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Results for “RNA Stability”

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PubMed1998

The tripartite leader sequence of subgroup C adenovirus major late mRNAs can increase the efficiency of mRNA export.

The subgroup C human adenoviruses induce selective export of newly synthesized viral mRNA from the nucleus to the cytoplasm, with concomitant inhibition of export of the majority of cellular mRNA species. Such posttranscriptional regulation of viral and cellular gene expression in infected cells requires viral E1B and E4 proteins. To facilitate the investigation of parameters that govern selective export in adenovirus-infected cells, we constructed a marked human beta-actin minigene under the control of the glucocorticoid-inducible enhancer-promoter of mouse mammary tumor virus and introduced it into the left end of the adenovirus type 5 (Ad5) genome. Transcription of this reporter gene (designated MA) as well as of a sibling, which differed only in the inclusion of a cDNA copy of the Ad2 major late tripartite leader sequence upstream of beta-actin sequences (termed MtplA), in recombinant virus-infected cells was strictly dependent on the addition of dexamethasone to the medium. When transcription of the MA gene was induced during the late phase of infection, newly synthesized MA RNA entered the cytoplasm. These transcripts, which contain no viral sequences, therefore reproduce the behavior of exceptional cellular mRNA species observed when transcription of their genes is activated during the late phase of infection (U.-C. Yang, W. Huang, and S. J. Flint, J. Virol. 70:4071-4080, 1996). Unexpectedly, however, higher concentrations of newly synthesized RNA accumulated in the cytoplasm when the tripartite leader sequence was present in the reporter RNA, despite equal rates of transcription of the two reporter genes. Examination of the partitioning of both newly synthesized and steady-state populations of MA and MtplA RNAs between nuclear and cytoplasmic compartments indicated that the tripartite leader sequence did not increase RNA stability in the cytoplasm. Comparison of nuclear and cytoplasmic reporter RNA species by Northern blotting, primer extension, and reverse transcription-PCR provided no evidence for altered processing induced by the tripartite leader sequence. We therefore conclude that the tripartite leader sequence, long known to facilitate the translation of mRNAs during the late phase of adenovirus infection, can also modulate mRNA export from the nucleus.

Actins↗

PubMed2004

Increased insulin translation from an insulin splice-variant overexpressed in diabetes, obesity, and insulin resistance.

Type 2 diabetes occurs when pancreatic beta-cells become unable to compensate for the underlying insulin resistance. Insulin secretion requires beta-cell insulin stores to be replenished by insulin biosynthesis, which is mainly regulated at the translational level. Such translational regulation often involves the 5'-untranslated region. Recently, we identified a human insulin splice-variant (SPV) altering only the 5'-untranslated region and conferring increased translation efficiency. We now describe a mouse SPV (mSPV) that is found in the cytoplasm and exhibits increased translation efficiency resulting in more normal (prepro)insulin protein per RNA. The RNA stability of mSPV is not increased, but the predicted secondary RNA structure is altered, which may facilitate translation. To determine the role of mSPV in insulin resistance and diabetes, mSPV expression was measured by quantitative real-time RT-PCR in islets from three diabetic and/or insulin-resistant, obese and nonobese, mouse models (BTBRob/ob, C57BL/6ob/ob, and C57BL/6azip). Interestingly, mSPV expression was significantly higher in all diabetic/insulin-resistant mice compared with wild-type littermates and was dramatically induced in primary mouse islets incubated at high glucose. This raises the possibility that the mSPV may represent a compensatory beta-cell mechanism to enhance insulin biosynthesis when insulin requirements are elevated by hyperglycemia/insulin resistance.

5' Untranslated Regions↗

PubMed1992

Preformed ribozyme destroys tumour necrosis factor mRNA in human cells.

Maintaining RNA stability is a major problem in the delivery of preformed inhibitory RNA to target cells. In this study, we delivered a hammerhead ribozyme directed against tumour necrosis factor alpha into human promyelocytic leukaemia cells by cationic liposome-mediated transfection. Delivering a ribozyme in this manner reduced by 90% and 85% tumour necrosis factor alpha mRNA and protein, respectively. A modified ribozyme with a bacteriophage T7 transcription terminator at its 3' end was more stable than one lacking this sequence. This indicates that ribozyme stability can be improved by the addition of terminal sequences expected to protect against cellular nucleases.

Base Sequence↗

PubMed2002

RNA trafficking and stabilization elements associate with multiple brain proteins.

Two of the best understood somatic cell mRNA cytoplasmic trafficking elements are those governing localization of beta-actin and myelin basic protein mRNAs. These cis-acting elements bind the trans-acting factors fibroblast ZBP-1 and hnRNP A2, respectively. It is not known whether these elements fulfil other roles in mRNA metabolism. To address this question we have used Edman sequencing and western blotting to identify six rat brain proteins that bind the beta-actin element (zipcode). All are known RNA-binding proteins and differ from ZBP-1. Comparison with proteins that bind the hnRNP A2 and AU-rich response elements, A2RE/A2RE11 and AURE, showed that AURE and zipcode bind a similar set of proteins that does not overlap with those that bind A2RE11. The zipcode-binding protein, KSRP, and hnRNP A2 were selected for further study and were shown by confocal immunofluorescence microscopy to have similar distributions in the central nervous system, but they were found in largely separate locations in cell nuclei. In the cytoplasm of cultured oligodendrocytes they were segregated into separate populations of cytoplasmic granules. We conclude that not only may there be families of trans-acting factors for the same cis-acting element, which are presumably required at different stages of mRNA processing and metabolism, but independent factors may also target different and multiple RNAs in the same cell.

Actins↗

PubMed1995

Comparison of Milli-Q PF plus water with DEPC-treated water in the preparation and analysis of RNA.

The Milli-Q PF Plus water polishing system is equipped with high-purity ion and organic removal media and a capillary fiber ultrafiltration device. The system produces ultrapure water practically free of ribonuclease contamination. The necessity for diethyl pyrocarbonate (DEPC) treated solutions in RNA molecular biological procedures was tested by preparing RNA from a variety of tissues and tissue cultured cells using either DEPC-treated, autoclaved solutions or pure Milli-Q PF water dispensed directly from the system. Tissue sources included rabbit brain, heart, lung, liver, kidney, and bladder as well as cultured human corpus cavernosum smooth muscle cells (HCCSMC). RNA was prepared by solubilization in guanidinium isothiocyanate, phenol/chloroform extraction, and isopropanol precipitation followed by Northern blot analysis. Hybridization with fibronectin (approximately 7.6kb) and glyceraldehyde-3-phosphate dehydrogenase (1.2kb) revealed that water from a Milli-Q PF water system performed as well as DEPC-treated, autoclaved solutions. RNA stability at 37 degrees C was examined for various times using rabbit lung RNA in either DEPC-treated water or Milli-Q PF water. Intact RNA was detected after 6 hours in total RNA and by Northern blots hybridized with fibronectin. There was no significant difference in RNA degradation between DEPC-treated water or Milli-Q PF water. We conclude that Milli-Q PF water is an acceptable substitute to DEPC-treated water for the preparation of RNA and Northern blot analysis.

Animals↗

PubMed2000

Hormonal regulation of mRNA stability and RNA-protein interactions in the pituitary.

Regulating gene expression from DNA to protein is a complex multistage process with multiple control mechanisms. Transcriptional regulation has been considered the major control point of protein production in eukaryotic cells; however, there is growing evidence of pivotal posttranscriptional regulation for many genes. This has prompted extensive investigations to elucidate the mechanisms controlling RNA processing, mRNA nuclear export and localization, mRNA stability and turnover, in addition to translational rates and posttranslational events. The regulation of mRNA stability has emerged as a critical control step in determining the cellular mRNA level, with individual mRNAs displaying a wide range of stability that has been linked to discrete sequence elements and specific RNA-protein interactions. This review will focus on current knowledge of the determinants of mRNA stability and RNA-protein interactions in the pituitary. This field is rapidly expanding with the identification of regulated cis-acting stability-modifying elements within many mRNAs, and the cloning and characterization of trans-acting proteins that specifically bind to their cognate cis elements. We will present evidence for regulation of multiple pituitary genes at the level of mRNA stability and some examples of the emerging data characterizing RNA-protein interactions.

Animals↗

PubMed2005

Metal ions and RNA folding: a highly charged topic with a dynamic future.

Metal ions are required to stabilize RNA tertiary structure and to begin the folding process. How different metal ions enable RNAs to fold depends on the electrostatic potential of the RNA and correlated fluctuations in the positions of the ions themselves. Theoretical models, fluorescence spectroscopy, small angle scattering and structural biology reveal that metal ions alter the RNA dynamics and folding transition states. Specifically coordinated divalent metal ions mediate conformational rearrangements within ribozyme active sites.

Animals↗

PubMed2005

Regulatory role of the 3' untranslated region (3'UTR) of rat 5' deiodinase (D1). effects on messenger RNA translation and stability.

The previous findings that both a long and a short type 1 deiodinase (D1) mRNA are present in different tissues and that the D1 gene contains two potential polyA signals suggest that the two mRNAs result from differential polyA signal usage. In this study, we examined the properties of the two D1 mRNAs generated in HEK 293 cells by the alternative use of each of the poly A signals in order to ascertain the potential regulatory role of the 3'UTR of this gene. Our results showed that the long mRNA is less stable, but that it is translated more efficiently than the short mRNA. The net result of these differences is a higher D1 activity with the long message. These data suggest that the D1 3'UTR may play an important role in regulating the stability and translational efficiency of the D1 mRNA, both of which could be physiologically relevant when the demand for D1 activity is high.

3' Untranslated Regions↗

PubMed1999

Sequence-specific binding of a hormonally regulated mRNA binding protein to cytidine-rich sequences in the lutropin receptor open reading frame.

In previous studies, a lutropin receptor mRNA binding protein implicated in the hormonal regulation of lutropin receptor mRNA stability was identified. This protein, termed LRBP-1, was shown by RNA gel electrophoretic mobility shift assay to specifically interact with lutropin receptor RNA sequences. The present studies have examined the specificity of lutropin receptor mRNA recognition by LRBP-1 and mapped the contact site by RNA footprinting and by site-directed mutagenesis. LRBP-1 was partially purified by cation-exchange chromatography, and the mRNA binding properties of the partially purified LRBP-1 were examined by RNA gel electrophoretic mobility shift assay and hydroxyl-radical RNA footprinting. These data showed that the LRBP-1 binding site is located between nucleotides 203 and 220 of the receptor open reading frame, and consists of the bipartite polypyrimidine sequence 5'-UCUC-X(7)-UCUCCCU-3'. Competition RNA gel electrophoretic mobility shift assays demonstrated that homoribopolymers of poly(rC) were effective RNA binding competitors, while poly(rA), poly(rG), and poly(rU) showed no effect. Mutagenesis of the cytidine residues contained within the LRBP-1 binding site demonstrated that all the cytidines in the bipartite sequence contribute to LRBP-1 binding specificity. Additionally, RNA gel electrophoretic mobility supershift analysis showed that LRBP-1 was not recognized by antibodies against two well-characterized poly(rC) RNA binding proteins, alphaCP-1 and alphaCP-2, implicated in the regulation of RNA stability of alpha-globin and tyrosine hydroxylase mRNAs. In summary, we show that partially purified LRBP-1 binds to a polypyrimidine sequence within nucleotides 203 and 220 of lutropin receptor mRNA with a high degree of specificity which is indicative of its role in posttranscriptional control of lutropin receptor expression.

3' Untranslated Regions↗

PubMed1989

Time of action of 4.5 S RNA in Escherichia coli translation.

A new class of suppressor mutants helps to define the role of 4.5 S RNA in translation. The suppressors reduce the requirement for 4.5 S RNA by increasing the intracellular concentration of uncharged tRNA. Suppression probably occurs by prolonging the period in which translating ribosomes have translocated but not yet released the uncharged tRNA, indicating that this is the point at which 4.5 S RNA enters translation. The release of 4.5 S RNA from polysomes is affected by antibiotics that inhibit protein synthesis. The antibiotic-sensitivity of this release indicates that 4.5 S RNA exits the ribosome following translocation and prior to release of protein synthesis elongation factor G. These results indicate that 4.5 S RNA acts immediately after ribosomal translocation. A model is proposed in which 4.5 S RNA stabilizes the post-translocation state by replacing 23 S ribosomal RNA as a binding site for elongation factor G. The 4.5 S RNA-requirement of mutants altered in 23 S ribosomal RNA support this model.

Anti-Bacterial Agents↗

PubMed2006

The diversity of RNA silencing pathways in plants.

RNA silencing was discovered in plants as a mechanism whereby invading nucleic acids, such as transgenes and viruses, are silenced through the action of small (20-26 nt) homologous RNA molecules. Our understanding of small RNA biology has significantly improved in recent years, and it is now clear that there are several cellular silencing pathways in addition to those involved in defense. Endogenous silencing pathways have important roles in gene regulation at the transcriptional, RNA stability and translational levels. They share a common core of small RNA generator and effector proteins with multiple paralogs in plant genomes, some of which have acquired highly specialized functions. Here, we review recent developments in the plant RNA silencing field that have identified components of specific silencing pathways and have shed light on the mechanisms and biological roles of RNA silencing in plants.

Gene Silencing↗

PubMed1996

The FinOP repressor system of plasmid R1: analysis of the antisense RNA control of traJ expression and conjugative DNA transfer.

A key determinant of the frequency of IncF plasmid-mediated DNA transfer between enterobacterial cells is the FinOP system. traJ, a positive regulator of the transfer (tra) genes is controlled at the post-transcriptional level by two negative elements, finP and finO. FinP is a plasmid-specific antisense RNA, whereas finO encodes a proteic co-repressor which is not plasmid specific but exchangeable among F-like plasmids. We designed a traJ-lacZ test system that allowed us to monitor the effects of FinP and various FinP mutants on traJ expression. Furthermore, the introduction of finO into the test system enabled us to assess the function of FinO in the interaction of FinP with its target, the traJ mRNA. In this test system, FinP, expressed from a single-copy plasmid, in the absence of FinO, repressed traJ expression six-fold. When expressed from a pBR322-derived multicopy plasmid FinP repressed traJ expression approx. 2000-fold. This result unambiguously demonstrated that FinP is sufficient to repress traJ expression in a gene dosage-dependent manner. Mutations of finP creating base exchanges either in loop I or loop II of the two stem-loop structures of the antisense RNA led to a dramatic decrease in the repressor activity. In a combined loop I-loop II mutation the repressor activity was almost completely lost, supporting the model that the first critical interaction between the two RNA molecules occurs via 'kissing' of both loops of the RNAs. Addition of finO to the test system enhanced the repression of traJ expression by FinP by up to two orders of magnitude. This effect of FinO on FinP activity in vivo might indicate that FinO, in addition to its function as an RNA stabilizer, promotes complex formation between the target mRNA and the antisense RNA. Such a function of FinO has recently been shown to exist in vitro (van Biesen and Frost (1994) Mol Microbiol 14: 427-436).

Bacterial Outer Membrane Proteins↗

PubMed1992

Molecular organization of Leishmania RNA virus 1.

The complete 5284-nucleotide sequence of the double-stranded RNA genome of Leishmania RNA virus 1 (LRV1) was determined and contains three open reading frames (ORFs) on the plus (+) (mRNA) strand. The predicted amino acid sequence of ORF3 has motifs characteristic of viral RNA-dependent RNA polymerases. ORF2, which may encode the major viral coat protein, overlaps ORF3 by 71 nucleotides, suggesting a +1 translational frameshift to produce a gag-pol type of fusion protein. Two alternative models for the frameshift are presented. The 5' splice leader sequence of kinetoplastid mRNAs is not in LRV1 RNA. This suggests that the 450-base region at the 5' end of the LRV1 (+)-strand, which contains ORF1 and is highly conserved among viral strains, does not encode protein but has a role in initiation of translation and/or RNA stability. The similarity of LRV1 genomic organization, replication cycle, and RNA-dependent RNA polymerase sequence to those of the yeast virus ScV L-A suggests a common ancestral origin. The possibility that LRV1 affects pathogenesis in leishmaniasis is intriguing.

Amino Acid Sequence↗

PubMed1999

FUNCTIONS OF THE 3'-UNTRANSLATED REGIONS OF POSITIVE STRAND RNA VIRAL GENOMES.

Positive strand RNA viral genomes are unique in the viral world in serving a dual role as mRNA and replicon. Since the origin of the minus-strand RNA replication intermediate is at the 3'-end of the genome, the 3'-untranslated region (UTR) clearly plays a role in viral RNA replication. The messenger role of this same RNA likely places functional demands on the 3'-UTR to serve roles typical of cellular mRNAs, including the regulation of RNA stability and translation. Current understanding indicates varied roles for positive strand RNA viral 3'-UTRs, with the dominant roles differing between viruses. Three case studies are discussed: turnip yellow mosaic virus RNA, whose 3' tRNA mimicry is thought to negatively regulate minus strand synthesis; brome mosaic virus, whose 3'-UTR contains a unique promoter element directing minus strand synthesis; and tobacco mosaic virus, whose 3'-UTR contains an enhancer of translational expression.

Journal Article↗

PubMed1975

Readenylation of polyadenylate-free globin messenger RNA restores its stability in vivo.

Using an ATP:RNA adenyltransferase from Escherichia coli, a polyadenylic sequence was resynthesized onto rabbit globin mRNA from which the poly (A) segment had been previously removed. Conditions for obtaining a homogenous reconstituted globin mRNA preparation containing 30 adenylic residues per message molecule were determined. The reconstituted globin mRNA was microinjected into Xenopus laevis oocytes. Its stability was very similar to that of native mRNA.

Adenine Nucleotides↗

PubMed2001

BSF binds specifically to the bicoid mRNA 3' untranslated region and contributes to stabilization of bicoid mRNA.

The early stages of Drosophila melanogaster development rely extensively on posttranscriptional forms of gene regulation. Deployment of the anterior body patterning morphogen, the Bicoid protein, requires both localization and translational regulation of the maternal bicoid mRNA. Here we provide evidence that the bicoid mRNA is also selectively stabilized during oogenesis. We identify and isolate a protein, BSF, that binds specifically to IV/V RNA, a minimal form of the bicoid mRNA 3' untranslated region that supports a normal program of mRNA localization during oogenesis. Mutations that disrupt the BSF binding site in IV/V RNA or substantially reduce the level of BSF protein lead to reduction in IV/V RNA levels, indicating a role for BSF in RNA stabilization. The BSF protein is novel and lacks all of the characterized RNA binding motifs. However, BSF does include multiple copies of the PPR motif, whose function is unknown but appears in other proteins with roles in RNA metabolism.

Amino Acid Sequence↗

PubMed1998

Cyclic AMP regulation of G(i alpha2) and G(i alpha3) mRNAs and proteins in astroglial cells.

Culture of rat astrocytes in the presence of 10 microM forskolin, isoproterenol, a nonselective beta-adrenergic agonist, or 8-bromo-cyclic AMP increased transiently in a time-dependent manner the levels of G(i alpha2) and G(i alpha3) mRNAs as measured by northern blot analysis. G(i alpha1) mRNA was not expressed in these cells. After 6-9 h of culture with forskolin or isoproterenol the amounts of G(i alpha2) and G(i alpha3) mRNAs were maximal (150-300% over control). In cells challenged with 8-bromo-cyclic AMP, the level of G(i alpha2) mRNA level was maximal after 1 h of culture, while the maximal for G(i alpha3) mRNA was reached after 6 h of culture. For prolonged exposure times (24 h) to these agents the levels of G(i alpha2) and G(i alpha3) mRNAs decreased to the values observed in control cells. The forskolin-induced increase in G(i alpha2) protein expression measured by western blot analysis was similar to the increase in G(i alpha2) mRNA amount. In contrast, forskolin induced only a modest increase in the quantity of G(i alpha3) protein (150% over control) despite a large increase of G(i alpha3) mRNA content. Transcription rates and RNA stability were measured simultaneously after isolation of newly synthesized mRNA was performed. The half-lives of G(i alpha) mRNAs were approximately 0.7 and 3 h for G(i alpha2) and G(i alpha3), respectively. Culturing astrocytes in the presence of forskolin resulted in an increase in the half-lives of G(i alpha2) mRNA and G(i alpha3) mRNA by five- and twofold, respectively. The relative transcription rate of the G(i alpha3) gene was slightly increased by approximately 1.3-1.5-fold in forskolin-treated cells but not that of G(i alpha2) gene. These results suggest that cyclic AMP enhanced G(i alpha2) and G(i alpha3) mRNA expression by both transcriptional and posttranscriptional mechanisms, with an increase of G(i alpha) mRNA stability as one of the major checkpoints.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2005

Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA.

MicroRNAs are small RNA molecules that regulate messenger RNA (mRNA) expression. MicroRNA 122 (miR-122) is specifically expressed and highly abundant in the human liver. We show that the sequestration of miR-122 in liver cells results in marked loss of autonomously replicating hepatitis C viral RNAs. A genetic interaction between miR-122 and the 5' noncoding region of the viral genome was revealed by mutational analyses of the predicted microRNA binding site and ectopic expression of miR-122 molecules containing compensatory mutations. Studies with replication-defective RNAs suggested that miR-122 did not detectably affect mRNA translation or RNA stability. Therefore, miR-122 is likely to facilitate replication of the viral RNA, suggesting that miR-122 may present a target for antiviral intervention.

Animals↗