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At least 1,153 records · Page 64Linked to original sources
PubMed2003

PNUTS, a protein phosphatase 1 (PP1) nuclear targeting subunit. Characterization of its PP1- and RNA-binding domains and regulation by phosphorylation.

PNUTS, Phosphatase 1 NUclear Targeting Subunit, is a recently described protein that targets protein phosphatase 1 (PP1) to the nucleus. In the present study, we characterized the biochemical properties of PNUTS. A variety of truncation and site-directed mutants of PNUTS was prepared and expressed either as glutathione S-transferase fusion proteins in Escherichia coli or as FLAG-tagged proteins in 293T cells. A 50-amino acid domain in the center of PNUTS mediated both high affinity PP1 binding and inhibition of PP1 activity. The PP1-binding domain is related to a motif found in several other PP1-binding proteins but is distinct in that Trp replaces Phe. Mutation of the Trp residue essentially abolished the ability of PNUTS to bind to and inhibit PP1. The central PP1-binding domain of PNUTS was an effective substrate for protein kinase A in vitro, and phosphorylation substantially reduced the ability of PNUTS to bind to PP1 in vitro and following stimulation of protein kinase A in intact cells. In vitro RNA binding experiments showed that a C-terminal region including several RGG motifs and a novel repeat domain rich in His and Gly interacted with mRNA and single-stranded DNA. PNUTS exhibited selective binding for poly(A) and poly(G) compared with poly(U) or poly(C) ribonucleotide homopolymers, with specificity being mediated by distinct regions within the domain rich in His and Gly and the domain containing the RGG motifs. Finally, a PNUTS-PP1 complex was isolated from mammalian cell lysates using RNA-conjugated beads. Together, these studies support a role for PNUTS in protein kinase A-regulated targeting of PP1 to specific RNA-associated complexes in the nucleus.

Amino Acid Motifs↗
PubMed1976

Mechanism of growth delay induced in Escherichia coli by near ultraviolet radiation.

Continuously growing cultures of E. coli B/r were irradiated with a fluence of broad-band near-ultraviolet radiation (315-405 nm) sufficient to cause extensive growth delay and complete cessation of net RNA synthesis. Chloramphenicol treatment was found to stimulate resumption of RNA synthesis, similar to that observed with chloramphenicol treatment after amino-acid starvation. E. coli strains in which amino-acid starvation does not result in cessation of RNA synthesis ("relaxed" or rel- strains) show no cessation of growth and only a slight effect on the rate of growth or of RNA synthesis. These findings show that such near-UV fluences do not inactivate the RNA synthetic machinery but affect the regulation of RNA synthesis, in a manner similat to that produced by amino-acid starvation. Such regulation is believed to be mediated through alterations in concentration of guanosine tetraphosphate (ppGpp), and our estimations of ppGpp after near-UV irradiation are consistent with such an interpretation. These data, combined with earlier published data, strongly suggest that the mechanism of near-UV-induced growth delay in E. coli involves partial inactivation of certain tRNA species, which is interpreted by the cell in a manner similar to that of amino-acid starvation, causing a rise in ppGpp levels, a shut-off of net RNA synthesis, and the induction of a growth delay.

Amino Acids↗
PubMed1992

Role of TAR RNA splicing in translational regulation of simian immunodeficiency virus from rhesus macaques.

The untranslated leader sequences of rhesus macaque simian immunodeficiency virus mRNAs form a stable secondary structure, TAR. This structure can be modified by RNA splicing. In this study, the role of TAR splicing in virus replication was investigated. The proportion of viral RNAs containing a spliced TAR structure is high early after infection and decreases at later times. Moreover, proviruses containing mutations which prevent TAR splicing are significantly delayed in replication. These mutant viruses require approximately 20 days to achieve half-maximal virus production, in contrast to wild-type viruses, which require approximately 8 days. We attribute this delay to the inefficient translation of unspliced-TAR-containing mRNAs. The molecular basis for this translational effect was examined in in vitro assays. We found that spliced-TAR-containing mRNAs were translated up to 8.5 times more efficiently than were similar mRNAs containing an unspliced TAR leader. Furthermore, these spliced-TAR-containing mRNAs were more efficiently associated with ribosomes. We postulate that the level of TAR splicing provides a balance for the optimal expression of both viral proteins and genomic RNA and therefore ultimately controls the production of infectious virions.

Animals↗
PubMed1994

Androgen regulation of the messenger RNA encoding diazepam-binding inhibitor/acyl-CoA-binding protein in the human prostatic adenocarcinoma cell line LNCaP.

To study the mechanisms by which androgens intervene in the regulation of growth and differentiation of human prostatic epithelial cells, cDNA clones encoding putative prostate-secreted proteins were characterized and tested as potential markers for androgen action. One of the isolated cDNAs expressed diazepam-binding inhibitor/acyl-CoA-binding protein (DBI/ACBP), suggesting that this polypeptide, that has been implicated in a large number of biochemical processes, is expressed and secreted by prostate cells. As demonstrated by Northern blot analysis, the mRNA encoding DBI/ACBP was expressed in prostate tissue and in the three human prostatic adenocarcinoma cell lines tested: LNCaP, PC-3 and DU-145. In androgen-sensitive LNCaP cells, the synthetic androgen R1881 stimulated the DBI/ACBP steady state mRNA levels with half maximal effects at a concentration of 0.2 nM. Increases were a maximal 12 h after addition of the synthetic hormone. DBI/ACBP mRNA levels could also be stimulated by the synthetic androgen mibolerone and by the natural androgens testosterone and dihydrotestosterone. In agreement with the altered steroid specificity of the androgen receptor in LNCaP cells, estradiol and progesterone also exerted a stimulatory effect. Cortisol and the synthetic glucocorticoid dexamethasone were without effect. Androgen stimulation of DBI/ACBP mRNA levels was abolished in the presence of the protein synthesis inhibitor cycloheximide, implying a role for labile or androgen-induced proteins in this androgen stimulation. This is in contrast to the androgen stimulation of the mRNA encoding prostate-specific antigen (PSA), suggesting that different mechanisms are involved in the androgen regulation of these two genes. Although further experiments are required to confirm that DBI/ACBP is secreted by prostatic epithelial cells, these data demonstrate that the mRNA encoding DBI/ACBP is expressed in prostate cells and is affected by androgens in androgen-responsive LNCaP cells.

Adenocarcinoma↗
PubMed1993

Interaction between the antisense and target RNAs involved in the regulation of IncB plasmid replication.

Physical analysis of RNA I, the small antisense RNA which regulates the replication of IncB miniplasmid pMU720, showed that it is a highly structured molecule containing an imperfectly paired stem closed by a 6-base hairpin loop. Mutational studies revealed that a 3-base sequence in the hairpin loop is critical to the interaction between RNA I and its complementary target in the RepA mRNA (RNA II). Furthermore, a 2-base interior loop in the upper stem was found to play an important role in facilitating effective binding between RNA I and RNA II. From these analyses, a model describing the molecular mechanism of binding between RNA I and RNA II is proposed.

Base Sequence↗
PubMed1996

RNA polymerase II holoenzyme contains SWI/SNF regulators involved in chromatin remodeling.

The RNA polymerase II holoenzyme contains RNA polymerase II, a subset of general transcription factors and SRB regulatory proteins. We report here that SWI and SNF gene products, previously identified as global gene regulators whose functions include remodeling chromatin, are also integral components of the yeast RNA polymerase II holoenzyme. The SWI/SNF proteins are components of the SRB complex, also known as the mediator, which is tightly associated with the RNA polymerase II C-terminal repeat domain. The SWI/SNF components provide the holoenzyme with the capacity to disrupt nucleosomal DNA and thus facilitate stable binding of various components of the transcription initiation complex at promoters.

Adenosine Triphosphate↗
PubMed2004

Regulation of Semliki Forest virus RNA replication: a model for the control of alphavirus pathogenesis in invertebrate hosts.

Alphavirus nonstructural proteins are translated as a polyprotein that is ultimately cleaved into four mature proteins called nsP1, nsP2, nsP3, and nsP4 from their order in the polyprotein. The role of this nonstructural polyprotein, of cleavage intermediates, and of mature proteins in synthesis of Semliki Forest virus (SFV) RNA has been studied using mutants unable to cleave one or more of the sites in the nonstructural polyprotein or that had the arginine sense codon between nsP3 and nsP4 changed to an opal termination codon. The results were compared with those obtained for Sindbis virus (SINV), which has a naturally occurring opal codon between nsP2 and nsP3. We found that (1) an active nonstructural protease in nsP2 is required for RNA synthesis. This protease is responsible for all three cleavages in the nonstructural polyprotein. (2) Cleavage between nsP3 and nsP4 (the viral RNA polymerase) is required for RNA synthesis by SFV. (3) SFV mutants that are able to produce only polyprotein P123 and nsP4 synthesize minus-strand RNA early after infection as efficiently as SF wild type but are defective in the synthesis of plus-strand RNA. The presence of sense or opal following nsP3 did not affect this result. At 30 degrees C, they give rise to low yields of virus after a delay, but at 39 degrees C, they are nonviable. (4) SFV mutants that produce nsP1, P23, nsP4, as well as the precursor P123 are viable but produce an order of magnitude less virus than wild type at 30 degrees C and two orders of magnitude less virus at 39 degrees C. The ratio of subgenomic mRNA to genomic RNA is much reduced in these mutants relative to the parental viruses. (5) At 30 degrees C, the variants containing an opal codon grow as well as or slightly better than the corresponding virus with a sense codon. At 39 degrees C, however, the opal variants produce significantly more virus. These results support the conclusion that SFV and SINV, and by extension all alphaviruses, regulate their RNA synthesis in the same fashion after infection. P123 and nsP4 form a minus-strand replicase that synthesizes plus-strand RNA only inefficiently, especially at the higher temperatures found in mammals and birds. A replicase containing nsP1, P23, and nsP4 can make both plus and minus strands, but prefers the promoter for genomic plus sense RNA to that for subgenomic mRNA. The fully cleaved replicase can make only plus-strand RNA, and prefers the promoter for subgenomic mRNA to that for genomic RNA. Alphaviruses alternate between infection of hematophagous arthropods and higher vertebrates. Although the infection of higher vertebrates is acute and often accompanied by disease, continuing transmission of the virus in nature requires that infection of arthropods be persistent and relatively asymptomatic. We propose that this mechanism for control of RNA synthesis evolved to moderate the pathogenicity of the viruses in their arthropod hosts.

Animals↗
PubMed2002

Transcript mutations of the alpha regulatory subunit of protein kinase A and up-regulation of the RNA-editing gene transcript in lupus T lymphocytes.

BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune disorder characterised by diverse dysfunctions of immune effector cells, including proliferation and cytotoxicity. In T cells from patients with SLE, activity of type 1 protein kinase A isozymes is greatly reduced because of decreased expression of the alpha and beta regulatory subunits (RI alpha and RI beta). We aimed to identify a molecular mechanism or mechanisms for this isozyme deficiency by assessing occurrence of mutations in transcripts of the RI alpha subunit in patients with SLE. METHODS: We cloned and sequenced cDNA of RI alpha and corresponding genomic DNA of the coding region to detect sequence changes from eight patients with SLE and six healthy controls. Because transcript editing is regulated by adenosine deaminases that act on RNA (ADAR), we quantified expression of ADAR1 transcripts in SLE and control T cells by competitive PCR. FINDINGS: Sequence analyses of cDNA showed heterogeneous transcript mutations, including deletions, transitions, and transversions. We identified 1.22 x 10(-3)/bp transcript mutations in SLE T cells-a frequency 7.5 times higher than that in control T cells. By contrast, we identified no genomic mutations. Two hotspots were identified in the RI alpha subunit transcripts from SLE T cells, one located adjacent to a pseudosubstrate site of the RI alpha subunit and the other a component of the cAMP binding A domain. ADAR1 mRNA content was 3.5 times higher in SLE cells than in control T cells (p=0.001). INTERPRETATION: An RNA-editing enzyme could be converting adenosine to inosine within double-stranded regions of RNA, resulting in transcript mutations. This process could be one mechanism resulting in mutations in the RI alpha subunit of type 1 protein kinase A.

Adenosine Deaminase↗
PubMed1987

Peptides regulate the activity of RNA polymerases in Tetrahymena.

An exponentially multiplying population of the protozoa Tetrahymena has a highly variable transcription rate when cultivated in a complex broth medium. It is shown that peptides in the medium specifically stimulate transcription and that the cells in response synthesize a peptide with a molecular weight of 3000-4000. This peptide inhibits transcription in vivo in cells with high rates of transcription and in in vitro transcription systems. We have partially purified radioactively labelled inhibitor peptide and found that cells stimulated to high transcription rate selectively accumulate inhibitor peptide in the nuclei. In vitro experiments have shown that the inhibitor peptide can reduce the RNA polymerase activity in the nuclei. The presence of inhibitor seems to lower the affinity of RNA-polymerases towards DNA and to increase their release during incubation of isolated nuclei. On the other hand, stimulatory peptides will compete with the inhibitor, and the polymerase activity in isolated nuclei is determined--at least partially--by the ratio between stimulatory and inhibitory peptide factors. It is suggested that regulatory peptides also are involved in regulation of transcription in vivo.

Animals↗
PubMed1990

Apolipoprotein B messenger RNA editing is developmentally regulated in pig small intestine: nucleotide comparison of apolipoprotein B editing regions in five species.

Apolipoprotein B (apo B) mRNA undergoes a posttranscriptional tissue-specific editing reaction which changes codon 2153 from glutamine (CAA) in apoB-100 mRNA to an in-frame stop codon (UAA) in apoB-48 mRNA. Small intestinal apo B mRNA was found to be predominantly (greater than 90%) unedited in fetal (40-day gestational age) piglets but greater than 95% edited in neonatal, suckling and adult animals. By contrast, both fetal, neonatal and adult pig liver contained greater than 99% unedited, apo B-100 mRNA. The nucleotide sequence spanning the edited region in apo B mRNA was found to be highly conserved. We speculate that the regulation of apo B mRNA editing may be developmentally modulated in pig small intestine.

Aging↗
PubMed2003

RsmY, a small regulatory RNA, is required in concert with RsmZ for GacA-dependent expression of biocontrol traits in Pseudomonas fluorescens CHA0.

In the plant-beneficial soil bacterium and biocontrol model organism Pseudomonas fluorescens CHA0, the GacS/GacA two-component system upregulates the production of biocontrol factors, i.e. antifungal secondary metabolites and extracellular enzymes, under conditions of slow, non-exponential growth. When activated, the GacS/GacA system promotes the transcription of a small regulatory RNA (RsmZ), which sequesters the small RNA-binding protein RsmA, a translational regulator of genes involved in biocontrol. The gene for a second GacA-regulated small RNA (RsmY) was detected in silico in various pseudomonads, and was cloned from strain CHA0. RsmY, like RsmZ, contains several characteristic GGA motifs. The rsmY gene was expressed in strain CHA0 as a 118 nt transcript which was most abundant in stationary phase, as revealed by Northern blot and transcriptional fusion analysis. Transcription of rsmY was enhanced by the addition of the strain's own supernatant extract containing a quorum-sensing signal and was abolished in gacS or gacA mutants. An rsmA mutation led to reduced rsmY expression, via a gacA-independent mechanism. Overexpression of rsmY restored the expression of target genes (hcnA, aprA) to gacS or gacA mutants. Whereas mutants deleted for either the rsmY or the rsmZ structural gene were not significantly altered in the synthesis of extracellular products (hydrogen cyanide, 2,4-diacetylphloroglucinol, exoprotease), an rsmY rsmZ double mutant was strongly impaired in this production and in its biocontrol properties in a cucumber-Pythium ultimum microcosm. Mobility shift assays demonstrated that multiple molecules of RsmA bound specifically to RsmY and RsmZ RNAs. In conclusion, two small, untranslated RNAs, RsmY and RsmZ, are key factors that relieve RsmA-mediated regulation of secondary metabolism and biocontrol traits in the GacS/GacA cascade of strain CHA0.

Bacterial Proteins↗
PubMed2004

Multiple domains control the subcellular localization and activity of ETR-3, a regulator of nuclear and cytoplasmic RNA processing events.

Embryonic lethal abnormal vision (ELAV) type RNA binding protein 3 (ETR-3; also called NAPOR, CUGBP2, or BRUNOL3) has been implicated in the regulation of nuclear and cytoplasmic RNA processing events, including alternative splicing, RNA editing, stability and translation. Here, we report that the ETR-3 protein contains multiple regions that control its subcellular localization and are important for its activity as a splicing regulator. We cloned ETR-3 from chicken heart and fused it to the C terminus of green fluorescent protein (GFPcETR3vL). GFPcETR3vL is found predominantly in the nucleus and is an active regulator of alternative splicing in cotransfection assays with a cardiac troponin T minigene. ETR-3 contains two N-terminal RNA recognition motifs (RRMs), a 210-amino acid divergent domain, and a C-terminal RRM. We demonstrate that the C terminus contains a strong nuclear localization signal overlapping the third RRM, which can confer nuclear localization on a normally cytoplasmic pyruvate kinase chimera. Additional deletions revealed nuclear localization and export activities in the divergent domain of ETR-3, as well as regions within the first two RRMs that are important for cytoplasmic localization. The nuclear export activity of the divergent domain is sensitive to leptomycin B, indicating that export to the cytoplasm is mediated via a CRM1-dependent pathway. The C terminus and a region within the divergent domain were also shown to be important for splicing activity of ETR-3. This is the first characterization of protein domains involved in mediating the subcellular localization and splicing activity of a member of the CELF family of RNA processing regulators.

Alternative Splicing↗
PubMed1991

Accumulation of MVM gene products is differentially regulated by transcription initiation, RNA processing and protein stability.

The accumulation and stability of minute virus of mice (MVMp) RNA and protein as well as comparative strengths of the two viral promoters have been analyzed in highly synchronous infections of murine A9 fibroblasts. Results indicate that there is a temporal phasing of the accumulation of MVM RNA and protein: the RNA products of the P4 promoter appear prior to the products of the P38 promoter and NS1 and NS2 appear prior to the capsid proteins. Total and cytoplasmic spliced RNA accumulate similarly, although there is a lag in cytoplasmic accumulation of about 2 hr. Total RNA contains abundant unspliced R1 and R3 which are confined to the nucleus. There is no detectable difference in the ratio of the various spliced versions of each RNA species throughout infection. R2 accumulates faster and in a greater amount than R1 in both total and cytoplasmic RNA even though they are generated from the same promoter. During this same period, however, NS1 and NS2 accumulate to similar levels during 1-hr pulses. The stabilities of all MVM RNA species produced at both 9 and 12 hr postrelease are equivalent. Late in infection R3 accumulates faster and in greater amounts than the combined products of the P4 promoter, by approximately two- to threefold. This increase can be accounted for by an increase in the frequency of initiation from the P38 promoter, relative to P4, as assayed by nuclear run-on experiments. Therefore, the steady-state levels of the individual viral proteins during infection is controlled by specific regulation at the level of the initiation of transcription, RNA processing, and protein stability.

Animals↗
PubMed1997

Chloroplast endoribonuclease p54 involved in RNA 3'-end processing is regulated by phosphorylation and redox state.

Chloroplast RNA-binding protein p54 is an endoribonuclease required for 3'end-processing of plastid precursor transcripts. We find that purified p54 can serve as a phosphate acceptor for protein kinases in vitro. Both the processing and RNA-binding activities of p54 are enhanced by phosphorylation and decreased by dephosphorylation. In addition, the enzyme is activated by the oxidized form of glutathione and inhibited by the reduced form, whereas other redox reagents that were tested showed no effect. Kinase treatment of p54 prior to oxidation by glutathione resulted in highest levels of activation, suggesting that phosphorylation and redox state act together to control p54 activity in vitro and possibly also in vivo.

Chloroplast Proteins↗
PubMed2004

RNA surveillance down-regulates expression of nonfunctional kappa alleles and detects premature termination within the last kappa exon.

Random V(D)J junctions would generate nonfunctional and/or out-of-frame sequences in about two-thirds of cases and result in abundant transcripts encoding truncated proteins. Although allelic exclusion at the DNA recombination level ensures that a single allele is functional, the frequent biallelic rearrangements need additional mechanisms to down-regulate aberrant transcripts in those cells with both a functionally and a nonfunctionally rearranged allele. The process of nonsense-mediated decay targets aberrantly rearranged Ig heavy-chain transcripts, but the situation of light-chain mRNAs is more complex, because they do not meet the usual requirements for nonsense-mediated decay and most often lack a spliceable intron downstream of the premature termination. We studied immunoglobulin heavy-chain -/- pro-B cells in which light chain genes get rearranged and expressed in the absence of any selection for the assembly of a functional B cell receptor. Using this model, we show that the whole kappa locus is accessible in pro-B cells and allows the assembly of a broad spectrum of VkappaJkappa segments, most of which are out-of-frame. This model provides an evaluation of the in vivo efficiency of RNA surveillance toward aberrant kappa mRNAs produced in pro-B cells. Our data show that nonfunctional kappa transcripts are excluded from the mature mRNA pool not only by detecting termination in an upstream exon but also by detecting changes in the position of termination within the last exon. Similar mechanisms efficiently down-regulate nonfunctional kappa transcripts arising in normal mature B cells due to the biallelic transcription of rearranged kappa genes.

Alleles↗
PubMed1984

The transcriptional regulation of Xenopus 5s RNA genes in chromatin: the roles of active stable transcription complexes and histone H1.

The properties of active and repressed 5S rna genes in somatic cell chromatin from Xenopus laevis cultured cells were studied by transcription in vitro. The somatic 5S RNA genes, which are active in vivo, are packaged in chromatin as active stable transcription complexes lacking only RNA polymerase III for transcription activity. The oocyte 5S RNA genes, which are inactive in somatic tissues, do not have transcription factors bound to them in purified chromatin and are prevented from binding these factors by a structure dependent on histone H1. In chromatin active stable transcription complexes protect 5S RNA genes from histone H1-mediated repression and H1 binding prevents the formation of stable active transcription complexes.

Animals↗