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PubMed2001

Involvement of a post-transcriptional mechanism in the inhibition of CYP1A1 expression by resveratrol in breast cancer cells.

Resveratrol decreases basal and induced CYP1A1 mRNA/protein levels in both in vitro and in vivo models, and some studies suggest that resveratrol acts as an aryl hydrocarbon receptor (AhR) antagonist. Treatment of T47D or MCF-7 cells with 10 microM resveratrol inhibited induction of CYP1A1 mRNA and CYP1A1-dependent activity after treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), as previously reported. In contrast, resveratrol did not inhibit TCDD-induced reporter gene activity in cells transfected with an Ah-responsive construct containing a human CYP1A1 gene promoter insert, whereas 3'-methoxy-4'-nitroflavone, a "pure" AhR antagonist, inhibited this response. Resveratrol induced transformation of the rat cytosolic AhR and, after treatment of T47D and MCF-7 cells with resveratrol, a transformed nuclear AhR complex was observed. In contrast to 3'-methoxy-4'-nitroflavone, resveratrol did not block TCDD-induced AhR transformation in vitro or nuclear uptake of the AhR complex in breast cancer cells. Thus, the action of resveratrol on the AhR was consistent with that of an AhR agonist; however, resveratrol did not exhibit functional AhR agonist or antagonist activities in breast cancer cells. Actinomycin D chase experiments in T47D cells showed that resveratrol and dehydroepiandrosterone both increased the rate of CYP1A1 mRNA degradation, whereas resveratrol did not affect CYP1A1-dependent activity in cells pretreated with TCDD for 18 hr. These data suggest that resveratrol inhibits CYP1A1 via an AhR-independent post-transcriptional pathway.

Antineoplastic Agents, Phytogenic↗
PubMed2000

Conservation of the 3'-untranslated region of the Rab1a gene in amniote vertebrates: exceptional structure in marsupials and possible role for posttranscriptional regulation.

The YPT1/RAB1 protein, a key regulator of the intracellular vesicle transport in eukaryotes, is highly conserved in function and amino acid sequence. Here we report that the most highly conserved nucleotide sequence of the Rab1a gene of amniote vertebrates corresponds to the 3'-untranslated region (3'-UTR) of the mRNA. Sequences of 27 species ranging from mammals to sauropsida are >91% identical in this region. Secondary structure prediction procedures applied to the 3'-UTR sequences between positions 750 and 984 and 1428 (mouse cDNA: Y00094), respectively, of the RAB1a mRNAs revealed families of alternative structures around nucleotide position 800 as recurrent features. The two hairpin loops are also predicted for marsupials, despite of their exceptional extension of the A-rich sequence in between. Yet, sequence conservation is much higher than required to conserve secondary structure. Implications for posttranscriptional regulation and protein binding are discussed.

3' Untranslated Regions↗
PubMedDate not supplied

A functional deadenylation assay identifies human CUG-BP as a deadenylation factor.

CUG-BP is a human nuclear and cytoplasmic RNA-binding protein. A role in the control of alternative splicing has been reported, but to date no cytoplasmic function for this protein has been demonstrated. A close sequence homolog of CUG-BP is EDEN-BP that is required for the specific cytoplasmic poly(A) tail shortening of certain mRNAs after fertilization of Xenopus eggs. Here, we show that human CUG-BP and Xenopus EDEN-BP have very similar RNA-binding specificities. In addition, we use a deadenylation assay to show that CUG-BP is able to act as a deadenylation factor. In contrast, a mutant form of CUG-BP, though still able to bind to RNA with a specificity similar to that of wild-type CUG-BP, does not act as a deadenylation factor. It is suggested that the CUG expansion associated with Type 1 myotonic dystrophy can affect the function or the activity of CUG-BP, leading to a trans-dominant effect on normal RNA processing. The results presented here identify CUG-BP-dependent deadenylation as a potential cytoplasmic target for this trans-dominant effect.

CELF1 Protein↗
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↗
PubMed2000

Tertiary core rearrangements in a tight binding transfer RNA aptamer.

Guided by an in vitro selection experiment designed to obtain tight binding aptamers of Escherichia coli glutamine specific tRNA (tRNAGln) for glutaminyl-tRNA synthetase (GlnRS), we have engineered a tRNA mutant in which the five-nucleotide variable loop sequence 5'-44CAUUC48-3' is replaced by 5'-44AGGU48-3'. This mutant tRNA binds to GlnRS with 30-fold improved affinity compared to the wild type. The 2.7 A cocrystal structure of the RNA aptamer-GlnRS complex reveals major rearrangements in the central tertiary core of the tRNA, while maintaining an RNA-protein interface identical to the wild type. The repacked RNA core features a novel hydrogen bonding arrangement of the trans Levitt pair G15-U48, a new sulfate binding pocket in the major groove, and increased hydrophobic stacking interactions among the bases. These data suggest that enhanced protein binding to a mutant globular RNA can arise from stabilization of RNA tertiary interactions rather than optimization of RNA-protein contacts.

Amino Acyl-tRNA Synthetases↗
PubMed2001

A collapsed state functions to self-chaperone RNA folding into a native ribonucleoprotein complex.

Most large RNAs achieve their active, native structures only as complexes with one or more cofactor proteins. By varying the Mg(2+) concentration, the catalytic core of the bI5 group I intron RNA can be manipulated into one of three states, expanded, collapsed or native, or into balanced equilibria between these states. Under near-physiological conditions, the bI5 RNA folds rapidly to a collapsed but non-native state. Hydroxyl radical footprinting demonstrates that assembly with the CBP2 protein cofactor chases the RNA from the collapsed state to the native state. In contrast, CBP2 also binds to the RNA in the expanded state to form many non-native interactions. This structural picture is reinforced by functional splicing experiments showing that RNA in an expanded state forms a non-productive, kinetically trapped complex with CBP2. Thus, rapid folding to the collapsed state functions to self-chaperone bI5 RNA folding by preventing premature interaction with its protein cofactor. This productive, self-chaperoning role for RNA collapsed states may be especially important to avert misassembly of large multi-component RNA-protein machines in the cell.

Animals↗
PubMed2004

Crystal structure of Dcp1p and its functional implications in mRNA decapping.

A major pathway of eukaryotic mRNA turnover begins with deadenylation, followed by decapping and 5'-->3' exonucleolytic degradation. A critical step in this pathway is decapping, which is carried out by an enzyme composed of Dcp1p and Dcp2p. The crystal structure of Dcp1p shows that it markedly resembles the EVH1 family of protein domains. Comparison of the proline-rich sequence (PRS)-binding sites in this family of proteins with Dcp1p indicates that it belongs to a novel class of EVH1 domains. Mapping of the sequence conservation on the molecular surface of Dcp1p reveals two prominent sites. One of these is required for the function of the Dcp1p-Dcp2p complex, and the other, corresponding to the PRS-binding site of EVH1 domains, is probably a binding site for decapping regulatory proteins. Moreover, a conserved hydrophobic patch is shown to be critical for decapping.

Amino Acid Sequence↗
PubMed2004

Molecular insights into the interaction of PYM with the Mago-Y14 core of the exon junction complex.

The exon junction complex (EJC) is deposited on mRNAs as a consequence of splicing and influences postsplicing mRNA metabolism. The Mago-Y14 heterodimer is a core component of the EJC. Recently, the protein PYM has been identified as an interacting partner of Mago-Y14. Here we show that PYM is a cytoplasmic RNA-binding protein that is excluded from the nucleus by Crm1. PYM interacts directly with Mago-Y14 by means of its N-terminal domain. The crystal structure of the Drosophila ternary complex at 1.9 A resolution reveals that PYM binds Mago and Y14 simultaneously, capping their heterodimerization interface at conserved surface residues. Formation of this ternary complex is also observed with the human proteins. Mago residues involved in the interaction with PYM have been implicated in nonsense-mediated mRNA decay (NMD). Consistently, human PYM is active in NMD tethering assays. Together, these data suggest a role for PYM in NMD.

Amino Acid Sequence↗
PubMed2004

DNA-selective hybridization and dual strand invasion of short double-stranded DNA using pyren-1-ylcarbonyl-functionalized 4'-C-piperazinomethyl-DNA.

Incorporation of a novel pyren-1-ylcarbonyl-functionalized 4'-C-piperazinomethyl-DNA monomer into oligodeoxynucleotides leads to increased thermal stability of duplexes with DNA complements but reduced thermal stability of duplexes with RNA complements. This DNA-selective hybridization is explored for recognition of double-stranded DNA by a novel dual strand invasion approach.

Base Sequence↗
PubMed2006

Gawky is a component of cytoplasmic mRNA processing bodies required for early Drosophila development.

In mammalian cells, the GW182 protein localizes to cytoplasmic bodies implicated in the regulation of messenger RNA (mRNA) stability, translation, and the RNA interference pathway. Many of these functions have also been assigned to analogous yeast cytoplasmic mRNA processing bodies. We have characterized the single Drosophila melanogaster homologue of the human GW182 protein family, which we have named Gawky (GW). Drosophila GW localizes to punctate, cytoplasmic foci in an RNA-dependent manner. Drosophila GW bodies (GWBs) appear to function analogously to human GWBs, as human GW182 colocalizes with GW when expressed in Drosophila cells. The RNA-induced silencing complex component Argonaute2 and orthologues of LSm4 and Xrn1 (Pacman) associated with 5'-3' mRNA degradation localize to some GWBs. Reducing GW activity by mutation or antibody injection during syncytial embryo development leads to abnormal nuclear divisions, demonstrating an early requirement for GWB-mediated cytoplasmic mRNA regulation. This suggests that gw represents a previously unknown member of a small group of genes that need to be expressed zygotically during early embryo development.

Animals↗
PubMed2000

Fragile X CGG repeat structures among African-Americans: identification of a novel factor responsible for repeat instability.

The cryptic CGG repeat responsible for the fragile X syndrome, located in the 5'-UTR of FMR1, is unique compared with the many other triplet repeat-causing diseases, making it ideal for identifying factors involved in repeat expansion that may be common to other triplet repeat diseases. To date, a number of factors have been identified which may influence repeat instability, including the number and position of interspersed AGGs, length of the 3' pure CGG repeat and haplotype background. However, nearly all such data were derived from studies of Caucasians. Using a large African-American population, we present the only comprehensive examination of factors associated with CGG repeat instability in a non-Caucasian population. Among Caucasians, susceptible alleles were thought to come from those in the intermediate repeat range (41-60 repeats); however, we find that susceptible alleles may come from a larger repeat pool (35-60 repeats) and are better defined by their pure CGG repeat and/or -presence of only one AGG interruption. These results demonstrate the existence of different susceptible alleles among world populations and may account for the similar prevalence of the fragile X syndrome in African-Americans compared with Caucasians despite the lower frequency of inter-mediate sized alleles in the African-American population. Finally, we show that repeat structures among unaffected African-Americans with the most frequent fragile X haplotype background are either pure or contain a single distal interruption. We propose that the lack of a proximal most interruption is a novel factor involved in CGG repeat instability.

5' Untranslated Regions↗
PubMed2000

Biochemical characterisation of cap-poly(A) synergy in rabbit reticulocyte lysates: the eIF4G-PABP interaction increases the functional affinity of eIF4E for the capped mRNA 5'-end.

The 5' cap and 3' poly(A) tail of eukaryotic mRNAs cooperate to synergistically stimulate translation initiation in vivo. We recently described mammalian cytoplasmic extracts which, following ultracentrifugation to partially deplete them of ribosomes and associated initiation factors, reproduce cap-poly(A) synergy in vitro. Using these systems, we demonstrate that synergy requires interaction between the poly(A)-binding protein (PABP) and the eukaryotic initiation factor (eIF) 4F holoenzyme complex, which recognises the 5' cap. Here we further characterise the requirements and constraints of cap-poly(A) synergy in reticulocyte lysates by evaluating the effects of different parameters on synergy. The extent of extract depletion and the amounts of different initiation factors in depleted extracts were examined, as well as the effects of varying the concentrations of KCl, MgCl(2) and programming mRNA and of adding a cap analogue. The results presented demonstrate that maximal cap-poly(A) synergy requires: (i) limiting concentrations of ribosome-associated initiation factors; (ii) precise ratios of mRNA to translation machinery (low concentrations of ribosome-associated initiation factors and low, non-saturating mRNA concentrations); (iii) physiological concentrations of added KCl and MgCl(2). Additionally, we show that the eIF4G-PABP interaction on mRNAs which are capped and polyadenylated significantly increases the affinity of eIF4E for the 5' cap.

Animals↗
PubMed2001

Rearrangement of structured RNA via branch migration structures catalysed by the highly related DEAD-box proteins p68 and p72.

RNA helicases, like their DNA-specific counterparts, can function as processive enzymes, unwinding RNA with a defined step size in a unidirectional fashion. Recombinant nuclear DEAD-box protein p68 and its close relative p72 are reported here to function in a similar fashion, though the processivity of both RNA helicases appears to be limited to only a few consecutive catalytic steps. The two proteins resemble each other also with regard to other biochemical properties. We have found that both proteins exhibit an RNA annealing in addition to their helicase activity. By using both these activities the enzymes are able in vitro to catalyse rearrangements of RNA secondary structures that otherwise are too stable to be resolved by their low processive helicase activities. RNA rearrangement proceeds via protein induced formation and subsequent resolution of RNA branch migration structures, whereby the latter step is dependent on ATP hydrolysis. The analysed DEAD-box proteins are reminiscent of certain DNA helicases, for example those found in bacteriophages T4 and T7, that catalyse homologous DNA strand exchange in cooperation with the annealing activity of specific single strand binding proteins.

Adenosine Triphosphatases↗
PubMed2006

Modulating role of RNA structure in alternative splicing of a critical exon in the spinal muscular atrophy genes.

Humans have two nearly identical copies of the survival motor neuron (SMN ) gene, SMN1 and SMN2. Homozygous loss of SMN1 causes spinal muscular atrophy (SMA). SMN2 is unable to prevent the disease due to skipping of exon 7. Using a systematic approach of in vivo selection, we have previously demonstrated that a weak 5' splice site (ss) serves as the major cause of skipping of SMN2 exon 7. Here we show the inhibitory impact of RNA structure on the weak 5' ss of exon 7. We call this structure terminal stem-loop 2 (TSL2). Confirming the inhibitory nature of TSL2, point mutations that destabilize TSL2 promote exon 7 inclusion in SMN2, whereas strengthening of TSL2 promotes exon 7 skipping even in SMN1. We also demonstrate that TSL2 negatively affects the recruitment of U1snRNP at the 5' ss of exon 7. Using enzymatic structure probing, we confirm that the sequence at the junction of exon 7/intron 7 folds into TSL2 and show that mutations in TSL2 cause predicted structural changes in this region. Our findings reveal for the first time the critical role of RNA structure in regulation of alternative splicing of human SMN.

Alternative Splicing↗
PubMed2004

Reverse Sanger sequencing of RNA by MALDI-TOF mass spectrometry after solid phase purification.

Several DNA/RNA sequencing strategies have been developed using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). In the reverse Sanger sequencing approach alpha-thiophosphate-containing NTPs are employed. Sequencing ladders are produced by the subsequent exonuclease cleavage, which is inhibited by the alpha-S-NTP at the 3' terminus. Here the reverse Sanger sequencing of RNA is described. The stability of RNA during the UV-MALDI process is higher relative to DNA, and RNA can be easily synthesized by transcription using bacteriophage RNA polymerase. alpha-S-rNTP was added to the reaction in a ratio of 1:3 to the native rNTPs and was incorporated statistically by the RNA polymerase. Four separate sequence ladders were produced, to avoid the problem of the only 1u mass difference between uridine and cytidine. However, it was shown that RNA transcription does not produce homogeneous transcripts. Therefore isolation of the full-length transcript is required to attain a non-ambiguous interpretation of cleavage spectra. This is achieved by the exclusive immobilization of the full-length transcript on a solid phase. The full-length transcripts were hybridized to magnetic beads, coated with short universal sequences, complementary to the in vitro RNA. After purification and isolation the RNA full-length transcript is cleaved by snake venom phosphodiesterase (SVP) and the obtained sequence ladder is analyzed by MALDI-MS.

RNA↗
PubMed2005

Activation of RNA metabolism-related genes in mouse but not human tissues deficient in SMN.

Mutations of the survival of motor neuron gene (SMN1) are responsible for spinal muscular atrophies (SMA), a frequent recessive autosomal motor neuron disease. SMN is involved in various processes including RNA metabolism. However, the molecular pathway linking marked deficiency of SMN to SMA phenotype remains unclear. Homozygous deletion of murine Smn exon 7 directed to neurons or skeletal muscle causes severe motor axonal or myofiber degeneration, respectively. With the use of cDNA microarrays, expression profiles of 8,400 genes were analyzed in skeletal muscle and spinal cord of muscular and neuronal mutants, respectively, and compared with age-matched controls. A high proportion of genes (20 of 429, 5%) was involved in pre-mRNA splicing, ribosomal RNA processing, or RNA decay, and 18 of them were upregulated in mutant tissues. By analyzing other neuromuscular disorders, we showed that most of them (14 of 18) were specific to the SMN defect. Quantitative PCR analysis of these transcripts showed that gene activation was an early adaptive response to the lack but not reduced amount of full-length SMN in mouse mutant tissues. In human SMA tissues, activation of this program was not observed, which could be ascribed to the reduction but not the absence of full-length SMN.

Animals↗
PubMed2002

Stable hepatitis C virus RNA detection by RT-PCR during four days storage.

BACKGROUND: Suboptimal specimen processing and storage conditions of samples which contain hepatitis C virus (HCV) RNA may result in a decline of HCV RNA concentration or false-negative results in the detection of HCV RNA in serum. We evaluated the stability of HCV RNA in serum and clotted blood samples stored at room temperature or at 4 degrees C for 4 days with the aim of optimizing the standard procedures of processing and storage of samples. METHODS: Blood from five HCV RNA positive patients was collected in tubes with and without separator gel, centrifuged 1 or 6 hours after collection. Samples were then left 6, 24, 48, 72 or 96 h at room temperature (21.5 - 25.4 degrees C) or at 4 degrees C before determining their HCV RNA level using the COBAS AMPLICOR HCV MONITOR Test, vs 2.0 (Roche Diagnostic Systems). RESULTS: The logarithm of the HCV RNA level measurements remained within a 0.3 value of the means for 4 days at both temperatures (room temperature or 4 degrees C). CONCLUSIONS: We conclude that blood samples may be collected and aliquoted within 6 h of collection and can be stored at 4 degrees C for 72 hours as proposed by the manufacturer without significant differences in measured HCV RNA level. Our results indicate that lapses in this scheme may still yield reliable results.

Adult↗