and F is intermediate at calving.ConclusionsThis manuscript describes a simplified RPA method that quantifies the abundance of six PC transcripts by using two riboprobes. The lack of uniformity in the pattern of PC 5′ UTR variants at calving suggests an additional complexity for control of bovine PC mRNA expression at calving that may be the result of transcriptional controls, variation in mRNA processing, or a combination of these processes.

In Vitro Evidence that Upstream of N-ras Participates in the Regulation of Parathyroid Hormone Messenger Ribonucleic Acid Stability

Calcium and phosphate regulate PTH gene expression posttranscriptionally through the binding of trans-acting factors to a defined cis-acting instability element in the PTH mRNA 3'-untranslated region (UTR). We have previously defined AU-rich binding factor 1 as a PTH mRNA binding and stabilizing protein. We have now identified, by affinity chromatography, Upstream of N-ras (Unr) as another PTH mRNA 3'-UTR binding protein. Recombinant Unr bound the PTH 3'-UTR transcript, and supershift experiments with antibodies to Unr showed that Unr is part of the parathyroid RNA binding complex. Finally, because there is no parathyroid cell line, the functionality of Unr in regulating PTH mRNA levels was demonstrated in cotransfection experiments in heterologous human embryonic kidney 293 cells. Depletion of Unr by small interfering RNA decreased simian virus 40-driven PTH gene expression in human embryonic kidney 293 cells transiently cotransfected with the human PTH gene. Overexpression of Unr increased the rat full-length PTH mRNA levels but not a PTH mRNA lacking the terminal 60-nucleotide cis-acting protein binding region. Unr also stabilized a chimeric GH reporter mRNA that contained the rat PTH 63-nucleotide cis-acting element but not a truncated PTH element. Therefore, Unr binds to the PTH cis element and increases PTH mRNA levels, as does AU-rich binding factor 1. Our results suggest that Unr, together with the other proteins in the RNA binding complex, determines PTH mRNA stability.

Ubiquitin-Proteasome-mediated Degradation, Intracellular Localization, and Protein Synthesis of MyoD and Id1 during Muscle Differentiation

Mammalian skeletal myogenesis results in the differentiation of myoblasts to mature syncytial myotubes, a process regulated by an intricate genetic network of at least three protein families: muscle regulatory factors, E proteins, and Id proteins. MyoD, a key muscle regulatory factor, and its negative regulator Id1 have both been shown to be degraded by the ubiquitin-proteasome system. Using C2C12 cells and confocal fluorescence microscopy, we showed that MyoD and Id1 co-localize within the nucleus in proliferating myoblasts. In mature myotubes, in contrast, they reside in distinctive subcellular compartments, with MyoD within the nucleus and Id1 exclusively in the cytoplasm. Cellular abundance of Id1 was markedly diminished from the very onset of muscle differentiation, whereas MyoD abundance was reduced to a much lesser extent and only at the later stages of differentiation. These reductions in MyoD and Id1 protein levels seem to result from a change in the rate of protein synthesis rather than the rate of degradation. In vivo protein stability studies revealed that the rates of ubiquitin-proteasome-mediated MyoD and Id1 degradation are independent of myogenic differentiation state. Id1 and MyoD were both rapidly degraded, each with a t 1/2 approximately = 1 h in myoblasts and in myotubes. Furthermore, relative protein synthesis rates for MyoD and Id1 were significantly diminished during myoblast to myotube differentiation. These results provide insight as to the interaction between MyoD and Id1 in the process of muscle differentiation and have implications for the involvement of the ubiquitin-proteasome-mediated protein degradation and protein synthesis in muscle differentiation and metabolism under abnormal and pathological conditions.

5′-UTR Structural Organization, Transcript Expression, and Mutational Analysis of the Human Rab Geranylgeranyl Transferase α-Subunit (RABGGTA) Gene

Hermansky-Pudlak syndrome (HPS) is a recessively inherited disease with dysfunction of several related subcellular organelles including platelet-dense granules, melanosomes, and lysosomes. Our recent identification of the mutation in murine Rab geranylgeranyl transferase α-subunit gene (Rabggta) in one mouse model of HPS, the gunmetal mouse, suggested that human patients with similar phenotypes might have mutations in the human orthologous RABGGTA gene. This prompted reanalysis of the 5′-untranslated structure of the human RABGGTA gene in normal individuals and in patients with deficiencies of platelet-dense granules (αδ-SPD), alpha granules (α-SPD or gray platelet syndrome, GPS) or alpha plus dense granules (αδ-SPD). We report the complete sequence of intron α of RABGGTA and demonstrate that exon α is immediately upstream of intron α. The exon/intron structural organization of the 5′-untranslated region (UTR) of human RABGGTA was found to be similar to that of the mouse Rabggta gene. However, exons α and introns α are not homologous between mouse and human. Features of the 5′-UTR of RABGGTA suggest it is a housekeeping gene. While obvious disease-causing mutations of human RABGGTA were not found in our existing SPD patients by sequencing its entire coding region, several polymorphisms of RABGGTA including a putative cryptic splicing mutation in intron 4 were identified. Knowledge of the 5′-UTR structure of RABGGTA and its common polymorphisms will be useful for mutation screening or linkage analysis in patients with albinism, thrombocytopenia, or platelet SPD.

Human CKIα L and CKIα S are encoded by both 2.4- and 4.2-kb transcripts, the longer containing multiple RNA-destablising elements

Casein kinase I (CKI) are a family of conserved second messenger-independent serine/threonine protein kinases found in all eukaryotes. The avian and mammalian CKI alpha isoform has four splice variants differing in the presence or absence of 28 amino acids (‘ L’ insertion) in the catalytic domain and/or 12 amino acids (‘ S’ insertion) in the regulatory domain. Here we report the isolation of cDNAs encoding human CKIα L and CKIα S. We find human CKIα L has a preference to phosphorylate phosvitin over casein, with a higher K m for casein than phosvitin, the reverse being the case for human CKIα S. Both human CKIα L, and CKIα S are derived from 4.2-kb mRNA transcripts and 2.4-kb transcripts, the latter probably generated by use of an alternate polyadenylation signal identified in the longer transcripts. The 4.2-kb transcripts contain six RNA-destabilising AU-rich element (ARE) motifs in the 3′-untranslated region (UTR), while the 2.4-kb transcripts contain a single ARE motif. In vitro analysis of CKI alpha 3′-UTR RNA sequences suggests that in HeLa cells, the longer 3′-UTR transcripts are likely to degrade approximately 13 times faster than the shorter 3′-UTR transcripts. This is the first report of a kinase mRNA containing multiple RNA-destabilising AREs in the longer of two mRNA transcripts.

Post-transcriptional Regulation of Vascular Endothelial Growth Factor by Hypoxia

The major control point for the hypoxic induction of the vascular endothelial growth factor (VEGF) gene is the regulation of the steady-state level of the mRNA. We previously demonstrated a discrepancy between the transcription rate and the steady-state mRNA level induced by hypoxia. This led us to examine the post-transcriptional regulation of VEGF expression. Actinomycin D experiments revealed that hypoxia increased VEGF mRNA half-life from 43 +/- 6 min to 106 +/- 9 min. Using an in vitro mRNA degradation assay, the half-life of VEGF mRNA 3'-untranslated region (UTR) transcripts were also found to be increased when incubated with hypoxic versus normoxic extracts. Both cis-regulatory elements involved in VEGF mRNA degradation under normoxic conditions and in increased stabilization under hypoxic conditions were mapped using this degradation assay. A hypoxia-induced protein(s) was found that bound to the sequences in the VEGF 3'-UTR which mediated increased stability in the degradation assay. Furthermore, genistein, a tyrosine kinase inhibitor, blocked the hypoxia-induced stabilization of VEGF 3'-UTR transcripts and inhibited hypoxia-induced protein binding to the VEGF 3'-UTR. These findings demonstrate a significant post-transcriptional component to the regulation of VEGF.

Degradation of unstable interleukin-1 alpha mRNA in a rabbit reticulocyte cell-free system. Localization of an instability determinant to a cluster of AUUUA motifs.

Labeled transcripts of interleukin-1 alpha (IL-1 alpha) cDNA were rapidly degraded in incubations with rabbit reticulocyte lysate (RRL). In contrast, a transcript of superoxide dismutase cDNA was stable in control incubations. A transcript of the 3'-untranslated region (UTR) of IL-1 alpha was rapidly degraded while that of the 5'-UTR and coding region was stable. This degradative activity was present in the post-ribosomal supernatant. Degradation of the 3'-UTR transcript was inhibited by the addition of a large excess of an 80-base RNA containing four AUUUA repeats, but not by the same RNA without such repeats. This suggested that AUUUA motifs were responsible for the instability of the 3'-UTR transcript. The 80-base RNA did not act as a competitive substrate for a nuclease since it was not degraded. Partial transcripts of IL-1 alpha 3'-UTR were incubated with RRL to localize instability determinants. Transcripts containing at least three clustered AUUUA motifs were rapidly degraded, while transcripts containing four scattered AUUUA motifs were stable. To study the mechanism of RNA degradation, the RRL was passed through an affinity column that retained AUUUA-binding proteins. The flow-through or the fraction eluted from such a column were inactive, but the two fractions together degraded the 3'-UTR transcript. This indicated that proteins bound by the affinity column did not have nuclease activity but targeted this RNA for degradation.

Nucleotide sequence and structural determinants of specific binding of coat protein or coat protein peptides to the 3' untranslated region of alfalfa mosaic virus RNA 4

The specific binding of alfalfa mosaic virus coat protein to viral RNA requires determinants in the 3' untranslated region (UTR). Coat protein and peptide binding sites in the 3' UTR of alfalfa mosaic virus RNA 4 have been analyzed by hydroxyl radical footprinting, deletion mapping, and site-directed mutagenesis experiments. The 3' UTR has several stable hairpins that are flanked by single-stranded (A/U)UGC sequences. Hydroxyl radical footprinting data show that five sites in the 3' UTR of alfalfa mosaic virus RNA 4 are protected by coat protein, and four of the five protected regions contain AUGC or UUGC. Electrophoretic mobility band shift results suggest four coat protein binding sites in the 3' UTR. A 3'-terminal 39-nucleotide RNA fragment containing four AUGC repeats bound coat protein and coat protein peptides with high affinity; however, coat protein bound poorly to antisense 3' UTR transcripts and poly(AUGC)10. Site-directed mutagenesis of AUGC865-868 resulted in a loss of coat protein binding and peptide binding by the RNA fragment. Alignment of alfalfa mosaic RNA sequences with those from several closely related ilarviruses demonstrates that AUGC865-868 is perfectly conserved; moreover, the RNAs are predicted to form similar 3'-terminal secondary structures. The data strongly suggest that alfalfa mosaic virus coat protein and ilavirus coat proteins recognize invariant AUGC sequences in the context of conserved structural elements.

Polyadenylation and deadenylation of maternal mRNAS during oocyte growth and maturation in the mouse

We previously showed that, during mouse oocyte maturation, specific maternal mRNAs (actins) are deadenylated, while others (hypoxanthine phosphoribosyltransferase:HPRT) are adenylated. As in other systems, these changes can be correlated with changes in translational activities. Maturation-specific polyadenylation in Xenopus depends on the presence of a U-rich cytoplasmic polyadenylation element (CPE) close to the 3' end of the RNA. RNAs that lack CPEs appear to be deadenylated by default when meiosis resumes. We show here that this default program also applies to maturing mouse oocytes. Microinjected beta- and gamma-actin 3' UTR (untranslated region) transcripts lacking CPEs but including polyA tails (100-200 N) behave as endogenous maternal actin mRNAs and are deadenylated by maturing oocytes. "Nonsense" transcripts that do not include CPEs, but that do contain polyA tails, are also deadenylated. beta- and gamma-Actin 3' UTRs with short polyA tails (50-80 N) are stable and exhibit no detectable change in adenylation when injected into growing, full-grown, or maturing oocytes. In contrast, HPRT 3' UTRs, which include the CPE UUUUAAAU and a short polyA tail (50 N), are polyadenylated during maturation. HPRT 3' UTR transcripts with long polyA tails (100-200 N) are more extensively deadenylated by growing and full-grown oocytes that retain germinal vesicles than by maturing oocytes. The presence of CPEs may be required for polyA tail shortening and translational inactivation of stable mRNAs during oocyte growth and subsequent selective readenylation and translation during meiotic maturation.(ABSTRACT TRUNCATED AT 250 WORDS)