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Comparison of mRNA and protein levels of four members of the protein 4.1 family: the type II brain 4.1/4.1B/KIAA0987 is the most predominant member of the protein 4.1 family in rat brain.

The human gene for the fourth member of the protein 4.1 family, KIAA0987, was recently identified by comprehensive cDNA analysis. To further characterize the corresponding gene and its product in rats, we cloned and sequenced rat KIAA0987 cDNA. RNA blot analyses revealed that the rat KIAA0987 gene was abundantly expressed only in the brain, kidney, and testis. Although we have previously reported that the third member of the protein 4.1 family, the KIAA0338 gene product, is predominantly expressed in rat brain, and thus was named brain 4.1, quantitative RNA blot analyses indicated that KIAA0987 should be called something other than brain 4.1 because the level of KIAA0987 mRNA was found to be of the same order as that of KIAA0338 mRNA. Our quantitative immunoblot analysis showed that the most predominant member of the protein 4.1 family at the protein level was the product of the KIAA0987 gene, not that of the KIAA0338 gene. Taking these results together, we consider it reasonable to name the KIAA0338 and KIAA0987 gene products 'type I brain 4.1' and 'type II brain 4.1,' respectively, because these two products were found to be more prominently produced in rat brain than the other two members of the protein 4.1 family, erythroid 4.1 and 4.1G.

Amino Acid Sequence↗

Trypanosome telomeres are protected by a homologue of mammalian TRF2.

Putative TTAGGG repeat-binding factor (TRF) homologues in the genomes of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major were identified. They have significant sequence similarity to higher eukaryotic TRFs in their C-terminal DNA-binding myb domains but only weak similarity in their N-terminal domains. T. brucei TRF (tbTRF) is essential and was shown to bind to duplex TTAGGG repeats. The RNA interference-mediated knockdown of tbTRF arrested bloodstream cells at G(2)/M and procyclic cells partly at S phase. Functionally, tbTRF resembles mammalian TRF2 more than TRF1, as knockdown diminished telomere single-stranded G-overhang signals. This suggests that tbTRF, like vertebrate TRF2, is essential for telomere end protection, and this also supports the hypothesis that TRF rather than Rap1 is the more ancient DNA-binding component of the telomere protein complex. Identification of the first T. brucei telomere DNA-binding protein and characterization of its function provide a new route to explore the roles of telomeres in pathogenesis of this organism. This work also establishes T. brucei as an attractive model for telomere biology.

Amino Acid Sequence↗

RNA-protein interactions that regulate pre-mRNA splicing.

Splicing of nuclear precursor messenger RNAs is an important and ubiquitous type of gene regulation in metazoans. Splicing joins the coding sequences called exons by removing the intervening noncoding sequences, introns, from primary transcripts. Alternative splicing generates an enormous repertoire of functional diversity by producing multiple RNAs and proteins from a single gene. In fact, recent genome sequences from several organisms suggest that splicing regulation is likely to provide an important source of functional diversity in more complex organisms. Because splice sites are short sequences at the ends of introns, the functional splice sites have to be distinguished from an excessively large number of sequences in the primary transcripts that resemble a splice site. Furthermore, alternative splice sites have to be correctly chosen at appropriate times. Thus, selection of proper splice sites remains a daunting biological problem. This review focuses on a few examples in which the molecular and biochemical basis for splice site selection is better understood.

Alternative Splicing↗

The thymocyte-specific MAR binding protein, SATB1, interacts in vitro with a novel variant of DNA-directed RNA polymerase II, subunit 11.

A yeast two-hybrid screen of a Jurkat (T cell) derived cDNA library, using SATB1 (a matrix attachment region binding protein) as the bait, yielded four independent isolates of a novel variant of the DNA directed RNA polymerase II, subunit 11 (RPB11). Absence of lysine-17 from the amino terminus of this variant cannot be explained by alternative mRNA splicing. Instead, allele-specific PCR, combined with GenBank database searches, suggests that a recent gene duplication event has resulted in distinct loci encoding three variant forms of RPB11. Differential splicing of mRNA transcripts accounts for unique carboxy termini among the RPB11 proteins. The exclusive association of SATB1 with one form of RPB11 is influenced primarily by the N-terminal amino acid disparity, as deletion of the entire C terminus does not alter interaction affinity. Association of RPB11 with SATB1 maps between amino acids 58 and 222 of SATB1, a region that includes a PDZ-like dimerization motif.

Base Sequence↗

Runx1/AML1/Cbfa2 mediates onset of mesenchymal cell differentiation toward chondrogenesis.

UNLABELLED: Runx proteins mediate skeletal development. We studied the regulation of Runx1 during chondrocyte differentiation by real-time RT-PCR and its function during chondrogenesis using overexpression and RNA interference. Runx1 induces mesenchymal stem cell commitment to the early stages of chondrogenesis. INTRODUCTION: Runx1 and Runx2 are co-expressed in limb bud cell condensations that undergo both cartilage and bone differentiation during murine development. However, the cooperative and/or compensatory effects these factors exert on skeletal formation have yet to be elucidated. MATERIALS AND METHODS: Runx1/Cbfa2 and Runx2/Cbfa1 were examined at different stages of embryonic development by immunohistochemistry. In vitro studies used mouse embryonic limb bud cells and assessed Runx expressions by immunohistochemistry and real-time RT-PCR in the presence and absence of TGFbeta and BMP2. Runx1 was overexpressed in mesenchymal cell progenitors using retroviral infection. RESULTS: Immunohistochemistry showed that Runx1 and Runx2 are co-expressed in undifferentiated mesenchyme, had similar levels in chondrocytes undergoing transition from proliferation to hypertrophy, and that there was primarily Runx2 expression in hypertrophic chondrocytes. Overall, the expression of Runx1 remained significantly higher than Runx2 mRNA levels during early limb bud cell maturation. Treatment of limb bud micromass cultures with BMP2 resulted in early induction of both Runx1 and Runx2. However, upregulation of Runx2 by BMP2 was sustained, whereas Runx1 decreased in later time-points when type X collagen was induced. Although TGFbeta potently inhibits Runx2 and type X collagen, it induces type II collagen mRNA and mildly but significantly inhibits Runx1 isoforms in the early stages of chondrogenesis. Virus-mediated overexpression of Runx1 in mouse embryonic mesenchymal cells resulted in a potent induction of the early chondrocyte differentiation markers but not the hypertrophy marker, type X collagen. Knockdown or Runx1 potently inhibits type II collagen, alkaline phosphatase, and Runx2 and has a late inhibitory effect on type X collagen. CONCLUSION: These findings show a distinct and sustained role for Runx proteins in chondrogenesis and subsequent chondrocyte maturation. Runx1 is highly expressed during chondrogenesis in comparison with Runx2, and Runx1 gain of functions stimulated this process. Thus, the Runx genes are uniquely expressed and have distinct roles during skeletal development.

Alcian Blue↗

Expression of the RNA binding proteins, Mel-N1, Mel-N2, and Mel-N3 in adipose cells.

Mel-N1 (murine embryonic lethal abnormal vision [ELAV]), a mammalian homolog of Drosophila ELAV, is an mRNA binding protein of the RNA Recognition Motif family. Studies with the human homolog, Hel-N1 have supported the hypothesis that Hel-N1, and its splice variant, Hel-N2 play a role in mRNA metabolism. Thus it becomes logical to extend this hypothesis to the murine variant Mel-N1 which has been described as a neuronal protein with a minor level of expression in the testis. Our current work expands the potential function for this protein through demonstration of expression of the full-length message and splice variants in adipose tissue as well as preadipocyte and adipocyte cell lines.

3T3 Cells↗

Expression of mRNAs of the aquaporin family in mouse oocytes and embryos.

The molecular basis of water and cryoprotectant permeability in mammalian oocytes and embryos is poorly understood. Therefore, we investigated the expression of mRNAs of water channel proteins (aquaporins) in mouse oocytes and embryos by RT-PCR. The total RNA of mouse oocytes at metaphase II and embryos at the 4-cell, morula, and blastocyst stages was isolated, reverse-transcribed, and subjected to nested PCR amplification. Aquaporins were expressed in both oocytes and embryos, but the types were different among the developmental stages: aquaporins 3 and 7 were expressed in oocytes and embryos at all stages examined, but aquaporins 8 and 9 were expressed only in blastocysts. On the other hand, aquaporins 1, 2, 4, 5, and 6 were not detected in any of the stages examined. The present study shows for the first time that aquaporins are expressed in mammalian oocytes and embryos. These aquaporins may play a role in water transport and conceivably also in cryoprotectant transport across the plasma membrane in these cells.

Animals↗

U6 snRNA variants isolated from the posterior silk gland of the silk moth Bombyx mori.

Five U6 small nuclear RNA (snRNA) isoforms were detected and characterized from the posterior silk gland (PSG) of the silk moth Bombyx mori (Nistari strain). Using the currently accepted U6 secondary structure model as a basis for comparison, the variants were analyzed for nucleotide differences across the sequence with a focus on known functional domains. Differences were observed primarily in single-stranded areas of which sixty percent were found in the highly conserved U4-U6 binding sites. In the Nistari strain, the U6A variant was found to be approximately four times more abundant as part of high molecular weight spliceosomal complexes when compared with U6A in the total unfractionated PSG cell lysate. Additionally, the European 703 B. mori strain total cell lysate U6 snRNA was analyzed and only the dominant U6A isoform initially identified in Nistari was found. Due to U6's essential role in pre-mRNA processing, variants may modulate assemblage of the catalytic core and in doing so potentially affect the rate of splicing. Phylogenetic analysis of the U6 snRNA sequences indicate an ancient divergence of U6 from the self-splicing group II intron module and a high degree of evolutionary conservation across species possibly due to functional constraints on the gene. Using in silico analysis, 35 full-length U6 variants were observed in the recently released Whole Genome Shotgun (WGS) database of the p50T strain. The consensus sequence of these U6 genes from p50T is identical to U6A identified in the Nistari strain. Furthermore p50T variant 1, which is represented in 14 genes, is equivalent to Nistari U6A.

Animals↗

Identification of alternative splicing forms of GLT-1 mRNA in the spinal cord of amyotrophic lateral sclerosis patients.

The glutamate transporter plays an essential role in regulating glutamate levels in the synaptic cleft. It has been postulated that the dysfunction of GLT-1, one subtype of glutamate transporter, may be etiologically related to amyotrophic lateral sclerosis (ALS). Two alternative splicing forms of GLT-1 messenger RNA (mRNA) were found in the cervical spinal cord of five ALS patients and three controls. Analysis with reverse transcription-polymerase chain reaction (RT-PCR) showed that the shorter mRNA was a result of exon 8 skipping. A truncated transcript containing an intronic sequence at the 3' end of exon 7 was also demonstrated. However, the incidence of both alternative mRNAs was not different between the five ALS patients and three controls. Interestingly, the mRNA were also found in the cerebral cortex of a control subject. These results suggest that alternative splicing forms of GLT-1 mRNAs do not play a pathogenetic role in ALS but rather a physiological one in the normal spinal cord and brain.

ATP-Binding Cassette Transporters↗

Complementary DNA analysis, expression and subcellular localization of hnRNP E2 gene in Xenopus laevis.

The cloning and sequencing of complementary DNAs corresponding to the two copies (a and b) of the Xenopus laevis gene for hnRNP E2 is presented. Comparison of the two sequences reveals that while they are somewhat divergent at the nucleotide level, they are very conserved at the amino acid level. The analysis also showed two transcripts of different length (alpha and beta), likely generated by alternative processing. There are indications that either gene copy can generate both type of transcripts. Northern blot analysis in oocytes and developing embryos showed that hnRNP E2 RNA is constantly present and that increases in amount at tadpole stage. A semiquantitative reverse transcriptase polymerase chain reaction analysis performed with RNA from developing embryos showed that long (alpha) transcript accumulation is constant during development, whereas the short one (beta) accumulation increases at later stages, thus determining the observed increase in total RNA. Nucleo-cytoplasm localization experiments indicated that in oocyte hnRNP E2 is exclusively cytoplasmic, whereas in somatic cells it is distributed in both compartments. Comparison of the amino acid sequence of the two X. laevis hnRNP E2 with the corresponding mammalian sequences shows a high homology along the molecule except for the region subjected to alternative splicing, which is completely different. Moreover, there are indications that the homologous of mammalian hnRNP E1 gene, very related to and derived from hnRNP E2 by retrotransposition, is not expressed or even not present in X. laevis, suggesting that mammalian hnRNP E1 gene may have originated after mammal/amphybia divergence.

Amino Acid Sequence↗

Interaction between 3' untranslated region of calcitonin receptor messenger ribonucleic acid (RNA) and adenylate/uridylate (AU)-rich element binding proteins (AU-rich RNA-binding factor 1 and Hu antigen R).

Our previous results in mouse osteoclasts suggested that calcitonin (CT) alters CT receptor (CTR) mRNA stability. The CTR mRNA transcript contains several adenylate/uridylate (AU)-rich destabilizing elements in the 3' untranslated region (3'UTR). When the 3'UTR of mouse CTR mRNA was labeled by [alpha-(32)P]-uridine 5-triphosphate, interactions were observed between the transcript and several cell extracts, including those from the osteoclast progenitor monocyte/macrophage cell line, RAW 264.7. The molecular masses of the interacting proteins ranged from approximately 35 to 50 kDa, similar to AU-rich RNA-binding factor 1 (AUF1) and Hu antigen R (HuR). Radiolabeled 3'UTR transcripts bound with a 40-kDa protein, which could be extracted from cells transfected with AUF1 p40. To confirm the binding specificity, a pSG5 vector construct, containing the AUF1 p40 with an hemagglutinin tag, was transiently transfected into NIH3T3 cells. The extracts were incubated with poly(A)-added CTR3'UTR. The reaction mixture was immunoprecipitated using an antihemagglutinin antibody and precipitated mRNA species were extracted and reverse transcribed using oligo-dT primers. It was found that PCR primers specific for the 3'UTR of CTR mRNA sequence generated a PCR signal. No signal was observed when mutated AUF1 p40 was transfected. In a manner similar to the AUF1 binding, HuR was also found to bind to the 3'UTR. Specific binding of AUF1 p40 and HuR was also found with RNA extracted from mouse osteoclasts. Treatment of osteoclasts with CT did not significantly affect the expression of AUF1 but decreased the levels of HuR and its mRNA. The role of CTR3'UTR in mRNA stability was further tested by expressing luciferase reporter constructs that did, or did not, contain the CTR3'UTR, under the control of the tetracycline-regulatory system. The results showed that the addition of 3'UTR considerably shortened the mRNA half-life of the luciferase reporter gene. These results suggest that AUF1 p40, HuR, and the 3'UTR of the CTR mRNA transcript could be involved in posttranscriptional regulation of CTR mRNA expression.

3' Untranslated Regions↗

Osmosensitive gene expression of taurine transporter and cyclin C in embryonic fibroblast cells.

Hypertonic conditions induce osmoregulatory activity. Molecular mechanism, however, remains to be further elucidated concerning the osmosensitive balancing activity. Using a differential display protocol, the genes of taurine transporter (TauT) and cyclin C were identified as candidate responding to the hypertonicity. When a quantitative PCR analysis was performed on the total RNA from KBEF cell treated under hypertonic conditions, the gene expressions of TauT and cyclin C were greatly increased. In terms of protein, the level of TauT expression increased up to 3.2-fold in response to the hypertonic treatment. Similarly to TauT, cyclin C protein also increased 2.4-fold compared to the control treatments. Under taurine-rich extracellular conditions, however, the level of TauT expression increased as little as 1.8-fold by hypertonicity treatment. Cyclin C expression was also lowered compared with low-taurine hypertonic treatment. Cysteine dioxygenase was shown to be highly responsive to the hypertonic treatment. Taken together, these results strongly indicate that the gene expressions of TauT and cyclin C are cooperatively regulated under hypotonic conditions.

Animals↗

Phosphorylation of progesterone receptor serine 400 mediates ligand-independent transcriptional activity in response to activation of cyclin-dependent protein kinase 2.

Human progesterone receptors (PR) are phosphorylated by cyclin-dependent protein kinase 2 (CDK2) at multiple sites, including Ser400. Herein, we have addressed the significance of phosphorylation of this residue. PR phospho-Ser400-specific antibodies revealed regulated phosphorylation of Ser400 in response to progestins and mitogens, and this correlated with increased CDK2 levels and activity. Expression of cyclin E elevated CDK2 activity and downregulated PR independently of ligand. Similarly, overexpression of activated mutant CDK2 increased PR transcriptional activity in the absence and presence of progestin. Mutation of PR Ser400 to alanine (S400A) blocked CDK2-induced PR activity in the absence, but not in the presence, of progestin. PR was unresponsive to activated CDK2 in breast cancer cells with elevated p27, and RNA interference knock-down of p27 partially restored CDK2-induced ligand-independent PR activation. Similarly, in p27(-/-) mouse embryonic fibroblasts, elevated CDK2 activity increased wild-type (wt) but not S400A PR transcriptional activity in the absence of progestin. CDK2 induced nuclear localization of unliganded wt but not S400A PR; liganded S400A PR exhibited delayed nuclear accumulation. These studies demonstrate that CDK2 regulates PR in the absence of progestins via phosphorylation of Ser400, thus revealing a novel mechanism for upregulated PR transcriptional activity in human breast cancer cells expressing altered cell cycle regulatory molecules.

Alanine↗

A slow RNA polymerase II affects alternative splicing in vivo.

Changes in promoter structure and occupation have been shown to modify the splicing pattern of several genes, evidencing a coupling between transcription and alternative splicing. It has been proposed that the promoter effect involves modulation of RNA pol II elongation rates. The C4 point mutation of the Drosophila pol II largest subunit confers on the enzyme a lower elongation rate. Here we show that expression of a human equivalent to Drosophila's C4 pol II in human cultured cells affects alternative splicing of the fibronectin EDI exon and adenovirus E1a pre-mRNA. Most importantly, resplicing of the Hox gene Ultrabithorax is stimulated in Drosophila embryos mutant for C4, which demonstrates the transcriptional control of alternative splicing on an endogenous gene. These results provide a direct proof for the elongation control of alternative splicing in vivo.

Adenoviridae↗

Human mediator enhances activator-facilitated recruitment of RNA polymerase II and promoter recognition by TATA-binding protein (TBP) independently of TBP-associated factors.

Mediator is a general cofactor implicated in the functions of many transcriptional activators. Although Mediator with different protein compositions has been isolated, it remains unclear how Mediator facilitates activator-dependent transcription, independent of its general stimulation of basal transcription. To define the mechanisms of Mediator function, we isolated two forms of human Mediator complexes (Mediator-P.5 and Mediator-P.85) and demonstrated that Mediator-P.5 clearly functions by enhancing activator-mediated recruitment of RNA polymerase II (pol II), whereas Mediator-P.85 works mainly by stimulating overall basal transcription. The coactivator function of Mediator-P.5 was not impaired when TATA-binding protein (TBP) was used in place of TFIID, but it was abolished when another general cofactor, PC4, was omitted from the reaction or when Mediator-P.5 was added after pol II entry into the preinitiation complex. Moreover, Mediator- P.5 is able to enhance TBP binding to the TATA box in an activator-dependent manner. Our data provides biochemical evidence that Mediator functions by facilitating activator-mediated recruitment of pol II and also promoter recognition by TBP, both of which can occur in the absence of TBP-associated factors in TFIID.

Cells, Cultured↗

Expression of a variant form of the glutamate transporter GLT1 in neuronal cultures and in neurons and astrocytes in the rat brain.

To identify glutamate transporters expressed in forebrain neurons, we prepared a cDNA library from rat forebrain neuronal cultures, previously shown to transport glutamate with high affinity and capacity. Using this library, we cloned two forms, varying in the C terminus, of the glutamate transporter GLT1. This transporter was previously found to be localized exclusively in astrocytes in the normal mature brain. Specific antibodies against the C-terminal peptides were used to show that forebrain neurons in culture express both GLT1a and GLT1b proteins. The pharmacological properties of glutamate transport mediated by GLT1a and GLT1b expressed in COS-7 cells and in neuronal cultures were indistinguishable. Both GLT1a and GLT1b were upregulated in astrocyte cultures by exposure to dibutyryl cAMP. We next investigated the expression of GLT1b in vivo. Northern blot analysis of forebrain RNA revealed two transcripts of approximately 3 and 11 kb that became more plentiful with developmental age. Immunoblot analysis showed high levels of expression in the cortex, hippocampus, striatum, thalamus, and midbrain. Pre-embedding electron microscopic immunocytochemistry with silver-enhanced immunogold detection was used to localize GLT1b in vivo. In the rat somatosensory cortex, GLT1b was clearly expressed in neurons in presynaptic terminals and dendritic shafts, as well as in astrocytes. The presence of GLT1b in neurons may offer a partial explanation for the observed uptake of glutamate by presynaptic terminals, for the preservation of input specificity at excitatory synapses, and may play a role in the pathophysiology of excitotoxicity.

Alternative Splicing↗

A novel growth differentiation factor-9 (GDF-9) related factor is co-expressed with GDF-9 in mouse oocytes during folliculogenesis.

Growth differentiation factor-9 (GDF-9) is a transforming growth factor-b (TGF-b) family member which is expressed in the oocytes in mouse ovaries (McGrath, S.A., Esquela, A.F., Lee, S.J., 1995. Oocyte-specific expression of growth/differentiation factor-9. Mol. Endocrinol. 9, 131-136). GDF-9 is indispensable for normal folliculogenesis since female mice deficient for the GDF-9 gene are infertile due to an arrest of follicular growth at the primary follicle stage (Dong, J., Albertini, D.F., Nishimori, K., Kumar, T.R. , Lu, N., Matzuk, M.M., 1996. Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature 383, 531-535). We searched the GenBank Expressed Sequence Tag (EST) database with the mouse GDF-9 cDNA sequence, and identified from a mouse 2-cell embryo library an EST cDNA that encodes a putative member of the TGF-b superfamily, and named it as GDF-9B. Northern blot hybridization analyses of mouse ovaries revealed a single transcript of approximately 4.0 kilobases (kb) for GDF-9B and of 2.0 kb for GDF-9. We cloned by reverse transcription-polymerase chain reaction from mouse ovarian RNA a partial 821-base pair GDF-9B cDNA that spans the sequence encoding the putative mature region of GDF-9B. The COOH-terminal region of GDF-9B appears to be 53% homologous to GDF-9. Moreover, like GDF-9, GDF-9B lacks the cysteine residue needed for the covalent dimerization of several TGF-b family members. Using in situ hybridization analysis, we demonstrate that GDF-9B and GDF-9 mRNAs are co-localized in the oocyte. We also show that GDF-9B and GDF-9 genes are co-ordinately expressed during follicular development.

Amino Acid Sequence↗

Coordinate regulation of secretory stress proteins (PSP/reg, PAP I, PAP II, and PAP III) in the rat exocrine pancreas during experimental acute pancreatitis.

BACKGROUND: Pancreatic stone protein (PSP/reg) is a constitutively secreted protein in pancreatic juice. Pancreatitis-associated protein (PAP) belongs to the same family of proteins. PAP is highly increased during acute pancreatitis, while no exact data exist regarding PSP/reg protein synthesis and secretion. Recently, an attempt to determine PSP/reg and PAP levels in sera of rats with acute pancreatitis showed a significant increase in PAP but failed to demonstrate changes in PSP/reg. Others reported that surgical manipulation of the pancreas, including sham controls, affected mRNA levels of PSP/reg. Neither report determined protein levels of PSP/reg. METHODS: Rats were treated intraperitoneally with a supramaximal dose of caerulein to induce pancreatitis, a physiological dose of caerulein, or a saline injection. Pancreata were analyzed for PAP and PSP/reg using ELISAs. RNA was extracted for Northern blot analysis of PAP I, II, and III and PSP/reg mRNA. RESULTS: Experimental induction of acute pancreatitis caused a coordinate increase in both PSP/reg and PAP. PAP showed an acute response and returned to low levels within 48 h while PSP/reg exhibited a more sustained response. Intraperitoneal application of a physiological dose of caerulein and even a saline injection caused an increase in PSP/reg. CONCLUSION: PSP/reg and PAP levels are increased through similar mechanisms by physiological and supramaximal doses of caerulein. However, PSP/reg regulation appears to sustain high levels while PAP levels are more transient. Since the regulation of this protein family is affected even under mild stress, we define them as secretory stress proteins.

Acute Disease↗