Changes In mRNA Stability Research Articles

Transcription of different exons 1 of the human neuronal nitric oxide synthase gene is dynamically regulated in a cell- and stimulus-specific manner.

An extensive screening of the human neuronal nitric oxide synthase (nNOS) mRNAs in various human tissues and cell lines unraveled an extreme complexity in the transcription of this gene. Using 5' rapid amplification of cDNA ends (5'-RACE), ten different exons 1 (named 1a-1l) were identified. They were spliced in a cell-specific manner to a common exon 2, which bears the translational start site. Three first exons (1d, 1g and 1f) were used predominantly for the transcription of the nNOS gene (146 out of 197 5'-RACE clones contained these exons). Exon 1k was found alone, but in many instances was interposed between exons 1b, 1d, 1g, 1i or 1j and the common exon 2. In addition to the cell-specific heterogeneity of human nNOS transcripts, nNOS is highly regulated at the transcriptional level. In resting A673 neuroepithelioma cells, the prevalent nNOS transcript was the exon 1g mRNA (with minor expression of exons 1d+1k and exon 1f mRNAs). When the cells were treated with dibutyryl-cAMP, nNOS mRNA was markedly upregulated. This upregulation was solely due to an increase in exon 1f mRNA, while the expression of the other mRNA species remained unchanged. Human HaCat keratinocyte-like cells expressed the exon 1i+1k and 1i nNOS transcripts under basal conditions. When stimulated with epidermal growth factor, only the exon 1i+1k transcript was upregulated. Although these nNOS transcripts do not differ in their translated region, the various mRNAs may trigger post-transcriptional effects such as changes in mRNA stability and translation efficiency.

The 3′ untranslated region of the new rat synaptic vesicle protein 2B mRNA transcript inhibits translational efficiency

Post-transcriptional regulatory mechanisms have been shown to play a major role in gene expression in eukaryotic cells. Sequences within the 3′-untranslated region (3′UTR) of mRNAs have been described as being important for enhancing or inhibiting message translation. Using fluorescence microscopy, Western blotting and the reverse transcription-polymerase chain reaction (RT-PCR) method, we demonstrate that the 3′UTR of the new rat transcript of synaptic vesicle protein 2B (SV2Bb) mRNA is involved in post-transcriptional regulation of SV2Bb translation. When fused to a reporter gene, this 3′UTR markedly inhibited protein synthesis in transiently transfected cells and this decreased translational efficiency did not occur through changes in mRNA stability. In conclusion, our study gives new insights into unraveling the molecular mechanisms involved in the post-transcriptional regulation of the SV2B gene.

The role of the 5' untranslated region (UTR) in glucose-dependent mRNA decay.

When S. cerevisiae are grown with glucose, SDH2 mRNA encoding the iron protein of the succinate dehydrogenase complex is unstable and present at low level. In yeast grown without glucose, SDH2 mRNA is stable and its level rises. Addition of glucose to a glucose-limited culture causes the SDH2 mRNA level to fall rapidly with a half-life of approximately 5-7 min. Previously the 5'UTR of the mRNA of SDH2 was shown to be necessary and sufficient to destabilize it in glucose (Lombardo et al., 1992). We now show that the SDH1 and SUC2 5'UTRs are capable of conferring glucose-sensitive mRNA instability. We also examine how changes in the SDH2 5'UTR affect glucose-triggered degradation. Finally, we show that changes in mRNA stability are correlated with changes in translational efficiency for these transcripts.

Picornavirus IRESes and the poly(A) tail jointly promote cap-independent translation in a mammalian cell-free system

In eukaryotic cells, efficient translation of most cellular mRNAs requires the synergistic interplay between the m7GpppN cap structure and the poly(A) tail during initiation. We have developed and characterized a cell-free system from human HeLa cells that recapitulates this important feature, displaying more than one order of magnitude of translational synergism between the cap structure and the poly(A) tail. The stimulation of cap-dependent translation by the poly(A) tail is length-dependent, but not mediated by changes in mRNA stability. Using this system, we investigated the effect of the poly(A) tail on the translation of picornaviral RNAs, which are naturally polyadenylated but initiate translation via internal ribosome entry sites (IRESs). We show that translation driven by the IRESs of poliovirus (PV), encephalomyocarditis virus (EMCV), and hepatitis A virus is also significantly augmented by a poly(A) tail, ranging from an approximately 3-fold stimulation for the EMCV-IRES to a more than 10-fold effect for the PV IRES. These results raise interesting questions concerning the underlying molecular mechanism(s). The cell-free system described here should prove useful in studying these questions as well as providing a general biochemical tool to examine the translation initiation pathway in a more physiological setting.

An Initial Characterization of the 3′ Untranslated Region of the EhPgp5 mRNA in Entamoeba histolytica

In Entamoeba histolytica , the multidrug resistance phenotype (MDR) is given by the overexpression of the EhPgp1 and EhPgp5 genes. Transcriptional regulation plays an important role in the mechanisms underlying the expression of these genes (1). However, the relevance of postranscriptional events has not yet been explored. In cultured rat hepatocytes, Pgp2 gene overexpression is primarily due to postranscriptional mechanisms, in which changes in mRNA stability appear to be critical (2). In addition, in rat liver tumors the overexpression of the Pgp1 , Pgp2 , and Pgp3 genes seems to be caused by a mechanism involving mRNA stabilization (3). The 3 9 untranslated region (3 9 UTR) sequence affects mRNA stability and its polyadenylation and translation, nuclear export, and efficiency processing. Changes in rates of turnover and RNA cleavage are affected by the formation of 3 9 UTR secondary structure motifs and by proteins binding to these structures. Postranscriptional events of E. histolytica EhPgp genes may be also regulated by RNA structure. As an initial step to study the postranscriptional mechanisms involved in EhPgp5 gene expression, we analyze here the relevant sequence features and the secondary structure of the 3 9 UTR of EhPgp5 mRNA.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Induces Binding of a 50 kDa Protein on the 3′ Untranslated Region of Urokinase-Type Plasminogen Activator mRNA

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a highly toxic compound that has recently attracted much attention as an environmental contaminant, elicits a variety of toxic responses. Most, if not all, of the toxic effects of TCDD are thought to result from alteration of gene expression. TCDD acts through both transcriptional and posttranscriptional mechanisms to alter gene expression of many genes. Transforming growth factor (TGF)-α and urokinase-type plasminogen activator (uPA) are examples of the genes up-regulated posttranscriptionally by TCDD by mRNA stabilization. While effects of TCDD on transcription have been extensively studied, the molecular mechanisms underlying the TCDD-induced changes in mRNA stability are poorly understood. In this study, we investigated the trans-acting factors involved in TCDD-dependent mRNA stabilization. UV-crosslinking study showed that a liver cytoplasmic protein of 50 kDa (p50) selectively recognized the 3′ UTR of the uPA mRNA in a TCDD-dependent manner. We also showed that the activation of p50 by TCDD is mediated through a protein phosphorylation cascade but not via de novo protein synthesis. This is the first study to show the presence of the TCDD-dependent RNA binding activity which may be involved in TCDD-dependent stabilization of mRNA.

HuR regulates cyclin A and cyclin B1 mRNA stability during cell proliferation.

Colorectal carcinoma RKO cells expressing reduced levels of the RNA-binding protein HuR (ASHuR) displayed markedly reduced growth. In synchronous RKO populations, HuR was almost exclusively nuclear during early G(1), increasing in the cytoplasm during late G(1), S and G(2). The expression and half-life of mRNAs encoding cyclins A and B1 similarly increased during S and G(2), then declined, indicating that mRNA stabilization contributed to their cell cycle-regulated expression. In gel-shift assays using radiolabeled cyclin RNA transcripts and RKO protein extracts, only those transcripts corresponding to the 3'-untranslated regions of cyclins A and B1 formed RNA-protein complexes in a cell cycle-dependent fashion. HuR directly bound mRNAs encoding cyclins A and B1, as anti-HuR antibodies supershifted such RNA-protein complexes. Importantly, the expression and half-life of mRNAs encoding cyclins A and B1 were reduced in ASHuR RKO cells. Our results indicate that HuR may play a critical role in cell proliferation, at least in part by mediating cell cycle-dependent stabilization of mRNAs encoding cyclins A and B1.

Post-transcriptional Control of Cyclooxygenase-2 Gene Expression: THE ROLE OF THE 3′-UNTRANSLATED REGION

The cyclooxygenase (COX)-2 enzyme is responsible for increased prostaglandin formation in inflammatory states and is the major target of nonsteroidal anti-inflammatory drugs. Normally COX-2 expression is tightly regulated, however, constitutive overexpression plays a key role in colon carcinogenesis. To understand the mechanisms controlling COX-2 expression, we examined the ability of the 3'-untranslated region of the COX-2 mRNA to regulate post-transcriptional events. When fused to a reporter gene, the 3'-untranslated region mediated rapid mRNA decay (t(1/2) = 30 min), which was comparable to endogenous COX-2 mRNA turnover in serum-induced fibroblasts treated with actinomycin D or dexamethasone. Deletion analysis demonstrated that a conserved 116-nucleotide AU-rich sequence element (ARE) mediated mRNA degradation. In transiently transfected cells, this region inhibited protein synthesis approximately 3-fold. However, this inhibition did not occur through changes in mRNA stability since mRNA half-life and steady-state mRNA levels were unchanged. RNA mobility shift assays demonstrated a complex of cytoplasmic proteins that bound specifically to the ARE, and UV cross-linking studies identified proteins ranging from 90 to 35 kDa. Fractionation of the cytosol showed differential association of ARE-binding proteins to polysomes and S130 fractions. We propose that these factors influence expression at a post-transcriptional step and, if dysregulated, may increase COX-2 protein as detected in colon cancer.

Hypoxia Increases Glyceraldehyde-3-phosphate Dehydrogenase Transcription in Rat Alveolar Epithelial Cells

Alveolar epithelial type II (ATII) cells are particularly hypoxia-tolerant in vitro. As one of the mechanisms of hypoxia tolerance is the induction of certain proteins, one of which is glyceraldehyde-3-phosphate dehydrogenase (GAPDH), we investigated whether hypoxia modified GAPDH expression in ATII cells. Hypoxia induced a time- and O2 concentration-dependent accumulation of GAPDH mRNA in cultured rat ATII cells (2- to 3-fold the normoxic value after 18 h in 0% O2), an effect completely reversed by reoxygenation. GAPDH mRNA induction was accounted for by an increase in GAPDH gene transcription during hypoxia with no change in mRNA stability. GAPDH protein synthesis increased 3- to 4-fold after 18 h of 0% O2, while the GAPDH protein steady-state level rose by 75%. GAPDH enzymatic activity in hypoxic cell homogenates increased by 45%. These results indicate that hypoxia induces GAPDH expression in ATII cells through an increase in transcription.

Suppression of Interleukin-12 Production by Human Monocytes After Preincubation With Lipopolysaccharide

Interleukin-12 (IL-12) is a potent proinflammatory and immunoregulatory cytokine skewing T lymphocytes to express a type 1 cytokine pattern. Optimal expression of IL-12 mRNA and bioactivity in vitro requires specific priming of monocytes by interferon-γ (IFN-γ) or granulocyte-macrophage colony-stimulating factor (GM-CSF) before lipopolysaccharide (LPS) stimulation. We show here for the first time that the production of IL-12 by IFN-γ– or GM-CSF–primed human monocytes can be completely suppressed by preincubation with LPS (fromEscherichia coli Serotype 055:B5) for 6 to 24 hours before the priming procedure. A dose-dependent suppression of IL-12p70 was measured on the levels of intracellular cytokine production and cytokine secretion. mRNA studies on the expression of p40 and p35 showed an LPS-induced downregulation of both subunits. The results of several different experimental approaches suggest that IL-12 downregulation was not due to endogenous IL-10, IL-4, prostaglandin E2 (PGE2), tumor necrosis factor- (TNF-), or nitric oxide (NO) production induced by LPS. Moreover, preincubation of monocytes with LPS did not lead to a downregulation of the CD14 antigen, which is an LPS receptor. LPS preincubation in this experimental setting did not result in a general hyporesponsiveness of the monocytes, as IL-6 production as well as IFN-γ–induced upregulation of CD54 did not decline. Downregulation of IL-12 was not due to changes in mRNA stability. These findings show that the immunoregulatory important cytokine, IL-12, underlies itself a complex regulation.

Suppression of Interleukin-12 Production by Human Monocytes After Preincubation With Lipopolysaccharide

AbstractInterleukin-12 (IL-12) is a potent proinflammatory and immunoregulatory cytokine skewing T lymphocytes to express a type 1 cytokine pattern. Optimal expression of IL-12 mRNA and bioactivity in vitro requires specific priming of monocytes by interferon-γ (IFN-γ) or granulocyte-macrophage colony-stimulating factor (GM-CSF) before lipopolysaccharide (LPS) stimulation. We show here for the first time that the production of IL-12 by IFN-γ– or GM-CSF–primed human monocytes can be completely suppressed by preincubation with LPS (fromEscherichia coli Serotype 055:B5) for 6 to 24 hours before the priming procedure. A dose-dependent suppression of IL-12p70 was measured on the levels of intracellular cytokine production and cytokine secretion. mRNA studies on the expression of p40 and p35 showed an LPS-induced downregulation of both subunits. The results of several different experimental approaches suggest that IL-12 downregulation was not due to endogenous IL-10, IL-4, prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), or nitric oxide (NO) production induced by LPS. Moreover, preincubation of monocytes with LPS did not lead to a downregulation of the CD14 antigen, which is an LPS receptor. LPS preincubation in this experimental setting did not result in a general hyporesponsiveness of the monocytes, as IL-6 production as well as IFN-γ–induced upregulation of CD54 did not decline. Downregulation of IL-12 was not due to changes in mRNA stability. These findings show that the immunoregulatory important cytokine, IL-12, underlies itself a complex regulation.

Region-Specific Regulation of RGS4 (Regulator of G-Protein–Signaling Protein Type 4) in Brain by Stress and Glucocorticoids:In VivoandIn VitroStudies

The present study demonstrates that the regulator of G-protein-signaling protein type 4 (RGS4) is differentially regulated in the locus coeruleus (LC) and the paraventricular nucleus (PVN) of the hypothalamus by chronic stress and glucocorticoid treatments. Acute or chronic administration of corticosterone to adult rats decreased RGS4 mRNA levels in the PVN but increased these levels in the LC. Similarly, chronic unpredictable stress decreased RGS4 mRNA levels in the PVN but had a strong trend to increase these levels in the LC. Chronic stress also decreased RGS4 mRNA levels in the pituitary. The molecular mechanisms of RGS4 mRNA regulation were further investigated in vitro in the LC-like CATH.a cell line and the neuroendocrine AtT20 cell line using the synthetic corticosterone analog dexamethasone. Consistent with the findings in vivo, dexamethasone treatment caused a dose- and time-dependent decrease in RGS4 mRNA levels in AtT20 cells but a dose- and time-dependent increase in CATH.a cells. RGS4 mRNA regulation seen in these two cell lines seems to be attributable, at least in part, to opposite changes in mRNA stability. The differential regulation of RGS4 expression in the LC and in key relays of the hypothalamic-pituitary-adrenal axis could contribute to the brain's region-specific and long-term adaptations to stress.

Cx32 mRNA in rat liver: effects of inflammation on poly(A) tail distribution and mRNA degradation.

Previous studies showed that the expression of connexin 32 (Cx32), the polypeptide subunit component of the major hepatic gap junction, is reduced in liver by changes in mRNA stability during bacterial lipopolysaccharide (LPS)-induced inflammation. In this study, we examined the distribution of Cx32 mRNA poly(A) tail lengths during LPS-induced inflammation, because this is considered the first step in the degradation of many mRNAs. During LPS treatment the first detectable change in Cx32 mRNA was a gradual shortening of its poly(A) tail, which reached a final size of approximately 20 nucleotides. However, the poly(A) tail did not disappear entirely before the bulk of Cx32 mRNA was degraded. Treatment with actinomycin D, which blocks the degradation of Cx32 mRNA after LPS administration, resulted in the appearance of a completely deadenylated mRNA, which otherwise could not be detected. On the contrary, treatment with cycloheximide resulted in a decrease in the stability of Cx32 mRNA without an apparent change of the poly(A) tail size. The effect of cycloheximide on Cx32 mRNA stability seems to be due indirectly to the induction of an inflammatory response by this drug. These results suggest that, similar for many mRNAs, shortening of the poly(A) tail is one of the first steps in the degradation of Cx32 mRNA during inflammation.

Regulation of Angiogenin Expression in Human HepG2 Hepatoma Cells by Mediators of the Acute-Phase Response

Angiogenin is a potent inducer of neovascularizationin vivo.However, like other angiogenic molecules, its specific physiologic roles and mechanisms regulating its expression remain to be elucidated. Angiogenin is a liver-derived component of normal serum whose concentration can increase in various disease states. This suggests that it might participate in the acute-phase response. In an initial study we showed that angiogenin protein and mRNA levels transiently increased in mice following an acute inflammatory stimulus. We now report that IL-6, a major inducer of acute-phase proteins, stimulates the synthesis and secretion of angiogenin protein in human HepG2 cells within 24 hr following treatment, an effect enhanced by dexamethasone. IL-6 also increases the amount of angiogenin mRNA without altering its half-life. This increase, suppressible by cycloheximide, peaks at 12 hr following stimulation and returns to basal levels by 48 hr. IL-1 alone slightly decreases the basal production of angiogenin protein and mRNA, but essentially abolishes the response to IL-6 in the absence or presence of dexamethasone. This antagonistic effect by IL-1 on IL-6 activity is not a result of changes in mRNA stability nor is it dependent on new protein synthesis. Thus, the combined effects of IL-6, IL-1, glucocorticoids, and perhaps other related factors may specifically control angiogenin expression. Since angiogenin is regulated in a manner similar to that of acute phase proteins bothin vitroandin vivo,it may play a role in the host response to injury.

Down-regulation of rat mitochondrial branched-chain 2-oxoacid dehydrogenase kinase gene expression by glucocorticoids

The mammalian mitochondrial branched-chain 2-oxoacid dehydrogenase (BCOD) complex is regulated by a reversible phosphorylation (inactivation)/dephosphorylation (activation) cycle. In the present study, the effects of glucocorticoids on the level of BCOD kinase mRNA were investigated in rat hepatoma cell lines (H4IIE and FTO-2B), as well as in the rat. In H4IIE cells, dexamethasone was found to significantly reduce steady-state concentrations of BCOD kinase mRNA after a 48 h culture, and this was correlated with a 2-fold increase in the dephosphorylated form of the BCOD complex. The half-life of the kinase mRNA in H4IIE cells was not affected by dexamethasone treatment. Therefore, the decrease in the steady-state kinase mRNA level resulting from dexamethasone treatment was not caused by changes in mRNA stability, which raised the possibility of regulation at the level of gene transcription. To identify the negative glucocorticoid-responsive element in the kinase promoter, nested deletion constucts in the 3.0 kb promoter region were examined in H4IIE cells cultured in the presence or absence of dexamethasone. No significant differences in promoter activity were observed on either transient or stable transfection. The data showed that the glucocorticoid-responsive element was located outside the 3. 0 kb promoter region. At the physiological level, hepatic BCOD kinase mRNA levels were reduced in rats injected intraperitoneally with dexamethasone. This effect was liver-specific, and was not detected in other tissues. These results suggest that the down-regulation of kinase gene expression by glucocorticoids is mediated through a liver-specific or -enriched transcription factor(s).

Down-regulation of rat mitochondrial branched-chain 2-oxoacid dehydrogenase kinase gene expression by glucocorticoids

The mammalian mitochondrial branched-chain 2-oxoacid dehydrogenase (BCOD) complex is regulated by a reversible phosphorylation (inactivation)/dephosphorylation (activation) cycle. In the present study, the effects of glucocorticoids on the level of BCOD kinase mRNA were investigated in rat hepatoma cell lines (H4IIE and FTO-2B), as well as in the rat. In H4IIE cells, dexamethasone was found to significantly reduce steady-state concentrations of BCOD kinase mRNA after a 48 h culture, and this was correlated with a 2-fold increase in the dephosphorylated form of the BCOD complex. The half-life of the kinase mRNA in H4IIE cells was not affected by dexamethasone treatment. Therefore, the decrease in the steady-state kinase mRNA level resulting from dexamethasone treatment was not caused by changes in mRNA stability, which raised the possibility of regulation at the level of gene transcription. To identify the negative glucocorticoid-responsive element in the kinase promoter, nested deletion constucts in the 3.0 kb promoter region were examined in H4IIE cells cultured in the presence or absence of dexamethasone. No significant differences in promoter activity were observed on either transient or stable transfection. The data showed that the glucocorticoid-responsive element was located outside the 3.0 kb promoter region. At the physiological level, hepatic BCOD kinase mRNA levels were reduced in rats injected intraperitoneally with dexamethasone. This effect was liver-specific, and was not detected in other tissues. These results suggest that the down-regulation of kinase gene expression by glucocorticoids is mediated through a liver-specific or -enriched transcription factor(s).

Glucocorticoids downregulate cyclooxygenase-1 gene expression and prostacyclin synthesis in fetal pulmonary artery endothelium.

Prostacyclin (prostaglandin I2 [PGI2]) is a key mediator of pulmonary vascular function during early postnatal life, and its production in the pulmonary vasculature rises markedly during that period because of increasing expression of cyclooxygenase type 1 (COX-1). The postnatal rise in COX-1 may be due to the release of inhibition by glucocorticoids, since plasma glucocorticoid levels fall after birth and glucocorticoids decrease PGI2 synthesis in certain nonpulmonary cell types. We therefore studied the direct effects of dexamethasone (DEX) on COX-1 expression in early-passage ovine fetal pulmonary-artery endothelial cells (PAECs). DEX (10(-10) to 10(-6) mol/L) caused a dose-related decrease in COX-1 mRNA expression that was evident by 24 hours, was maximal at 10(-6) mol/L (50% inhibition), and was not due to changes in mRNA stability. There was a parallel decline in COX-1 protein expression. COX-1 protein rose following DEX withdrawal, and DEX blunted the stimulatory effect of 17beta-estradiol on COX-1 expression. DEX alone (10(-8) mol/L for 48 hours) caused a 93% fall in basal PGI2 production, and bradykinin- and A23187-stimulated PGI2 were diminished 96% and 94%, respectively. Similarly, PGI2 synthesis from arachidonic acid fell 86% with DEX; all of the above effects are consistent with COX-1 downregulation. The glucocorticoid receptor (GR) antagonist mifepristone (RU-486; 10(-6) mol/L) blocked the inhibitory effect of DEX, and GR expression was evident by immunoblot analysis. These findings indicate that glucocorticoids downregulate COX-1 expression and PGI2 synthesis in fetal PAECs through the activation of PAEC GR and effects on COX-1 gene transcription. This mechanism may modulate pulmonary PGI2 production in the perinatal period, and it may also play a role in the effects of glucocorticoids on the systemic circulation at a variety of ages.

Transcriptional Pulsing Approaches for Analysis of mRNA Turnover in Mammalian Cells

Modulation of mRNA stability provides a powerful means for controlling gene expression during the cell cycle, cell differentiation, the immune response, as well as many other physiological transitions. Through the years, many different methods have been developed for measuring mRNA stability. Frequently mRNA stability is studied indirectly by analyzing the steady-state level of mRNA. Therefore by inference, changes in mRNA abundance are thought to affect only the stability of the mRNA, an assumption that is not always correct. Alternatively, direct measurements of mRNA decay are performed in a number of ways, including kinetic labeling techniques and administration of transcriptional inhibitors. Due to the nature of these techniques, they either are technically demanding or introduce a significant change in cell physiology. In addition, many critical mechanistic issues as to deadenylation kinetics, decay intermediates, and precursor–product relationships cannot be readily addressed by these methods. Here, we describe and discuss in detail two different transcriptional pulsing methods based on the c-fosserum-inducible promoter and the tetracycline-regulated promoter systems as an effort to better elucidate the mechanistic steps and regulation underlying differential and selective mRNA turnover in mammalian cells. Both systems allow unequivocal monitoring of deadenylation and decay kinetics as well as determination of precursor–product relationship. In addition, decay rate constants and half-lives are determined and used in both methods to quantitatively denote the mRNA stability. Thus, they provide a reliable way to determine subtle yet physiologically meaningful changes in mRNA stability. Application of one method or the other covers the study of mRNA turnover in most mammalian cell types under a wide range of physiological conditions.

Cell Cycle-dependent Usage of Transcriptional Start Sites: A NOVEL MECHANISM FOR REGULATION OF CYCLIN B1

Cyclin B1 mRNA is expressed temporally throughout the cell cycle with peak expression in G2 and M phase. Both transcriptional and posttranscriptional controls are important for this cell cycle-dependent regulation of cyclin B1 mRNA. In this study, we observed that cyclin B1 has two major transcripts: (a) a constitutively expressed transcript, and (b) a cell cycle-regulated transcript expressed predominantly during G2-M phase. These different transcripts are due to alternative start sites. The constitutively expressed transcript starts 65 bases upstream from the cell cycle-regulated message. Changes in mRNA stability did not appear to control the expression of the cell cycle-specific transcript, but we were able to identify a 24-base pair region of the cyclin B1 promoter spanning the start site of the cell cycle-regulated transcript that was critical for its cell cycle-regulated promoter activity. This suggests that transcriptional regulation is responsible for controlling the presence of each message. The 24-base pair sequence required for cell cycle regulation was notable for containing the nucleotides GGCT repeated three times. The possibility that these two transcripts might be physiologically distinct was raised when the cell cycle-specific transcript was found to be translated more efficiently in vitro than the constitutively expressed transcript. These results characterize a novel mechanism for the regulation of cyclin B1 throughout the cell cycle that is dependent upon the use of different transcriptional start sites.

Identification of a nuclear protein in Trypanosoma brucei with homology to RNA-binding proteins from cis-splicing systems

Gene expression in trypanosomes is controlled at the level of pre-mRNA maturation via trans-splicing and polyadenylation and through changes in mRNA stability. To identify the trans- acting factors involved in this regulation, we have used a degenerate PCR approach to clone genes encoding the RNA recognition motif (RRM) consensus. We have identified a single-copy gene encoding a protein (designated RRM1) which contains three consensus RRM motifs, two tandem copies of a retroviral gag- like CCHC ‘zinc finger’ and an arginine-serine (RS) rich region. Western blotting indicates that RRM1 is expressed in both procyclic and bloodstream-form trypanosomes and has an apparent mobility on SDS-PAGE of ca. 70 Kd. RRM1 is localized in the trypanosome nucleus in substructures which may be functionally analogous to the ‘speckles’ associated with cis-splicing in higher eukaryotic cells. The structure of RRM1, its pattern of expression and its intracellular location suggest that it may play a role in trans-splicing.