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At least 1,099 records · Page 61Linked to original sources
PubMed2003

An expressed pseudogene regulates the messenger-RNA stability of its homologous coding gene.

A pseudogene is a gene copy that does not produce a functional, full-length protein. The human genome is estimated to contain up to 20,000 pseudogenes. Although much effort has been devoted to understanding the function of pseudogenes, their biological roles remain largely unknown. Here we report the role of an expressed pseudogene-regulation of messenger-RNA stability-in a transgene-insertion mouse mutant exhibiting polycystic kidneys and bone deformity. The transgene was integrated into the vicinity of the expressing pseudogene of Makorin1, called Makorin1-p1. This insertion reduced transcription of Makorin1-p1, resulting in destabilization of Makorin1 mRNA in trans by way of a cis-acting RNA decay element within the 5' region of Makorin1 that is homologous between Makorin1 and Makorin1-p1. Either Makorin1 or Makorin1-p1 transgenes could rescue these phenotypes. Our findings demonstrate a specific regulatory role of an expressed pseudogene, and point to the functional significance of non-coding RNAs.

Alleles↗
PubMed2000

Germ plasm and molecular determinants of germ cell fate.

One mechanism for the specification of cell types during embryonic development is the cytoplasmic localization of determinants in the egg into certain blastomeres. Primordial germ cell (PGC) development in many organisms is characterized by the inheritance of germ plasm, a cytologically distinct assembly of mitochondria and electron-dense germinal granules. This chapter reviews the structure of germ plasm and the experimental evidence for its importance in PGC specification in Caenorhabditis elegans, Drosophila, and Xenopus. It then compares and contrasts recent data on the identification of germ plasm components in these organisms. Many components are potentially RNA-binding proteins, implicating the regulation of RNA metabolism, transport, and translation as critical processes in PGC development. Germ plasm components also mediate transcriptional repression, regulate migration, and control mitotic divisions in PGCs. The chapter concludes with a discussion on the general roles of germ plasm components and how they might act to specify PGC fate.

Animals↗
PubMed2002

Two isoforms of the Drosophila RNA binding protein, how, act in opposing directions to regulate tendon cell differentiation.

Differential RNA metabolism regulates a wide array of developmental processes. Here, we describe a mechanism that controls the transition from premature Drosophila tendon precursors into mature muscle-bound tendon cells. This mechanism is based on the opposing activities of two isoforms of the RNA binding protein How. While the isoform How(L) is a negative regulator of Stripe, the key modulator of tendon cell differentiation, How(S) isoform elevates Stripe levels, thereby releasing the differentiation arrest induced by How(L). The opposing activities of the How isoforms are manifested by differential rates of mRNA degradation of the target stripe mRNA. This mechanism is conserved, as the mammalian RNA binding Quaking proteins may similarly affect the levels of Krox20, a regulator of Schwann cell maturation.

3' Untranslated Regions↗
PubMed1976

Role of ribosomal RNA methylases in the regulation of ribosome production in mammalian cells.

The activity of rRNA methylases was stimulated by high-energy precursors of RNA (ribonucleoside triphosphates) and inhibited by degradation products of RNA (ribonucleotides and oligoribonucleotides). The response of methylases from rat Novikoff ascites tumor and liver to these metabolites was strikingly different. The highly active tumor enzymes responded preferentially to inhibition by catabolic metabolites, whereas the less active liver enzymes responded exclusively to stimulation by anabolic metabolites. When the activity of rRNA methylases was assayed in response to increasing concentration of S-adenosylmethionine, the tumor enzymes responded with a hyperbolic substrate dependence curve and the liver enzymes with a sigmoidal curve. In the presence of an inhibitory dinucleotide, ApA, the tumor enzymes responded with a sigmoidal curve; in the presence of a stimulator, adenosine 5'-triphosphate, the liver enzymes responded with a hyperbolic substrate concentration curve. When normal rats were subject to a series of treatments by thioacetamide, a hepatocarcinogen, the liver nucleolar rRNA methylases became responsive to inhibition by ApA and relatively unresponsive to stimulation by adenosine 5'-triphosphate. When tumor-bearing rats were treated with polyinosinate:polycytidylate, an antitumor agent, the tumor nucleolar rRNA methylases became unresponsive to inhibition by ApA and more responsive to stimulation by adenosine 5'-triphosphate. A correlation was noted between increased methylation efficiency in vivo and increased stability of nucleolar RNA during incubation in vitro, or vice versa. These results are interpreted to indicate that rRNAmethylases are regulated by cellular metabolites during the nucleolar biosynthesis of ribosomes and that rRNA methylases may provide a favorable site for selective action by cancer chemotherapeutic agents.

Adenosine Triphosphate↗
PubMed1997

A RNA polymerase III-based two-hybrid system to study RNA polymerase II transcriptional regulators.

In a previous study, we explored the mechanisms of SNR6 gene activation by grafting a heterologous DNA-binding domain, GAL4-(1-147), to various components of the yeast RNA polymerase III transcription system. Here, we demonstrate that a modified SNR6 gene harboring GAL4-binding sites (UAS(G)-SNR6) can be efficiently activated via an intervening, unrelated protein-protein interaction, thus laying the foundations of a RNA polymerase III-based two-hybrid system. In a model system, the interacting proteins recruiting TFIIIC to DNA were PRP21 and PRP9 or PRP21 and PRP11. Mutations affecting the interaction between PRP21 and PRP9, or PRP21 and PRP11 decreased UAS(G)-SNR6 activation level proportionally. RNA polymerase II transcriptional activators, like GAL4, VP16 or p53, fused to GAL4 DNA-binding domain, did not activate the UAS(G)-SNR6 gene. However, GAL4 strongly activated UAS(G)-SNR6 when GAL80, an interacting protein, was fused to TFIIIC. This result indicates that this two-hybrid system can be used to assess the interactions between RNA polymerase II regulatory proteins and their partners.

DNA-Binding Proteins↗
PubMed1993

Regulation of myeloblastin messenger RNA expression in myeloid leukemia cells treated with all-trans retinoic acid.

Retinoic acid is known to induce differentiation of human myeloid leukemia cells in vitro. Recently, all-trans retinoic acid has been used to induce remissions in patients with acute promyelocytic leukemia, probably through differentiation of the leukemia cells. Myeloblastin (mbn) is a protease that has been identified in the human leukemia cell line HL-60. Downregulation of this protease can inhibit proliferation and induce differentiation of HL-60-derived leukemia cells. Here we have investigated the regulation of mbn messenger RNA (mRNA) expression in two human leukemia cell lines, HL-60 and NB4, treated with all-trans retinoic acid. Under this treatment, downregulation of mbn mRNA was observed in both cell lines, but was considerably delayed in NB4 cells that carry the t(15;17) translocation characteristic of acute promyelocytic leukemia. We have found that multiple mechanisms were involved in the control of mbn mRNA expression. These mechanisms were different in HL-60 and NB4 cells. Our results show that in HL-60 cells, all-trans retinoic acid rapidly decreased transcription of mbn. In contrast, in the t(15;17)-positive NB4 cells treated with all-trans retinoic acid, upregulation of mbn mRNA expression was followed by a late downregulation, both achieved via posttranscriptional mechanisms.

Cell Cycle↗
PubMed2002

Transcriptional and post-transcriptional regulation of tyrosine hydroxylase messenger RNA in PC12 cells during persistent stimulation by VIP and PACAP38: differential regulation by protein kinase A and protein kinase C-dependent pathways.

VIP and PACAP38 are closely related peptides that are released in the adrenal gland and sympathetic ganglia and regulate catecholamine synthesis and release. We used PC12 cells as a model system to examine receptor and second messenger pathways by which each peptide stimulates transcriptional and post-transcriptional mechanisms that regulate the level of the mRNA for tyrosine hydroxylase (TH), the rate-limiting enzymatic step in catecholamine synthesis. Concentration-response studies revealed that PACAP38 had both greater efficacy and potency than VIP. The specific PAC1 receptor antagonist PACAP[6-38] blocked the effects of each peptide on TH mRNA content while the PACAP/VIP type II receptor antagonist (N-AC-Tyr(1)-D-Phe(2))-GRF-(1-29)-NH(2) was without effect. At equipotent concentrations, each peptide stimulated a transient increase in TH gene transcription lasting less than 3h. Continuous VIP treatment stimulated a transient increase in TH mRNA lasting less than 24h. In contrast, continuous exposure to PACAP38 stimulated a stable increase in TH mRNA that persisted for 2 days in the absence of elevated transcription, pointing to different post-transcriptional effects of the two peptides. PACAP38 alone had no effect on the magnitude of TH gene transcription or TH mRNA in A126-1B2 PKA-deficient PC12 cells. However, when combined with dexamethasone, PACAP38 produced a synergistic increase in TH mRNA in the absence of PACAP38-stimulated TH gene transcription. In contrast, VIP had no effect on either TH mRNA content or TH gene transcription in this model. PACAP38, but not VIP, stimulated PKC activity. Calphostin C antagonized the effect of PACAP38 on the persistent post-transcriptional elevation in TH mRNA. Thus, the results support the conclusion that VIP and PACAP38 each stimulate PAC1 receptors to increase TH gene transcription through a PKA-controlled pathway, but their divergent post-transcriptional effects result at least partly from differing abilities to stimulate PKC.

Animals↗
PubMed1996

A tightly regulated expression system in Escherichia coli with SP6 RNA polymerase.

A tightly regulated gene-expression system was developed using SP6 RNA polymerase (RpoSP6). The RpoSp6-encoding gene (rpoSP6) was inserted into a mini-F plasmid (mini-F) and expression was controlled by the lactose promoter (P(lac)) and operator (O(lac)) on the plasmid. Therefore, a controlled expression system for the target genes can easily be constructed in various host strains by co-transformation of the system plasmid pFSP6 with other vector plasmids containing the genes linked to the SP6 promoters (P(SP6)). Using the lac gene linked to P(SP6) as a reporter, we evaluated the regulation of expression in this system in various host strains. Low-level expression of lac was detected in Escherichia coli harboring this expression system when RpoSP6 was uninduced, although very low activities of beta-galactosidase (beta-Gal) were observed which were independent of the presence of pFSP6. This basal level of beta-Gal activity was possibly derived, because the P(SP6) element has very weak activity for E. coli RNA polymerase (Rpo). These results showed that RpoSP6 seemed to be produced at very low levels in uninduced cells. Beta-GAl activity increased about 18-32-fold when the expression of rpoSP6 was induced, as compared with the beta-Gal activity when uninduced. The tight regulation of this system is superior to that of other known systems and it has a considerable advantage for gene expression in E. coli.

Base Sequence↗
PubMed2004

Selection of stable RNA molecules that can regulate the channel-opening equilibrium of the membrane-bound gamma-aminobutyric acid receptor.

The gamma-aminobutyric acid (GABA(A)) receptor belongs to a superfamily of membrane-bound proteins that regulate signal transmission between cells in the nervous system. It is the target of convulsants such as picrotoxin and is mutated in some forms of epilepsy, a disease affecting approximately 50 million people worldwide. In picrotoxin inhibition and in one form of epilepsy, a decrease in the channel-opening equilibrium of a GABA(A) receptor is responsible for receptor dysfunction. Here we identify compounds that can regulate the channel-opening equilibrium of the GABA(A) receptor. Fluorinated RNA polymers containing a 40-nucleotide region with a randomized sequence were used to select those that can displace picrotoxin from the membrane-bound GABA(A) receptor in the rat forebrain. After 11 selection rounds, two classes of RNA molecules that bind to the GABA(A) receptor with nanomolar affinity were isolated and sequenced. Class I and class II molecules have different consensus sequences and different binding affinities for the receptor. A transient kinetic technique, the cell-flow method, was employed in combination with the whole-cell current-recording technique to determine the affinity of the selected RNA aptamers for the GABA(A) receptor. Class I molecules have a higher affinity for the closed-channel form than for the open-channel receptor form and inhibit the receptor; class II aptamers bind with equal or higher affinity to the open-channel form and alleviate picrotoxin inhibition.

Animals↗
PubMed2002

The role of the carboxyterminal domain of RNA polymerase II in regulating origins of DNA replication in Saccharomyces cerevisiae.

MCM (minichromosome maintenance) proteins function as a replication licensing factor (RLF-M), which contributes to limiting initiation of DNA replication to once per cell cycle. In the present study we show that a truncation of the pol II CTD in a S. cerevisiae strain harboring a mutation in mcm5 partially reverses its ts phenotype and improves maintenance of CEN/ARS minichromosomes. We correlate this phenotype to effects on DNA replication rather than to effects on transcription or specific gene expression. We also demonstrate that a similar truncation of the CTD reduces minichromosome stability and impairs stimulation of DNA replication by trans-activators and that tethering of recombinant pol II CTD to an origin of replication has a significant stimulatory effect on minichromosome stability. Furthermore, we show that pol II is recruited to ARS1. We propose that in S. cerevisiae a mechanism of coordinating pol II transcription and DNA replication is mediated by the CTD of pol II.

Cell Cycle Proteins↗
PubMed2003

Csm, a cardiac-specific isoform of the RNA helicase Mov10l1, is regulated by Nkx2.5 in embryonic heart.

Nkx2.5 (also called Csx) is an evolutionarily conserved cardiac transcription factor of the homeobox gene family. Nkx2.5 is required for early heart development, because Nkx2.5 null mice die before completion of cardiac looping. To identify genes regulated by Nkx2.5 in the developing heart, we performed differential screening in combination with suppression subtractive hybridization using RNA isolated from wild-type and Nkx2.5 null hearts at embryonic day 8.5. One gene that we found to be markedly down-regulated in the hearts from Nkx2.5 null embryos is an isoform of Mov10 like-1 (Mov10l1), a putative RNA helicase expressed in testis. We named this novel isoform as Csm (cardiac-specific isoform of Mov10l1). Csm is identical with the 3' region of the Mov10l1 gene, but its transcript starts from the exon 16 of Mov10l1. The conceptual protein encoded by Csm cDNA contains a helicase motif as well as ATPase and RNA interaction motifs. Csm is expressed specifically in the heart, and its expression in the heart is restricted to cardiac myocytes. Csm potentiated phenylephrine-induced hypertrophic response in cardiac myocytes. Furthermore, transient cotransfection analysis showed that Nkx2.5 transactivates the Csm promoter, suggesting that Nkx2.5 is essential for embryonic Csm expression.

Amino Acid Sequence↗
PubMed1994

Translational regulation of nanos by RNA localization.

Localization of the maternally synthesized nanos (nos) RNA to the posterior pole of the Drosophila embryo provides the source for a posterior-to-anterior gradient of Nos protein. Correct spatial regulation of nos activity is essential for normal pattern formation. High local concentrations of Nos protein in the posterior of the embryo are necessary to inhibit translation of the transcription factor Hunchback in this region, and thus permit expression of genes required for abdomen formation (see ref. 5 for review). By contrast, misexpression of Nos protein at the anterior of the embryo prevents translation of the anterior morphogen Bicoid, suppressing head and thorax development. Posterior localization of nos RNA is mediated by sequences within the nos 3' untranslated region (3'UTR) and requires the function of eight genes of the 'posterior group'. Although the unlocalized nos RNA is stable in embryos from females mutant for any of the posterior group genes, these embryos appear to lack nos activity because they develop the abdominal defects characteristic of embryos produced by nos mutant females. We report here that unlocalized nos RNA is translationally repressed. Translational repression is mediated by the nos 3'UTR and can be alleviated either by replacement of the 3'UTR with heterologous 3'UTR sequences or by posterior localization. Thus, RNA localization provides a novel mechanism for translational regulation.

Animals↗
PubMed1998

RNA-mediated trans-activation of transcription from a viral RNA.

The red clover necrotic mosaic virus genome is composed of two single-stranded RNA components, RNA-1 and RNA-2. The viral capsid protein is translated from a subgenomic RNA (sgRNA) that is transcribed from genomic RNA-1. Here, a 34-nucleotide sequence in RNA-2 is shown to be required for transcription of sgRNA. Mutations that prevent base-pairing between the RNA-1 subgenomic promoter and the 34-nucleotide trans-activator prevent expression of a reporter gene. A model is proposed in which direct binding of RNA-2 to RNA-1 trans-activates sgRNA synthesis. This RNA-mediated regulation of transcription is unusual among RNA viruses, which typically rely on protein regulators.

Base Composition↗
PubMed2005

Genomic mapping of RNA polymerase II reveals sites of co-transcriptional regulation in human cells.

BACKGROUND: Transcription by RNA polymerase II is regulated at many steps including initiation, promoter release, elongation and termination. Accumulation of RNA polymerase II at particular locations across genes can be indicative of sites of regulation. RNA polymerase II is thought to accumulate at the promoter and at sites of co-transcriptional alternative splicing where the rate of RNA synthesis slows. RESULTS: To further understand transcriptional regulation at a global level, we determined the distribution of RNA polymerase II within regions of the human genome designated by the ENCODE project. Hypophosphorylated RNA polymerase II localizes almost exclusively to 5' ends of genes. On the other hand, localization of total RNA polymerase II reveals a variety of distinct landscapes across many genes with 74% of the observed enriched locations at exons. RNA polymerase II accumulates at many annotated constitutively spliced exons, but is biased for alternatively spliced exons. Finally, RNA polymerase II is also observed at locations not in gene regions. CONCLUSION: Localizing RNA polymerase II across many millions of base pairs in the human genome identifies novel sites of transcription and provides insights into the regulation of transcription elongation. These data indicate that RNA polymerase II accumulates most often at exons during transcription. Thus, a major factor of transcription elongation control in mammalian cells is the coordination of transcription and pre-mRNA processing to define exons.

Chromatin Immunoprecipitation↗
PubMed2002

The downstream DNA jaw of bacterial RNA polymerase facilitates both transcriptional initiation and pausing.

Regulation of RNA polymerase during initiation, elongation, and termination of transcription is mediated in part by interactions with intrinsic regulatory signals encoded in the RNA and DNA that contact the enzyme. These interactions include contacts to an 8-9-bp RNA:DNA hybrid within the active-site cleft of the enzyme, contacts to the melted nontemplate DNA strand in the vicinity of the hybrid, contacts to exiting RNA upstream of the hybrid, and contacts to approximately 20 bp of duplex DNA downstream of the active site. Based on characterization of an amino acid substitution (G1161R) and a deletion (Delta1149-1190) in the jaw domain of the bacterial RNA polymerase largest subunit (beta'), we report here that contacts of the jaw domain to downstream DNA at the leading edge of the transcription complex contribute to regulation during all three phases of transcription. The results provide insight into the role of the jaw domain-downstream DNA contact in transcriptional initiation and pausing and suggest possible explanations for the previously reported isolation of the jaw mutants based on reduced ColEI plasmid replication.

Amino Acid Sequence↗
PubMed2003

DNA damage-binding proteins and heterogeneous nuclear ribonucleoprotein A1 function as constitutive KCS element components of the interferon-inducible RNA-dependent protein kinase promoter.

Protein kinase regulated by RNA (PKR) plays important roles in many cellular processes including virus multiplication and cell growth, differentiation, and apoptosis. The promoter of the PKR gene possesses a novel 15-bp element designated KCS, positioned upstream of a consensus interferon (IFN)-stimulated response element, that is required for both basal and interferon-inducible transcription. Protein binding to the KCS element is not dependent upon IFN treatment and correlates with transcriptional activity of the PKR promoter. The identity of KCS-binding proteins (KBP) that selectively bind at the KCS element is largely unknown, except for the transcription factor Sp1. We now have purified KBP from HeLa cell nuclear extracts by ion-exchange and DNA-affinity chromatography steps and then identified four constituent proteins of the KBP complex by mass spectrometry and immunochemistry: KBP120 and KBP45 are the damaged DNA-binding protein subunits, p127 DDB1 and p48 DDB2, respectively; KBP100 is the transcription factor Sp1; and KBP35 is the heterogeneous nuclear ribonucleoprotein A1. The steady-state levels of these four KCS-binding proteins in human cells are not altered by IFN treatment. Components of the KBP complex bind selectively and constitutively to the KCS element in the absence of IFN treatment, both in vitro as measured by competition electrophoretic mobility shift assay (EMSA) and DNA pull-down assays and in vivo as measured by chromatin immunoprecipitation assays. Depletion of DDB2 by antisense strategy reduces KBP complex formation by EMSA. These results provide new insight into the biochemical identity and activity of proteins involved in PKR promoter function.

Amino Acid Sequence↗
PubMed1996

Cleavage of RNA hairpins mediated by a developmentally regulated CCCH zinc finger protein.

Control of RNA turnover is a major, but poorly understood, aspect of gene regulation. In multicellular organisms, progress toward dissecting RNA turnover pathways has been made by defining some cis-acting sequences that function as either regulatory or cleavage targets (J. G. Belasco and G. Brawerman, Control of Messenger RNA Stability, 1993). However, the identification of genes encoding proteins that regulate or cleave target RNAs has been elusive (C. A. Beelman and R. Parker, Cell 81:79-183, 1995); this gap in knowledge has made it difficult to identify additional components of RNA turnover pathways. We have utilized a modified expression cloning strategy to identify a developmentally regulated gene from Drosophila melanogaster that encodes a RNase that we refer to as Clipper (CLP). Significant sequence matches to open reading frames encoding unknown functions identified from the Caenorhabditis elegans and Saccharomyces cerevisiae genome sequencing projects suggest that all three proteins are members of a new protein family conserved from lower eukaryotes to invertebrates. We demonstrate that a member of this new protein family specifically cleaves RNA hairpins and that this activity resides in a region containing five copies of a previously uncharacterized CCCH zinc finger motif. CLP's endoribonucleolytic activity is distinct from that associated with RNase A (P. Blackburn and S. Moore, p. 317-433, in P. D. Boyer, ed., The Enzymes, vol. XV, part B, 1982) and is unrelated to RNase III processing of rRNAs and tRNAs (J. G. Belasco and G. Brawerman, Control of Messenger RNA Stability, 1993, and S. A. Elela, H. Igel, and M. Ares, Cell 85:115-124, 1995). Our results suggest that CLP may function directly in RNA metabolism.

Amino Acid Sequence↗