RNA secondary structure and in vitro translation efficiency

Cell-free protein synthesis is a promising technology featuring many advantages compared to in vivo expression techniques. However, most proteins are still synthesized in vivo due to relatively low protein yields commonly achieved in vitro, especially in the batch mode of reaction. In Escherichia coli S30 extract-based cell-free systems protein yields are supposed to be partially limited by a secondary structure formation of the mRNA. In this study we checked promising members of various classes of RNA chaperones and several different RNA helicases on their ability to enhance in vitro translation. The data clearly show that the addition of none of these factors provides a general solution to the problem. However, protein yields can be increased in presence of a microRNA hybridizing with the 5′ untranslated region of mRNAs, possibly by inducing structural changes improving accessibility of the Shine Dalgarno sequence for the ribosomes.     

Specificity of reticulocyte initiation factors for the translation of globin messenger RNA

Initiation factors from rabbit reticulocytes can select globin mRNA for translation in an ascites cell-free system in the presence of either encephalomyocarditis viral RNA or endogenous ascites mRNAs. It appears that the viral RNA cannot compete for either α- or β-globin-specific factors.     

Recognition of messenger RNA during translational initiation in Escherichia coli

The structural aspects of recognition by E. coli ribosomes of translational initiation regions on homologous messenger RNAs have been reviewed. Also discussed is the location of initiation region on mRNA, its confines, typical nucleotide sequences responsible for initiation signal, and the influence of RNA macrostructure on protein synthesis initiation. Most of the published DNA nucleotide sequences surrounding the start of various E. coli genes and those of its phages have been collected.     

Partially edited RNAs are intermediates of RNA editing in plant mitochondria

RNA editing in flowering plant mitochondria addresses several hundred specific C nucleotides in individual sequence contexts in mRNAs and tRNAs. Many of the in vivo steady state RNAs are edited at some sites but not at others. It is still unclear whether such incompletely edited RNAs can either be completed or are aborted. To learn more about the dynamics of the substrate recognition process, we investigated in vitro RNA editing at a locus in the atp4 mRNA where three editing sites are clustered within four nucleotides. A single cis-element of about 20 nucleotides serves in the recognition of at least two sites. Competition with this sequence element suppresses in vitro editing. Surprisingly, unedited and edited competitors are equally effective. Experiments with partially pre-edited substrates indicate that indeed the editing status of a substrate RNA does not affect the binding affinity of the specificity factor(s). RNA molecules in which all editing sites are substituted by either A or G still compete, confirming that editing site recognition can occur independently of the actual editing site. These results show that incompletely edited mRNAs can be substrates for further rounds of RNA editing, resolving a long debated question.     

In vitro translation of human pheochromocytoma messenger RNAs: characterization of tyrosine-hydroxylase and dopamine-beta-hydroxylase

mRNAs extracted from human pheochromocytoma were translated in vitro in a lysate of a rabbit reticulocytes. Two enzymes of the biosynthetic pathway of the catecholamines, tyrosine-hydroxylase (TH) and dopamine-beta-hydroxylase (DBH), were characterized as translation products after immunoprecipitation by specific antisera and electrophoretic analysis. The precursor of TH is a polypeptide having a molecular mass of 62,000 identical to that found for the mature protein. The molecular mass of the precursor of DBH 73,000 while that of the mature form is 79,000. TH and DBH have been translated from mRNAs having sedimentation coefficients of 22S and 25S, respectively.     

Variability in messenger RNA levels in human umbilical vein endothelial cells of different lineage and time in culture

Summary Levels of seven messenger RNA species were compared in human umbilical vein endothelial cells of different lineage and time in culture. Specifically, cells obtained from the American Type Culture Collection (ATCC) and subcultured were compared to early passage cells from cultures produced in our laboratory. Messenger RNA for tissue plasminogen activator, plaminogen activator inhibitor 1, urokinase, and thrombomodulin were expressed at higher levels in the ATCC cells. Thrombospondin, von Willebrand's Factor, and protein S messenger RNA were expressed at higher levels in the cells that we isolated. In addition, in the ATCC cells a shift in the proportion of plasminogen activator inhibitor messenger RNA from the 3.4 to the 2.4 kilobase species was found. We conclude that specific messenger RNA levels can vary considerably between cultured human umbilical vein endothelial cells. The large variation in mRNA levels which we describe has important implications for experiments involving gene expression in cultured endothelium.     

Localization of the initiation of translation in messenger RNAs of prokaryotes by learning techniques

Learning processes are applied to the recognition of protein coding regions in prokaryotes. Non-contradictory, statistical and logical rules are deduced from a set of known examples of coding sequences. These rules enable to build characteristic patterns on the m-RNA upstream of the initiating codon. These rules are applied with success to recognize more than 180 coding sequences and to detect and/or eliminate hypothetical reading frames or unknown genes.     

Protein-coding structured RNAs: A computational survey of conserved RNA secondary structures overlapping coding regions in drosophilids

Functional RNA elements can be embedded also within exonic sequences coding for functional proteins. While not uncommon in viruses, only a few examples of this type have been described in some detail for eukaryotic genomes. Here we use RNAz and RNAcode, two comparative genomics methods that measure signatures of stabilizing selection acting on RNA secondary structure and peptide sequence, resp., to survey the fruit fly genomes. We estimate that there might be on the order of 1000 loci that are subject to dual selection pressure. The used genome-wide screens also expose the limitations of the currently available methods.     

Comparison of fungal mitochondrial introns reveals extensive homologies in RNA secondary structure

The complete sequences of nine Saccharomyces cerevisiae mitochondrial introns, six of which carry long open reading frames, have already been published. We have recently determined the sequence of an intron in the large ribosomal mitochondrial RNA of Kluyveromyces thermotolerans (Jacquier et al., in preparation), which we found to be closely related to its S. cerevisiae counterpart. This latter result prompted us to undertake a systematic search for possible homologous elements in the other, available sequences with the help of an original computer program. A previously unsuspected wealth of evolutionarily conserved sequences and secondary structures was thus uncovered. Seven at least of the available sequences may be folded up into elaborate secondary structure models, the cores of which are nearly identical. These models result in bringing together the exon-intron junctions into relatively close spatial proximity and looping out either all or most of the sequences in open reading frame, when present. These results and their possible implications with respect to the mechanism of splicing are discussed in the light of available genetic and biochemical data.     

Differential regulation by dexamethasone of glucocorticoid receptor messenger RNA concentrations in neuronal cultures derived from fetal rat hypothalamus and cerebral cortex

1. Differential regulation, by dexamethasone, of glucocorticoid receptor gene expression was studied in three different neuronal cultures derived from hypothalamus amygdala, and cerebral cortex. 2. Cellular glucocorticoid receptor (GR) mRNA concentration was measured by hybridization using a 32P-labeled RNA probe complementary to a 2.2-kb fragment of the glucocorticoid receptor mRNA. Changes in the amount of GR mRNA were evaluated in relation to the content of beta-actin mRNA. 3. In cells derived from either hypothalamus or cerebral cortex, we observed a complex pattern of GR mRNA concentrations which were characterized by cyclic variations of GR mRNA content during continuous treatment with dexamethasone for up to 72 hr. 4. In contrast to cells derived from the hypothalamus where a persistent 30-40% reduction in GR mRNA levels was seen for up to a least 72 hr, we observed, in cells derived from the cerebral cortex, a sustained increased (1.4-fold) of the GR mRNA at this same time interval.     

The translation of the messenger for the poly(A)-binding protein-associated with translated mRNA is suppressed. A case of cytoplasmic repression in duck erythroblasts

In vivo protein synthesis in duck erythroblasts was compared to in vitro translation of polyribosomal and free cytoplasmic mRNA. The in vivo study showed the absence of de novo synthesis of the Mr 73 000 poly(A)-binding protein found associated with all polyribosomal mRNA. In vitro translation demonstrated that the mRNA for this protein is absent from the polyribosomal mRNA fraction but constitutes a medium frequency messenger among the repressed free mRNA. This result confirms the existence of a qualitative translational control in terminal differentiating duck erythroblasts leading eventually to the arrest of the protein synthesizing machinery.