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PubMed2006

The 5' stem-loop and its role in mRNA stability in maize S cytoplasmic male sterility.

The co-transcribed orf355-orf77 region of the mitochondrial genome is associated with S cytoplasmic male sterility (CMS-S) in maize; the amounts of its 1.6- and 2.8-kb transcripts were previously shown to be greatly reduced in fertility-restored microspores relative to the amounts in sterile plants. To investigate the mechanism underlying this reduction, detailed analysis of the 5' and 3' termini of these transcripts was conducted. Using 3' RACE analysis, the polyadenylation sites of the 1.6- and 2.8-kb transcripts were mapped adjacent to a 3' stem-loop, which may play an important role in stabilizing their 3' ends. No difference was found between the polyadenylation sites in sterile and fertility-restored microspores that could account for the differences in orf355-orf77 transcript levels. The 5' terminus of the 1.6-kb transcript was further studied by primer extension; the result revealed that there was a deletion of nine nucleotides only in fertility-restored microspores, and that this deletion eliminated a 5' stem-loop sequence. We propose that the elimination of the 5' stem-loop in the fertility-restored microspores could be the cause of the degradation of the 1.6-kb transcript. Because the 2.8-kb transcript can be cleaved to generate the 1.6-kb transcript, the amount of the 2.8-kb transcript is also reduced in fertility-restored microspores.

Base Sequence↗
PubMed1992

Coordination of expression of Zymomonas mobilis glycolytic and fermentative enzymes: a simple hypothesis based on mRNA stability.

Although Zymomonas mobilis is prototrophic, glycolytic and fermentative enzymes (ethanologenic enzymes) constitute over half of the cytoplasmic protein. In this study, transcript stability, functional message pools, and the abundance of cytoplasmic products were compared for genes encoding eight of these essential enzymes. The transcripts of all were very stable, with half-lives ranging from 8 to 18 min. This transcript stability is proposed as an important feature in Z. mobilis that may distinguish highly expressed genes for energy generation from biosynthetic genes, which are required at much lower levels. The evolution of multiple promoters to enhance transcription from single-copy genes, of structural features that alter translational efficiency, and of differences in protein turnover is hypothesized to serve a subordinate role in the regulation of Z. mobilis gene expression. Among the eight ethanologenic genes examined, differences in transcript stability were found to directly correlate with differences in functional message pools and cytoplasmic protein levels. These differences in transcript stability are hypothesized to have evolved as a primary mechanism to balance the levels of individual enzymes within the glycolytic and fermentative pathways.

Bacterial Proteins↗
PubMed1995

The product of the pleiotropic Escherichia coli gene csrA modulates glycogen biosynthesis via effects on mRNA stability.

The carbon storage regulator gene, csrA, modulates the expression of genes in the glycogen biosynthesis and gluconeogenesis pathways in Escherichia coli and has been cloned, mapped and sequenced (T. Romeo, M. Gong, M.Y. Liu, and A.M. Brun-Zinkernagel, J. Bacteriol. 175:4744-4755, 1993; T. Romeo and M. Gong, J. Bacteriol. 175:5740-5741, 1993). We have now conducted experiments that begin to elucidate a unique mechanism for csrA-mediated regulation. Steady-state levels of glgC transcripts, encoding ADP-glucose pyrophosphorylase, were elevated by up to sixfold in a csrA::kanR mutant and were less than 6.5% of wild-type levels in a strain containing pCSR10 (csrA+), as shown by S1 nuclease protection analysis. The rate of chemical decay of these transcripts after adding rifampin to cultures was dramatically reduced by the csrA::kanR mutation. Deletion studies of a glgC'-'lacZ translational fusion demonstrated that the region surrounding the initiation codon was important for csrA-mediated regulation and indicated that neither csrA-mediated regulation nor stationary phase induction of glgC expression originates at the level of transcript initiation. Cell-free (S-200) extracts containing the CsrA gene product potently and specifically inhibited the in vitro transcription-translation of glg genes. The deduced amino acid sequence of CsrA was found to contain the KH motif, which characterizes a subset of diverse RNA-binding proteins. The results indicate that CsrA accelerates net 5'-to-3' degradation of glg transcripts, potentially through selective RNA binding.

Amino Acid Sequence↗
PubMed2004

The expression of HSP83 genes in Leishmania infantum is affected by temperature and by stage-differentiation and is regulated at the levels of mRNA stability and translation.

BACKGROUND: Exposure of Leishmania promastigotes to the temperature of their mammalian hosts results in the induction of a typical heat shock response. It has been suggested that heat shock proteins play an important role in parasite survival and differentiation. RESULTS: Here we report the studies on the expression of the heat shock protein 83 (HSP83) genes of Leishmania infantum. Confirming previous observations for other Leishmania species, we found that the L. infantum HSP83 transcripts also show a temperature-dependent accumulation that is controlled by a post-transcriptional mechanism involving sequences located in the 3'-untranslated region (3'-UTR). However, contrary to that described for L. amazonensis, the accumulation of the HSP83 transcripts in L. infantum is dependent on active protein synthesis. The translation of HSP83 transcripts is enhanced during heat shock and, as first described in L. amazonensis, we show that the 3'-UTR of the L. infantum HSP83 gene is essential for this translational control. Measurement of the steady-state levels of HSP83 transcripts along the promastigote-to-amastigote differentiation evidenced a specific profile of HSP83 RNAs: after an initial accumulation of HSP83 transcripts observed short after (2 h) incubation in the differentiation conditions, the amount of HSP83 RNA decreased to a steady-state level lower than in undifferentiated promastigotes. We show that this transient accumulation is linked to the presence of the 3'-UTR and flanking regions. Again, an 8-fold increase in translation of the HSP83 transcripts is observed short after the initiation of the axenic differentiation, but it is not sustained after 9 h. CONCLUSIONS: This transient expression of HSP83 genes could be relevant for the differentiation of Leishmania, and the underlying regulatory mechanism may be part of the developmental program of this parasite.

3' Untranslated Regions↗
PubMed2005

Failure of the PAXgene Blood RNA System to maintain mRNA stability in whole blood.

In multicentre studies of malignant and inflammatory diseases, whole blood, cell or tissue samples are often collected for analyses of gene expression to predict or monitor treatment effects. For correct analysis, sample stability during handling and transport is crucial. In developing the logistics for multicentre studies in malignant melanoma and inflammatory bowel disease, we found poor stability of a number of transcripts using the PAXgene Blood RNA System, which was advertised to maintain RNA stability for several days at room temperature. The results indicate that general statements on sample stability are not reliable and have to be verified for the specific transcripts of interest.

Artifacts↗
PubMed1998

hnRNP C increases amyloid precursor protein (APP) production by stabilizing APP mRNA.

We have previously shown that heterogeneous nuclear ribonucleoprotein C (hnRNP C) and nucleolin bound specifically to a 29 nt sequence in the 3'-untranslated region of amyloid precursor protein (APP) mRNA. Upon activation of peripheral blood mononuclear cells, hnRNP C and nucleolin acquired APP mRNA binding activity, concurrent with APP mRNA stabilization. These data suggested that the regulated interaction of hnRNP C and nucleolin with APP mRNA controlled its stability. Here we have directly examined the role of the cis element and trans factors in the turnover and translation of APP mRNA in vitro . In a rabbit reticulocyte lysate (RRL) translation system, a mutant APP mRNA lacking the 29 nt element was 3-4-fold more stable and synthesized 2-4-fold more APP as wild-type APP mRNA. Therefore, the 29 nt element functioned as an APP mRNA destabilizer. RNA gel mobility shift assays with the RRL suggested the presence of endogenous nucleolin, but failed to show hnRNP C binding activity. However, wild-type APP mRNA was stabilized and coded for 6-fold more APP when translated in an RRL system supplemented with exogenous active hnRNP C. Control mRNAs lacking the 29 nt element were unaffected by hnRNP C supplementation. Therefore, occupancy of the 29 nt element by hnRNP C stabilized APP mRNA and enhanced its translation.

Amyloid beta-Protein Precursor↗
PubMed2005

High NaCl increases TonEBP/OREBP mRNA and protein by stabilizing its mRNA.

Hypertonicity increases mRNA and protein abundance of the transcription factor tonicity-responsive enhancer/osmotic response element binding protein (TonEBP/OREBP), contributing to increased transcription of downstream osmoprotective genes. Previously, this was attributed to increased transcription of TonEBP/OREBP because no change was found in its mRNA stability. However, there is no direct evidence for increased transcription, and the 3'-untranslated region (UTR) of TonEBP/OREBP contains numerous adenylate/uridylate-rich elements, which can modulate RNA stability. Therefore, we have reinvestigated the effect of hypertonicity on TonEBP/OREBP mRNA stability. We find that, in mouse inner medullary collecting duct cells, raising osmolality from 300 to 500 mosmol/kgH(2)O by adding NaCl increases TonEBP/OREBP mRNA to a peak of 2.3-fold after 4 h, followed by a decline. TonEBP/OREBP protein increases to a sustained peak of 3.0-fold at 8 h. To determine the stability of TonEBP/OREBP mRNA, we measured the rate of its decrease after inhibiting transcription with actinomycin D, finding that it is stabilized for 6 h after addition of NaCl. This stabilization is sufficient to explain the increase in mRNA without any change in transcription. To investigate how hypertonicity stabilizes TonEBP/OREBP mRNA, we tested luciferase reporters containing parts of the TonEBP/OREBP mRNA UTR. Inclusion of both the 5'- and 3'-UTR increases reporter activity, consistent with mRNA stabilization. Surprisingly, however, it is the 5'-UTR that stabilizes; the 3'-UTR, by itself, decreases reporter activity. We concluded that 1) hypertonicity stabilizes TonEBP/OREBP mRNA, contributing to its increase, and 2) stabilization depends on the presence of the 5'-UTR.

3' Untranslated Regions↗
PubMed1993

Angiotensin II stimulates the synthesis of angiotensinogen in hepatocytes by inhibiting adenylylcyclase activity and stabilizing angiotensinogen mRNA.

Angiotensin II stimulates the hepatic synthesis and secretion of angiotensinogen, the substrate of renin. In the present study performed on freshly isolated rat hepatocytes we demonstrate that this effect of angiotensin II is mainly related to a transient inhibition of adenylylcyclase. Agents known to decrease intracellular cAMP (angiotensin II, vasopressin, guanfacine) or the cAMP-antagonist Rp-adenosine-3',5'-cyclic phosphothioate stimulated, whereas cAMP-stimulating agents (isoproterenol, forskolin, glucagon) or the cAMP-agonist Sp-adenosine-3',5'-cyclic phosphothioate inhibited angiotensinogen synthesis. In contrast, all agents known to affect intracellular concentrations of calcium, as confirmed in Fura-2-loaded hepatocytes (Bay K 8644, calcimycin, calmidazolium, ionomycin, or methoxamine) failed to influence the synthesis of angiotensinogen. The inhibitory effect of angiotensin II as well as the stimulatory effect of glucagon on cAMP were inversely related to angiotensinogen mRNA and angiotensinogen secretion over a wide concentration range of both peptides. Both the angiotensin II-dependent inhibition of cAMP and the angiotensin II-induced increase in angiotensinogen mRNA were abolished by a pertussis toxin pretreatment. In hepatocyte membranes, pertussis toxin ADP-ribosylated a single protein (approximately 41 kDa) probably representing the alpha-subunit of the Gi-protein, coupling inhibitory receptors to adenylylcyclase. We further show that the increase of angiotensinogen mRNA and secretion mainly represents the result of mRNA stabilization, since in a nuclear run-on assay, angiotensin II pretreatment of hepatocytes does not significantly alter the rate of [32P]UTP incorporation into angiotensinogen mRNA, whereas angiotensin II prolonged the half-life of angiotensinogen mRNA in transcription-arrested as well as in [3H]uridine pulse-labeled hepatocytes about 2.5-fold from 80 to 190 min. It is concluded that angiotensin II induces an increase in angiotensinogen synthesis in hepatocytes by stabilizing of angiotensinogen mRNA and that this effect is mediated through inhibition of adenylylcyclase.

Adenylyl Cyclase Inhibitors↗