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PubMed2000

The role of platelet alpha-granular proteins in the regulation of thrombopoietin messenger RNA expression in human bone marrow stromal cells.

Thrombopoietin (TPO), the specific cytokine that regulates platelet production, is expressed in human bone marrow (BM), kidney, and liver. There appears to be no regulation of TPO in the kidney and liver, but TPO messenger RNA (mRNA) expression can be modulated in the stromal cells of the BM. In this study, we used primary human BM stromal cells as a model to study the regulation of TPO mRNA expression in response to various platelet alpha-granular proteins. We showed that platelet-derived growth factor (PDGF) BB and fibroblast growth factor (FGF) 2 stimulated TPO mRNA expression in both a dose-dependent and time-dependent manner. The addition of 50 ng/mL of PDGF and 20 ng/mL of FGF resulted in maximal induction of TPO mRNA expression in 4 hours. We also found that platelet factor 4 (PF4), thrombospondin (TSP), and transforming growth factor-beta (TGF-beta) are negative modulators of megakaryocytopoiesis. We observed suppression in TPO mRNA expression with 1 microg/mL of both PF4 and TSP and 50 ng/mL of TGF-beta, with maximal suppression occurring 4 hours after the addition of these proteins. Finally, the addition of whole-platelet lysate produced a dose-dependent inhibition of TPO expression. On the basis of these findings, we propose that the platelet alpha-granular proteins studied may regulate TPO gene expression in BM stromal cells by means of a feedback mechanism.

Bone Marrow↗
PubMed1987

Determination of the RNA secondary structure that regulates lysis gene expression in bacteriophage MS2.

The lysis gene of the RNA bacteriophage MS2 is not expressed unless translation of the overlapping coat gene takes place. To understand the molecular basis for this translational coupling the RNA secondary structure around the lysis gene start was analyzed with structure-specific enzymes and chemicals. The existence of a hairpin between nucleotides 1636 and 1707 is in agreement with the structural mapping data and also with the conservation of base-pairing in the related M12 phage. In this hairpin, the G residues in the Shine and Dalgarno region and start codon are inaccessible to RNase T1, which is consistent with the fact that ribosomal access to the lysis gene is blocked when there is no coat gene translation. Deletions or point mutations that are predicted to destabilize the hairpin give rise to lysis protein synthesis that is independent of coat gene translation. Base substitutions that are not expected to weaken the helix do not lead to independent lysis gene expression. Finally, nucleotide changes that strengthen the hairpin lead neither to uncoupled nor to coupled synthesis of the lysis protein. Structural analysis of mutant MS2 RNA shows that small changes in the stability of the secondary structure lead to substantial differences in translation initiation. The function of the hairpin structure in coupling lysis gene to coat gene translation requires that its stability is kept within narrow limits.

Bacteriophages↗
PubMed2004

Enhancer-dependent splicing of FGFR1 alpha-exon is repressed by RNA interference-mediated down-regulation of SRp55.

The FGFR1 gene transcript is alternatively processed to produce functionally different receptor forms. Previously, we identified a 69-nucleotide exonic splicing enhancer (ESE) required for alpha-exon inclusion in JEG3 cells. In the present study, we found that this sequence is composed of three independent elements, two smaller ESE sequences flanking an exonic splicing silencer sequence. Ultraviolet cross-linking and immunoprecipitation identified ESE-specific binding of the splicing regulator SRp55. A RNA interference-mediated decrease in SRp55 confirmed the significance of this interaction. There was a 6- to 14-fold decrease in exon inclusion on ablation of SRp55. In SNB19 glioblastoma cells, which normally skip this exon, SRp55 was also demonstrated to play a role in exon inclusion after the removal of intronic splicing silencer sequences. These observations indicate that SRp55 plays a major role in maintaining normal FGFR1 alpha-exon inclusion, which is subject to dominant intronic splicing silencer-mediated and exonic splicing silencer-mediated inhibition in SNB19 cells.

Alternative Splicing↗
PubMed1985

Site-specific phosphorylation of avian retrovirus nucleocapsid protein pp12 regulates binding to viral RNA. Evidence for different protein conformations.

Phosphorylation of serine 40 of the major nucleocapsid protein of avian retroviruses, pp12, regulates binding to viral RNA (Leis, J., Johnson, S., Collins, L. S., and Traugh, J. A. (1984) J. Biol. Chem. 259, 7726-7732). The phosphorylation state of the protein can be altered in vitro, resulting in the interconversion of the protein between a state of high affinity for single-stranded RNA and low affinity for single- or double-stranded RNA. The reversible phosphorylation of serine 40 is accompanied by a change in the conformation of the protein as demonstrated by quenching of intrinsic tryptophan fluorescence and chemical modification studies. Quenching of fluorescence of the sole tryptophan residue, Trp 80, by poly(U), KI, and CsCl indicates that the microenvironment of this residue is more positive in pp12 than in p12. Chemical modification studies indicate that the 3 lysine residues at positions 36, 37, and 39 of pp12 react with 2,4,6-trinitrobenzenesulfonic acid, while only 1 of these residues reacts in p12. The addition of single-stranded, but not double-stranded RNA, to pp12 protects 2 of the 3 lysine residues from chemical modification, suggesting that the two protected lysyl groups are required for binding to single-stranded viral RNA. In contrast to the phosphorylation of serine 40, phosphorylation of serine 43, catalyzed by protease-activated kinase II in vitro, does not induce changes in the protein conformation nor does it alter the RNA binding properties of the protein.

Amino Acid Sequence↗
PubMed1998

Global regulation by the small RNA-binding protein CsrA and the non-coding RNA molecule CsrB.

Csr (carbon storage regulator) is a recently discovered global regulatory system that controls bacterial gene expression post-transcriptionally. Its effector is a small RNA-binding protein referred to as CsrA or, in phytopathogenic Erwinia species, RsmA (repressor of stationary phase metabolites). Numerous genes whose expression occurs in the stationary phase of growth are repressed by csrA/rsmA, and csrA activates certain exponential-phase metabolic pathways. Glycogen synthesis and catabolism, gluconeogenesis, glycolysis, motility, cell surface properties and adherence are modulated by csrA in Escherichia coli, while the production of several secreted virulence factors, the plant hypersensitive response elicitor HrpN(Ecc) and, potentially, other secondary metabolites are regulated by rsmA in Erwinia carotovora. CsrA represses glycogen synthesis by binding to and destabilizing glgCAP mRNA and is hypothesized to repress other genes by a similar mechanism. The second component of the Csr system is CsrB (AepH in Erwinia species), a non-coding RNA molecule that forms a large globular ribonucleoprotein complex with approximately 18 CsrA subunits and antagonizes the effects of CsrA in vivo. Highly repeated sequence elements found within the loops of predicted stem-loops and other single-stranded segments of CsrB RNA may facilitate CsrA binding. Current information supports a model in which CsrA exists in an equilibrium between CsrB and CsrA-regulated mRNAs, which predicts that CsrB levels may be a key determinant of CsrA activity in the cell. The presence of csrA homologues in phylogenetically diverse species further suggests that this novel kind of regulatory system is likely to play a broad role in modulating eubacterial gene expression.

Amino Acid Sequence↗
PubMed1991

Down regulation of poliovirus receptor RNA in HeLa cells resistant to poliovirus infection.

A line of HeLa cells (SOFIA) was previously isolated that is resistant to poliovirus infection and does not express functional virus binding sites at the cell surface. The expression of the poliovirus receptor (PVR) gene in SOFIA cells was examined to determine the molecular basis for the failure of these cells to express PVRs. Southern blot analysis of genomic DNA revealed that the PVR gene in SOFIA cells did not contain gross alterations. However, PVR transcripts were not detected in Northern (RNA) blot analysis of SOFIA cell RNA. In vitro nuclear run-on analysis showed that transcription of PVR-specific RNA was reduced in SOFIA cells. Treatment of SOFIA cells with 5-azacytidine restored susceptibility to poliovirus infection, which correlated with the appearance of PVRs at the cell surface, as detected with anti-PVR monoclonal antibody D171. PVR RNA was detected in clones derived from 5-azacytidine-treated SOFIA cells. SOFIA cells were converted to poliovirus sensitivity at a rate of 5 to 7%, suggesting that down regulation of PVR expression involved few cellular targets. Resistance of SOFIA cells to poliovirus infection therefore appears to result from down regulation of PVR RNA, leading to lack of PVR expression at the cell surface. Methylation may play a role in regulating the expression of the PVR gene, which is not essential for survival of HeLa cells.

Azacitidine↗
PubMed2005

MicroInspector: a web tool for detection of miRNA binding sites in an RNA sequence.

Regulation of post-transcriptional gene expression by microRNAs (miRNA) has so far been validated for only a few mRNA targets. Based on the large number of miRNA genes and the possibility that one miRNA might influence gene expression of several targets simultaneously, the quantity of ribo-regulated genes is expected to be much higher. Here, we describe the web tool MicroInspector that will analyse a user-defined RNA sequence, which is typically an mRNA or a part of an mRNA, for the occurrence of binding sites for known and registered miRNAs. The program allows variation of temperature, the setting of energy values as well as the selection of different miRNA databases to identify miRNA-binding sites of different strength. MicroInspector could spot the correct sites for miRNA-interaction in known target mRNAs. Using other mRNAs, for which such an interaction has not yet been described, we discovered frequently potential miRNA binding sites of similar quality, which can now be analysed experimentally. The MicroInspector program is easy to use and does not require specific computer skills. The service can be accessed via the MicroInspector web server at http://www.imbb.forth.gr/microinspector.

Base Pairing↗
PubMed2006

trans regulation of cap-independent translation by a viral subgenomic RNA.

Many positive-strand RNA viruses generate 3'-coterminal subgenomic mRNAs to allow translation of 5'-distal open reading frames. It is unclear how viral genomic and subgenomic mRNAs compete with each other for the cellular translation machinery. Translation of the uncapped Barley yellow dwarf virus genomic RNA (gRNA) and subgenomic RNA1 (sgRNA1) is driven by the powerful cap-independent translation element (BTE) in their 3' untranslated regions (UTRs). The BTE forms a kissing stem-loop interaction with the 5' UTR to mediate translation initiation at the 5' end. Here, using reporter mRNAs that mimic gRNA and sgRNA1, we show that the abundant sgRNA2 inhibits translation of gRNA, but not sgRNA1, in vitro and in vivo. This trans inhibition requires the functional BTE in the 5' UTR of sgRNA2, but no translation of sgRNA2 itself is detectable. The efficiency of translation of the viral mRNAs in the presence of sgRNA2 is determined by proximity to the mRNA 5' end of the stem-loop that kisses the 3' BTE. Thus, the gRNA and sgRNA1 have "tuned" their expression efficiencies via the site in the 5' UTR to which the 3' BTE base pairs. We conclude that sgRNA2 is a riboregulator that switches off translation of replication genes from gRNA while permitting translation of structural genes from sgRNA1. These results reveal (i) a new level of control of subgenomic-RNA gene expression, (ii) a new role for a viral subgenomic RNA, and (iii) a new mechanism for RNA-mediated regulation of translation.

3' Untranslated Regions↗
PubMed1998

Characterization of the Escherichia coli RNA 3'-terminal phosphate cyclase and its sigma54-regulated operon.

The RNA 3'-terminal phosphate cyclase catalyzes the ATP-dependent conversion of the 3'-phosphate to the 2',3'-cyclic phosphodiester at the end of various RNA substrates. Recent cloning of a cDNA encoding the human cyclase indicated that genes encoding cyclase-like proteins are conserved among Eucarya, Bacteria, and Archaea. The protein encoded by the Escherichia coli gene was overexpressed and shown to have the RNA 3'-phosphate cyclase activity (Genschik, P., Billy, E., Swianiewicz, M., and Filipowicz, W. (1997) EMBO J. 16, 2955-2967). Analysis of the requirements and substrate specificity of the E. coli protein, presented in this work, demonstrates that properties of the bacterial and human enzymes are similar. ATP is the best cofactor (Km = 20 microM), whereas GTP (Km = 100 microM) and other nucleoside triphosphates (NTPs) act less efficiently. The enzyme undergoes nucleotidylation in the presence of [alpha-32P]ATP and, to a lesser extent, also in the presence of other NTPs. Comparison of 3'-phosphorylated oligoribonucleotides and oligodeoxyribonucleotides of identical sequence demonstrated that the latter are at least 300-fold poorer substrates for the enzyme. The E. coli cyclase gene, named rtcA, forms part of an uncharacterized operon containing two additional open reading frames (ORFs). The ORF positioned immediately upstream, named rtcB, encodes a protein that is also highly conserved between Eucarya, Bacteria, and Archaea. Another ORF, called rtcR, is positioned upstream of the rtcA/rtcB unit and is transcribed in the opposite direction. It encodes a protein having features of sigma54-dependent regulators. By overexpressing the N-terminally truncated form of RtcR, we demonstrate that this regulator indeed controls expression of rtcA and rtcB in a sigma54-dependent manner. Also consistent with the involvement of sigma54, the region upstream of the transcription start site of the rtcA/rtcB mRNA contains the -12 and -24 elements, TTGCA and TGGCA, respectively, characteristic of sigma54-dependent promoters. The cyclase gene is nonessential as demonstrated by knockout experiments. Possible functions of the cyclase in RNA metabolism are discussed.

Amino Acid Sequence↗
PubMed1997

Nuclear antisense RNA induces extensive adenosine modifications and nuclear retention of target transcripts.

Antisense RNA may regulate the expression of a number of eukaryotic genes, but little is known about its prevalence or mechanism of action. We have used a model system in which antisense control can be studied both genetically and biochemically. Late in polyoma virus infection, early-strand mRNA levels are down-regulated by nuclear antisense RNA from the late strand. Analysis of early-strand transcripts isolated late in infection revealed extensive base modifications. In many transcripts almost half of the adenosines were altered to inosines or guanosines. These results suggest modification of RNA duplexes by double-stranded RNA adenosine deaminase or a related enzyme. Probes that detect only modified RNAs revealed that these molecules are not highly unstable, but accumulate within the nucleus and are thus inert for gene expression. Antisense-induced modifications can account for most or all of the observed regulation, with the lowered levels of early-strand RNAs commonly observed late in infection resulting from the fact that many transcripts are invisible to standard hybridization probes. This work suggests that similar antisense-mediated control mechanisms may also operate under physiological conditions in uninfected eukaryotic cells, and leads to the proposal that there is a novel pool of nuclear RNAs that cannot be seen with many molecular probes heretofore used.

3T3 Cells↗
PubMed2000

Two trans-acting rat-brain proteins, MARTA1 and MARTA2, interact specifically with the dendritic targeting element in MAP2 mRNAs.

Different isoforms of the microtubule-associated protein 2 (MAP2) are somatodendritic components of neurons that seem to regulate the stability of the dendritic cytoskeleton. MAP2 localization into dendrites appears to be a complex multicausal mechanism that involves the specific recruitment of MAP2 mRNAs into dendritic compartments. Recently, we have functionally characterized a 640-nucleotide dendritic targeting element (DTE) in the 3' untranslated region (3' UTR) of MAP2 transcripts that mediates extrasomatic mRNA localization in primary neurons (Blichenberg et al. , 1999). In analogy to molecular mechanisms regulating cytoplasmic RNA translocation in other cell systems, we propose that, in vivo, the cis-acting MAP2-DTE interacts with specific protein factors present in neurons. To identify putative trans-acting DTE-binding proteins, we performed in vitro ultraviolet crosslinking assays. Using this experimental system, two 90-kDa and 65-kDa MAP2-RNA trans-acting proteins, MARTA1 and MARTA2, were identified in rat-brain extracts. Both MARTAs bind with high affinity to the MAP2-DTE, but not to other investigated regions of MAP2 transcripts or the somatically restricted alpha-tubulin mRNA. Moreover, MARTA1 and MARTA2 do not bind significantly to other dendritically localized transcripts encoding vasopressin and arg3.1, nor to a dendritic trafficking element from the mRNA encoding the alpha-subunit of the Ca(2+)/calmodulin-dependent protein kinase II. Binding of MARTA1 and MARTA2 to the MAP2-DTE occurs with an affinity in the nanomolar range. Whereas MARTA1 is clearly detectable in crude lysates, cytosolic and ribosomal salt-wash fractions, and in nuclear extracts, MARTA2 is preferentially found in the ribosomal salt-wash preparation. Neither MARTA is restricted to rat brain, and both are present in a number of other rat tissues. Thus, both proteins may be involved in a variety of nuclear and cytoplasmic events that regulate RNA metabolism in different cell types.

3' Untranslated Regions↗
PubMed1982

Deficiency of functional messenger RNA for a developmentally regulated beta-crystallin polypeptide in a hereditary cataract.

The messenger RNA for a beta-crystallin polypeptide with a molecular size of 27 kilodaltons, first detected 5 to 10 days after birth in the normal mouse lens and the Nakano mouse cataract, was not detected in the Philly mouse cataract with translation in vitro. The heterozygous Philly lens had intermediate levels of the 27-kilodalton beta-crystallin polypeptide and exhibited delayed onset of the cataract. The deficiency of functional 27-kilodalton beta-crystallin messenger RNA is the earliest lesion reported yet for the Philly lens and points to a transcriptional or posttranscriptional developmental defect in this hereditary cataract.

Animals↗
PubMed2005

The RNA-binding protein Musashi-1 regulates neural development through the translational repression of p21WAF-1.

RNA-binding proteins regulate cell fate decisions during nervous system development. The Msi family of RNA-binding proteins is expressed in multipotential neural progenitors, and is required for maintaining cells in a proliferative state. We demonstrate that Msi-1's ability to regulate progenitor maintenance is through the translational inhibition of the cyclin-dependent kinase inhibitor p21WAF-1. Msi-1 ectopic expression increases the proliferation rate and the capacity to regulate p21WAF-1 protein expression, independent of p53. The 3' untranslated region (UTR) of the native p21(WAF-1) mRNA contains a Msi-1 consensus-binding site that permits Msi-1 to directly repress the translation of p21WAF-1 protein. Reduction of Msi-1 through antisense leads to aberrant p21WAF-1 expression, which significantly impairs neural differentiation. A double knockdown for p21WAF-1 and Msi-1 rescues the production of mature MAP+ neurons. Our results further elucidate the symbiotic relationship between cell cycle withdrawal and the onset of differentiation in the developing nervous system, as well as increasing the understanding of the influence that RNA-binding proteins serve in regulating these processes.

Cell Cycle↗
PubMed1979

Regulation of growth hormone messenger RNA.

In cultured rat pituitary cells, glucocorticoids regulate growth hormone production by modulating the number of growth hormone messenger RNA molecules. The effect is quite specific, since only a few other mRNAs are affected by the hormones. This response is demonstrated by assays involving cell-free mRNA translation and cDNA-RNA hybridization. Furthermore, the inducibility by the glucocorticoids is regulated by at least one other class of hormones, thyroid hormone. Thus, this system serves as a model for studying not only the glucocorticoid regulation of specific mRNA, but also the control of this regulation by other factors in the target tissue.

Animals↗
PubMed2005

Structure of an RNA polymerase II-RNA inhibitor complex elucidates transcription regulation by noncoding RNAs.

The noncoding RNA B2 and the RNA aptamer FC bind RNA polymerase (Pol) II and inhibit messenger RNA transcription initiation, but not elongation. We report the crystal structure of FC(*), the central part of FC RNA, bound to Pol II. FC(*) RNA forms a double stem-loop structure in the Pol II active center cleft. B2 RNA may bind similarly, as it competes with FC(*) RNA for Pol II interaction. Both RNA inhibitors apparently prevent the downstream DNA duplex and the template single strand from entering the cleft after DNA melting and thus interfere with open-complex formation. Elongation is not inhibited, as nucleic acids prebound in the cleft would exclude the RNA inhibitors. The structure also indicates that A-form RNA could interact with Pol II similarly to a B-form DNA promoter, as suggested for the bacterial transcription inhibitor 6S RNA.

Base Sequence↗
PubMed2025

Hexokinase 2 is an RNA-binding protein that regulates mRNA translation independently of glycolysis and induces melanoma cell proliferation.

Although metabolic benefits of glycolysis have been extensively described in tumor cells, the extra-metabolic functions linked to this energetic pathway in tumor growth and cell proliferation have not been clearly established yet. Recently, some key glycolytic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase 2, were reported to regulate mRNA translation. Translational control of gene expression is considered as a critical effector in cancer biology, representing a highly promising area of research. Here, we report that Hexokinase 2 (HK2), a glucose kinase that catalyzes the first step of glycolysis at the outer mitochondrial membrane (OMM), is an RNA-binding protein (RBP) that regulates mRNA translation in melanoma cell lines. Polysome profiling experiments followed by RNA sequencing indicate that the translational regulation exerted by HK2 is partly independent of the metabolic status or the glycolytic pathway. We found that HK2 specifically regulates translation of the mRNA encoding SOX10, a transcription factor implicated in the regulation of tumor initiation, maintenance, and progression in melanoma. RNA-protein interaction assays, including CrossLinking ImmunoPrecipitation (CLIP), indicate that HK2 is an RBP whose interaction with RNA is independent of its enzymatic activity, its ability to bind glucose or its association with the OMM. HK2 directly interacts with the 5' untranslated region (5'UTR) of the SOX10 mRNA through a stem-loop RNA secondary structure. Using RNA-protein proximity ligation assays and a fluorescence-based ribosome-bound mRNA mapping method, we found that high glucose conditions, which promote the release of HK2 from the OMM, induce an increase in HK2-SOX10 mRNA interaction and SOX10 mRNA translation in the cytoplasm. We further showed that HK2-dependent SOX10 mRNA translation is involved in melanoma cell proliferation and colony formation. Collectively, our data highlight a nonmetabolic function of HK2 acting as an RBP and translation regulator.

Hexokinase↗
PubMed2000

The antisense RNA of the par locus of pAD1 regulates the expression of a 33-amino-acid toxic peptide by an unusual mechanism.

The par stability determinant of the Enterococcus faecalis plasmid pAD1 is the first antisense RNA-regulated post-segregational killing system (PSK) identified in a Gram-positive organism. Par encodes two small, convergently transcribed RNAs, designated RNA I and RNA II, which are the toxin and antidote of the par PSK system respectively. RNA I encodes an open reading frame of 33 codons designated fst. The results presented here demonstrate that the peptide encoded by fst is the par toxin. The fst sequence was shown to be sufficient for cell killing, and removal of the final codon inactivated the toxin. In vitro translation reactions of purified RNA I transcript produced a product of the expected size for the fst-encoded peptide. This product was not produced when purified RNA II transcript was added to the translation reaction. Toeprint analysis demonstrated that purified RNA II was able to inhibit ribosome binding to RNA I. These data suggest that fst expression is regulated by RNA II via an antisense RNA mechanism. In vitro translation studies and toeprint analyses also indicated that fst expression is internally regulated by a stem-loop structure at the 5' end of RNA I. Removal of this structure resulted in better ribosome binding to RNA I and a 300-fold increase in production of the fst-encoded peptide. Finally, RNA II was shown to be less stable than RNA I in vivo, providing a basis for the selective expression of fst in plasmid-free cells.

Bacterial Toxins↗
PubMed1984

The role of RNA polymerase initiation and elongation in control of total RNA and histone mRNA synthesis in sea urchin embryos.

The involvement of RNA polymerase initiations in regulating total RNA synthesis and the synthesis of the early histone mRNAs was investigated. Nuclei were isolated from developing sea urchin embryos from 4- to 600-cell stages, and the transcription of already initiated polymerase complexes was studied in a "run-off," or elongation, assay; this assay was optimized by using high levels of ribonucleoside triphosphates. Under these conditions the relative levels of RNA synthesis in isolated nuclei from different stages closely paralleled the known rates of synthesis in vivo. However, if sarkosyl is included in the elongation assay, the nuclei of older stages display greatly stimulated synthesis while early cleavage stage nuclei are not stimulated. Sarkosyl does not reveal any elongated transcripts from the early histone genes in nuclei from later stages of development. This has been interpreted to mean that there are many initiated polymerase II complexes that do not elongate rapidly at later stages, but the early histone genes are inactive at later stages because they do not possess any productively initiated polymerases.

Animals↗