Role In mRNA Stability Research Articles

CRM 1‐Mediated Nucleocytoplasmic Export of Adrenergic Receptor mRNAs: Role in mRNA Stability and Agonist‐Mediated Down‐Regulation

The β1-adrenergic receptor (β1-AR) mRNAs are post-transcriptionally regulated at the level of mRNA stability and undergo accelerated agonist-mediated degradation. Treatment of cells with leptomycin B, an inhibitor of the nuclear export receptor CRM 1, results in a dramatic enhancement of β1-AR mRNA stability. To assess whether CRM 1 or other nuclear export receptors play direct roles in β1-AR mRNA down-regulation, we used cell-permeable peptides containing the leucine-rich nuclear export signal (NES) recognized by CRM 1, to serve as inhibitors of CRM 1-mediated nuclear export. When DDT1MF2 transfectants ectopically expressing β1-AR mRNAs were concurrently treated with isoproterenol and peptide inhibitors, only the co-addition of the NES inhibitor reversed the isoproterenol-induced reduction of β1-AR mRNA levels. Using UV-crosslinking/immunoprecipitation analyses, we have verified the interaction of CRM 1 – HuR complexes with the β1-AR mRNA. CRM 1 is nearly absent in neonatal rat cortical neurons (RCNs), which express abundant β1-AR mRNAs but do not undergo agonist-mediated β1-AR mRNA down-regulation. Upon transfection with a CRM 1 expression recombinant, the neonatal RCNs exhibit agonist-mediated β1-AR mRNA down-regulation and decreased β1-AR transcript stability. We conclude that agonist-mediated β1-AR mRNA down-regulation and degradation utilize the CRM 1-mediated nucleocytoplasmic export pathway for deposition of transcripts into the cytoplasm. Supported by NIH MH071317 and MH63137.

Role of mRNA Stability during Genome-wide Adaptation of Lactococcus lactis to Carbon Starvation

The stability of mRNA was investigated for the first time at the genomic scale during carbon starvation adaptation of Lactococcus lactis IL1403. In exponential phase, mRNA half-lives were correlated positively to open reading frame length. A polypurine sequence, AGGAG, was identified as a putative 5'-stabilizer and inverted repeated sequences as a 3'-destabilizer. These original findings suggested that multiple pathways of mRNA degradation should coexist: internal cleavage, endonuclease cleavage initiated at the 5'-end, and exonuclease attack at the 3'-end. During carbon starvation adaptation, mRNA stability globally increased, but specific mechanisms allowing a wide range of stabilization factors between genes and differential kinetic evolution were involved. A formal method allowing the quantification of the relative influences of transcription and degradation on the mRNA pool control was developed and applied in L. lactis. Gene expression was mostly controlled by altered transcription prior to carbon source exhaustion, while the influence of mRNA stability increased during the starvation phase. This study highlighted that stability modulation in response to adverse growth conditions can govern gene regulation to the same extent as transcription in bacteria.

The role of mRNA stability in airway remodelling

As a consequence of long-term exposure to inflammatory mediators, the airways of asthmatics become remodelled. Airway fibrosis becomes apparent, with thickening of the lamina recticularis and increased interstitial matrix deposition being typical features of an asthmatic airway. Mucus hypersecretion occurs, airway smooth muscle mass is increased and neovascularization is evident in the subepithelial mucosa. As development of a remodelled airway is correlated with deterioration of lung function in asthmatics, there is an urgent need for therapies that reduce airway inflammation and reverse structural changes in a remodelled airway. However, in order to design efficacious anti-remodelling agents we first need a greater understanding of the molecular mechanism/s underlying the development of airway remodelling. To date, however, most studies have primarily focused on the transcriptional regulation of genes that promote airway remodelling. Post-transcriptional mechanisms, such as control of mRNA stability, remain largely unexplored. Levels of cellular mRNA transcripts are regulated by controlling the rate at which the mRNA decays, thus investigation into the mechanisms underlying mRNA stability in asthma are of critical importance. Therefore, this review will present an overview of the control of mRNA stability and examine how mRNA stability may play a role in the development of airway remodelling in asthma.

Nitric oxide post-transcriptionally up-regulates LPS-induced IL-8 expression through p38 MAPK activation.

Nitric oxide (NO(.-)) contributes to vascular collapse in septic shock and regulates inflammation. Here, we demonstrate in lipopolysaccharide (LPS)-stimulated human THP-1 cells and monocytes that NO(.-) regulates interleukin (IL)-8 and tumor necrosis factor alpha (TNF-alpha) by distinct mechanisms. Dibutyryl-cyclic guanosine 5'-monophosphate (cGMP) failed to simulate NO(.-)-induced increases in TNF-alpha or IL-8 production. In contrast, dibutyryl-cyclic adenosine monophosphate blocked NO(.-)-induced production of TNF-alpha (P=0.009) but not IL-8. NO(.-) increased IL-8 (5.7-fold at 4 h; P=0.04) and TNF-alpha mRNA levels (2.2-fold at 4 h; P=0.037). However, nuclear run-on assays demonstrated that IL-8 transcription was slightly decreased by NO(.-) (P=0.08), and TNF-alpha was increased (P=0.012). Likewise, NO(.-) had no effect on IL-8 promoter activity (P=0.84) as measured by reporter gene assay. In THP-1 cells and human primary monocytes treated with actinomycin D, NO(.-) had no effect on TNF-alpha mRNA stability (P>0.3 for both cell types) but significantly stabilized IL-8 mRNA (P=0.001 for both cell types). Because of its role in mRNA stabilization, the p38 mitogen-activated protein kinase (MAPK) pathway was examined and found to be activated by NO(.-) in LPS-treated THP-1 cells and human monocytes. Further, SB202190, a p38 MAPK inhibitor, was shown to block NO(.-)-induced stabilization of IL-8 mRNA (P<0.02 for both cell types). Thus, NO(.-) regulates IL-8 but not TNF-alpha post-transcriptionally. IL-8 mRNA stabilization by NO(.-) is independent of cGMP and at least partially dependent on p38 MAPK activation.

Identification and characterization of proteins that interact with the carboxy terminus of poly(A)-binding protein and inhibit translation in vitro.

Poly(A)-binding proteins (PABPs) are multifunctional proteins that play important roles in mRNA stability and protein translation. Two cucumber ( Cucumis sativus L.) proteins, PCI6 (PABP-CT-interacting) and PCI243 were identified based on ability to interact with the carboxy terminus (CT) of PABP in yeast two-hybrid and in vitro binding assays. PCI6 and PCI243 share a conserved amino acid domain (SxLnpnApxFxP) in common with human PABP-CT interactors, and with Arabidopsis ERD15 (early-responsive to dehydration). Deletion analysis and point mutations indicate that presence of this domain is necessary for the interaction, and tests with ERD15 demonstrate that it is predictive of interaction. Other plant proteins possessing this domain fall into two categories: small, acidic proteins like PCI6, PCI243 and ERD15, and larger neutral proteins that also include an RNA recognition motif. PCI6 is expressed in a range of tissues, e.g., leaves, roots, stems and flowers, and follows a diurnal pattern of expression, increasing during light hours and declining overnight. In wheat germ and mouse ascites Krebs-2 in vitro translation systems, PCI6 inhibited translation whereas the non-interacting mutant, PCI6-23A, did not or had a greatly reduced effect. The activity of PCI6, therefore, is reminiscent of that of human PABP-interacting protein 2 (Paip2). These results demonstrate a novel interaction between PABP and several plant proteins sharing a SxLnpxApxFxP motif, with possible implications for translational regulation.

Identification of a 43-kDa protein in human liver cytosol that binds to the 3′-untranslated region of CYP2A6 mRNA

Hepatic expression of cytochrome P450 2A6 (CYP2A6) varies widely in humans and is induced during hepatitis; however, the mechanism regulating CYP2A6 has not been established. The murine orthologue Cyp2a5 is regulated post-transcriptionally by mRNA stabilization. A 43-kDa protein that binds to the 3′-untranslated region (3′-UTR) of Cyp2a5 mRNA has been identified, but its role in mRNA stabilization is unclear. We hypothesized that similar interactions occur between cytosolic proteins in human liver and CYP2A6 3′-UTR mRNA. We identified, by RNA electrophoretic mobility shift assay, an hepatic cytosolic protein that binds specifically to sequences in the 3′-UTR of CYP2A6. Complexes did not form with denatured proteins and were eliminated with proteinase K digestion. Complex formation was inhibited with a molar excess of unlabeled CYP2A6 RNA but not by non-specific competitor RNA. Protein-mRNA interactions were not affected by probe denaturation, suggesting that RNA secondary structure is not essential for binding. UV cross-linking of complexes revealed RNA-binding proteins in both human and mouse liver cytosols with molecular masses of approximately 43 kDa. Using truncated RNA probes corresponding to various lengths of CYP2A6 mRNA, the protein-binding site was localized to a 50-nucleotide region between bases 1478 and 1527 of the 3′-UTR. Complex formation with hepatic cytosolic protein from four human subjects correlated with levels of hepatic CYP2A6 microsomal protein, suggesting a possible regulatory role. Further characterization of the RNA-binding protein, the primary binding site, and the influence of this interaction on CYP2A6 mRNA stability will help to elucidate the relevance of these findings to the post-transcriptional control of CYP2A6.

Role of mRNA stability and translation in the expression of cytochrome c oxidase during mouse myoblast differentiation: instability of the mRNA for the liver isoform of subunit VIa.

The role of mRNA stability and translation in mediating the expression of selected subunits of cytochrome c oxidase (COX) was examined during the differentiation of mouse myoblasts into myotubes in cell culture. The expression of the liver (L) and heart (H) isoforms of COX VIa, which undergo an isoform switch during muscle development, as well as of the Va subunit, which is expressed in all tissues, was analysed. The translational efficiencies of COX Va, VIa-L and VIa-H, as well as of mitochondrially encoded COX mRNAs, were inferred from their distribution in polysome gradients. These experiments suggest that the translational efficiencies of these mRNAs do not change during myoblast differentiation, although the nuclear mRNAs for COX Va, VIa-L and VIa-H are translated more efficiently than the mitochondrial mRNAs. Analysis of mRNA stability using the tetracycline-repressible promoter system and/or actinomycin D indicates that COX VIa-L mRNA decays with a half-life of approximately 5-6 h in both myoblasts and myotubes, whereas COX VIa-H and Va mRNAs decay with half-lives of > 15 h in myotubes. This relative instability of COX VIa-L mRNA serves to limit the accumulation of COX VIa-L mRNA in these myogenic cells, as compared with mRNAs for other COX subunits. Deletion/replacement mapping experiments suggest that the COX VIa-L 3' untranslated region contains a destabilization element. Analysis of the rate of poly(A) tail shortening on COX VIa-L and stable alpha-globin mRNAs suggests that the overall rate of poly(A) shortening per se is not rate limiting for the degradation of COX VIa-L mRNA.

Role of mRNA stability and translation in the expression of cytochrome c oxidase during mouse myoblast differentiation: instability of the mRNA for the liver isoform of subunit VIa

The role of mRNA stability and translation in mediating the expression of selected subunits of cytochrome c oxidase (COX) was examined during the differentiation of mouse myoblasts into myotubes in cell culture. The expression of the liver (L) and heart (H) isoforms of COX VIa, which undergo an isoform switch during muscle development, as well as of the Va subunit, which is expressed in all tissues, was analysed. The translational efficiencies of COX Va, VIa-L and VIa-H, as well as of mitochondrially encoded COX mRNAs, were inferred from their distribution in polysome gradients. These experiments suggest that the translational efficiencies of these mRNAs do not change during myoblast differentiation, although the nuclear mRNAs for COX Va, VIa-L and VIa-H are translated more efficiently than the mitochondrial mRNAs. Analysis of mRNA stability using the tetracycline-repressible promoter system and/or actinomycin D indicates that COX VIa-L mRNA decays with a half-life of ∼ 5–6h in both myoblasts and myotubes, whereas COX VIa-H and Va mRNAs decay with half-lives of &amp;gt; 15h in myotubes. This relative instability of COX VIa-L mRNA serves to limit the accumulation of COX VIa-L mRNA in these myogenic cells, as compared with mRNAs for other COX subunits. Deletion/replacement mapping experiments suggest that the COX VIa-L 3´ untranslated region contains a destabilization element. Analysis of the rate of poly(A) tail shortening on COX VIa-L and stable α-globin mRNAs suggests that the overall rate of poly(A) shortening per se is not rate limiting for the degradation of COX VIa-L mRNA.

HuR regulates cyclin A and cyclin B1 mRNA stability during cell proliferation.

Colorectal carcinoma RKO cells expressing reduced levels of the RNA-binding protein HuR (ASHuR) displayed markedly reduced growth. In synchronous RKO populations, HuR was almost exclusively nuclear during early G(1), increasing in the cytoplasm during late G(1), S and G(2). The expression and half-life of mRNAs encoding cyclins A and B1 similarly increased during S and G(2), then declined, indicating that mRNA stabilization contributed to their cell cycle-regulated expression. In gel-shift assays using radiolabeled cyclin RNA transcripts and RKO protein extracts, only those transcripts corresponding to the 3'-untranslated regions of cyclins A and B1 formed RNA-protein complexes in a cell cycle-dependent fashion. HuR directly bound mRNAs encoding cyclins A and B1, as anti-HuR antibodies supershifted such RNA-protein complexes. Importantly, the expression and half-life of mRNAs encoding cyclins A and B1 were reduced in ASHuR RKO cells. Our results indicate that HuR may play a critical role in cell proliferation, at least in part by mediating cell cycle-dependent stabilization of mRNAs encoding cyclins A and B1.

Evolutionary significance of an unusual chloroplast DNA inversion found in two basal angiosperm lineages.

Two basal lineages of flowering plants possess an intergenic inversion in the chloroplast inverted repeat (IR), a region of the genome from which there have been few previous reports of this class of structural mutation. The size of the inversion (approximately 200 bp) places it in a class not previously seen in the plastid genome. The two lineages with the rearrangement, representatives of the orders Laurales and Nymphaeales, are not closely related and the inversion therefore probably arose independently in each group. The inversion is bordered by short, but highly conserved, inverted repeat motifs that were most likely associated with the inversion process. A stem-loop structure that involves these motifs may play a functional role in mRNA stability. It is seen in all optimal or nearly optimal predicted RNA foldings of the intergenic region.

Assays for Analyzing Exonucleasesin Vitro

Ribonucleases play essential roles in cell growth, differentiation, and the response to stress. This article deals with exoribonucleases, enzymes that degrade RNAs beginning at either the 5′ or 3′ end and proceed down the length of the RNA. The preparation of a crude extract of a mammalian 3′-to-5′ exonuclease is described. Assay conditions for both 5′-to-3′ and 3′-to-5′ exonucleases are given. One of these is a yeast enzyme that is known to be involved in mRNA decay. Others are vertebrate exonucleases that are presumed to have a role in mRNA stability but have not yet been proven to do so.

Identification and developmental expression of a 5′–3′ exoribonuclease from Drosophila melanogaster

In multicellular organisms, very little is known about the role of mRNA stability in development, and few proteins involved in degradation pathways have been characterized. We have identified the Drosophila homologue of XRN1, which is the major cytoplasmic 5′–3′ exoribonuclease in Saccharomyces cerevisiae. The protein sequence of this homologue ( pacman) has 59% identity to S. cerevisiae XRN1 and 67% identity to the mouse homologue (mXRN1p) in certain regions. Sequencing of this cDNA revealed that it includes a trinucleotide repeat (CAG) 9 which encodes polyglutamine. By directly measuring pacman exoribonuclease activity in yeast, we demonstrate that pacman can complement the yeast XRN1 mutation. Northern blots show a single transcript of approximately 5.2 kb which is abundant only in 0–8-h embryos and in adult males and females. In situ hybridization analysis revealed that the pcm transcripts are maternally derived, and are expressed at high levels in nurse cells. During early embryonic syncytial nuclear divisions, pcm transcripts are homogenously distributed. pcm mRNA is expressed abundantly and ubiquitously throughout the embryo during gastrulation, with high levels in the germ band and head structures. After germ band retraction, pcm transcripts are present at much lower levels, in agreement with the Northern results. Our experiments provide the first example of an exoribonuclease which is differentially expressed throughout development.

C-jun N-terminal kinase is involved in AUUUA-mediated interleukin-3 mRNA turnover in mast cells.

Whereas signalling pathways involved in transcriptional control have been studied extensively, the pathways regulating mRNA turnover remain poorly understood. We are interested in the role of mRNA stability in cell activation and oncogenesis using PB-3c mast cells as a model system. In these cells the short-lived interleukin-3 (IL-3) mRNA is stabilized by ionomycin treatment and following oncogenesis. To identify the signalling pathways involved in these mechanisms, we analysed the effect of different kinase inhibitors. SB202190 and wortmannin were shown to antagonize ionomycin-induced IL-3 mRNA stabilization in PB-3c cells in the presence of actinomycin D, and this effect coincided with their ability to inhibit c-jun N-terminal kinase (JNK) activation by ionomycin. Moreover, transfection of activated MEKK1 amplified ionomycin-induced IL-3 mRNA expression at the post-transcriptional level, and a dominant-negative mutant of JNK counteracted mRNA stabilization by ionomycin. Taken together, these data indicate that JNK is involved in the regulation of IL-3 mRNA turnover in mast cells. In addition, transfection experiments revealed that the cis-acting AU-rich element in the 3' untranslated region of IL-3 mRNA is necessary and sufficient to confer JNK-dependent mRNA stabilization in response to cell activation.

Association between uncoupling protein polymorphisms (UCP2-UCP3) and energy metabolism/obesity in Pima indians.

The UCP2-UCP3 gene cluster maps to chromosome 11q13 in humans, and polymorphisms in these genes may contribute to obesity through effects on energy metabolism. DNA sequencing of UCP2 and UCP3 revealed three polymorphisms informative for association studies: an Ala-->Val substitution in exon 4 of UCP2, a 45 bp insertion/deletion in the 3'-untranslated region of exon 8 of UCP2 and a C-->T silent polymorphism in exon 3 of UCP3. Initially, 82 young (mean age = 30 +/- 7 years), unrelated, full-blooded, non-diabetic Pima Indians were typed for these polymorphisms by direct sequencing. The three sites were in linkage disequilibrium ( P < 0.00001). The UCP2 variants were associated with metabolic rate during sleep (exon 4, P = 0.007; exon 8, P = 0.016) and over 24 h (exon 8, P = 0.038). Heterozygotes for UCP2 variants had higher metabolic rates than homozygotes. The UCP3 variant was not significantly associated with metabolic rate or obesity. In a further 790 full-blooded Pima Indians, there was no significant association between the insertion/deletion polymorphism and body mass index (BMI). However, when only individuals >45 years of age were considered, heterozygotes (subjects with the highest sleeping metabolic rate) had the lowest BMI (P = 0.04). The location of the insertion/deletion polymorphism suggested a role in mRNA stability; however, it appeared to have no effect on skeletal muscle UCP2 mRNA levels in a subset of 23 randomly chosen Pima Indians. In conclusion, these results suggest a contribution from UCP2 (or UCP3) to variation in metabolic rate in young Pima Indians which may contribute to overall body fat content later in life.

Appropriate expression of filamentous phage f1 DNA replication genes II and X requires RNase E-dependent processing and separate mRNAs.

The products of in-frame overlapping genes II and X carried by the filamentous phage f1 genome are proteins with required but opposing functions in phage DNA replication. Their normal relative levels are important for continuous production of phage DNA without killing infected Escherichia coli hosts. Here we identify several factors responsible for determining the relative levels of pII and pX and that, if perturbed, alter the normal distribution of the phage DNA species in infected hosts. Translation of the two proteins is essentially relegated to separate mRNAs. The mRNAs encoding genes II and X are also differentially sensitive to cleavage dependent on rne, the gene encoding the only E. coli endo-RNase known to have a global role in mRNA stability. Whereas pII levels are limited at the level of mRNA stability, normal pX levels require transcription in sufficient amounts from the promoter for the smaller mRNA encoding only pX.

Translational control of puf operon expression in Rhodobacter sphaeroides 2.4.1.

The puf operon of Rhodobacter sphaeroides 2.4.1 encode the beta- and alpha-polypeptides of the B875 complex, the L and M polypeptides of the reaction centre and the pufX gene product. A previous report from the authors' laboratory indicated the potential existence of a 20-codon open reading frame (orfK, now designated pufK) located immediately upstream of the pufB structural gene. It is now demonstrated that pufK is translated in vivo and that the specific levels or nature of the rare codons within pufK affect the expression of pufK. Using a series of pufK-specific mutations, both in trans as lacZ translational fusions and incorporated into the genome in single copy, evidence has been obtained that translation initiation through pufK may be essential to translation of pufB. Further, the abundance, quality and distribution of rare codons within pufK may serve to 'gate' the entry of ribosomes at pufB. The data also suggest that translation of pufB is uncoupled from that of pufA, with the latter capable of being produced in excess of the former. It is also revealed that the secondary structure at the 5' end of the large and small puf transcripts may play a role in mRNA stability and that stability of the small puf transcript is independent of translation.

Ribosomal Association of Poly(A)-binding Protein in Poly(A)-deficient Saccharomyces cerevisiae

Poly(A)-binding protein, the most abundant eukaryotic mRNP protein, is known primarily for its association with polyadenylate tails of mRNA. In the yeast, Saccharomyces cerevisiae, this protein (Pabp) was found to be essential for viability and has been implicated in models featuring roles in mRNA stability and as an enhancer of translation initiation. Although the mechanism of action is unknown, it is thought to require an activity to bind poly(A) tails and an additional capacity for an interaction with 60 S ribosomal subunits, perhaps via ribosomal protein L46 (Rpl46). We have found that a significant amount of Pabp in wild-type cells is not associated with polyribosome complexes. The remaining majority, which is found in these complexes, maintains its association even in yeast cells deficient in polyadenylated mRNA and/or Rpl46. These observations suggest that Pabp may not require interaction with poly(A) tails during translation. Further treatment of polyribosome lysates with agents known to differentially disrupt components of polyribosomes indicated that Pabp may require contact with some RNA component of the polyribosome, which could be either non-poly(A)-rich sequences of the translated mRNA or possibly a component of the ribosome. These findings suggest that Pabp may possess the ability to bind to ribosomes independently of its interaction with poly(A). We discuss these conclusions with respect to current models suggesting a multifunctional binding capacity of Pabp.

Posttranscriptional regulation of gene expression in liver regeneration: role of mRNA stability

The rentry of hepatocytes and nonparenchymal cells from the normal quiescent G0 phase into the cell cycle during liver regeneration after 70% partial hepatectomy results in the discrete modulation of mRNA transcripts for many different genes. The modulation of steady-state levels of transcripts for genes involved in hepatocyte growth and replication during liver regeneration indicates that gene expression is regulated not only transcriptionally but also posttranscriptionally. In fact, posttranscriptional control appears to be the primary mechanism of regulating gene expression after the first 3 h after partial hepatectomy. Alteration in transcript stability is a key posttranscriptional regulatory mechanism used by the regenerating liver to modulate the steady-state transcript levels of multiple genes. Even genes that are transcriptionally activated during liver regeneration exhibit posttranscriptional control at the level of transcript stability. Moreover, the abundance of mRNA binding proteins, as well as translational activity and rate of poly(A) tail removal, are modulated and appear to influence transcript stability during liver regeneration. However, alteration of transcript stability is not the sole posttranscriptional mechanism regulating steady-state levels. Posttranscriptional control also occurs at the level of alternative splicing, stabilization of heterogeneous nuclear (hn) RNA, and hnRNA processing. Moreover, the role of nucleocytoplasmic transport of mature mRNA during liver regeneration is still undefined. Thus, during liver regeneration gene expression is regulated at multiple levels after the initial synthesis of hnRNA. By understanding the role of posttranscriptional mechanisms in regulating steady-state transcript levels in an in vivo model of normal growth, we will begin to appreciate its role in the genesis of abnormal growth.

Identification and characterization of a 44 kDa protein that binds specifically to the 3'-untranslated region of CYP2a5 mRNA: inducibility, subcellular distribution and possible role in mRNA stabilization.

Stabilization of mRNA is important in the regulation of CYP2a5 expression but the factors involved in the process are not known [Aida and Negishi (1991) Biochemistry 30, 8041-8045]. In this paper, we describe, for the first time, a protein that binds specifically to the 3'-untranslated region of CYP2a5 mRNA and which is inducible by pyrazole, a compound known to increase the half-life of CYP2a5 mRNA. We also demonstrate that pyrazole treatment causes an elongation of the CYP2a5 mRNA poly(A) tail, and that phenobarbital, which is transcriptional activator of the CYP2a5 gene that does not affect the mRNA half-life, neither induces the RNA-binding protein nor affects the poly(A) tail size. SDS/PAGE of the UV-cross-linked RNA-protein complex demonstrated that the RNA-binding protein has an apparent molecular mass of 44 kDa. The protein-binding site was localized to a 70-nucleotide region between bases 1585 and 1655. Treatment of cytoplasmic extracts with an SH-oxidizing agent, diamide, an SH-blocking agent, N-ethylmaleimide or potato acid phosphatase abolished complex-formation, suggesting that the CYP2a5 mRNA-binding protein is subject to post-translational regulation. Subcellular fractionation showed that the 44 kDa protein is present in polyribosomes and nuclei, and that its apparent induction is much stronger in polyribosomes than in nuclear extracts. We propose that this 44 kDa RNA-binding protein is involved in the stabilization of CYP2a5 mRNA by controlling the length of the poly(A) tail.

Differential contributions of two elements of rho-independent terminator to transcription termination and mRNA stabilization

The hallmark features of rho-independent transcription terminators are a G+C-rich dyad symmetry sequence followed by a run of T residues on a sense strand. Both of these structural elements are required for efficient transcription termination. Besides its primary function, rho-independent terminators are also known to enhance expression of an upstream gene by stabilizing RNA in a few cases. The Escherichia coli crp gene encoding cAMP receptor protein (CRP) contains a typical rho-independent terminator. To gain further insight into the roles of the G+C-rich dyad symmetry sequence and the poly(T) tract both in transcription termination and mRNA stabilization, we constructed a series of variant crp terminators and analyzed their abilities regarding these two functions. Disruption of the G+C-rich dyad symmetry sequence almost completely eliminated terminator activity while disruption of the poly(T) tract reduced terminator activity significantly but not completely. Thus, the contribution of the G+C-rich dyad symmetry sequence to transcription termination is larger than that of the poly(T) tract. Disruption of the G+C-rich dyad symmetry region reduced expression of the upstream crp gene by accelerating the rate of mRNA degradation. However, disruption of the poly(T) sequence had no effect on the stability of the crp mRNA, indicating that the poly(T) tract plays no role in mRNA stabilization. When the crp terminator was replaced by terminators derived from other genes, the fusion genes expressed the crp mRNA at the same level as did the native crp gene, suggesting that the mRNA stabilization effect is probably a general nature of rho-independent terminators.