scholarly journals Human mineralocorticoid receptor A and B protein forms produced by alternative translation sites display different transcriptional activities

2004 ◽  
pp. 585-590 ◽  
Author(s):  
L Pascual-Le Tallec ◽  
C Demange ◽  
M Lombes
Keyword(s):  
Translation Initiation ◽  
Mineralocorticoid Receptor ◽  
Single Gene ◽  
In Vitro Transcription ◽  
Targeted Mutagenesis ◽  
In Vitro Translation ◽  
Terminal Fragment ◽  
Initiation Of Translation ◽  
Alternative Translation

OBJECTIVE: Aldosterone binds the mineralocorticoid receptor (MR) and is involved in the regulation of ionic transport, mainly sodium retention. MR is encoded by one single gene transcribed into various messengers that are thought to be translated into a unique 107 kDa protein. The aim of this study was to identify and characterize translation initiation variants of the human MR protein. METHODS: For this purpose we analyzed the extreme N-terminal fragment of various MR species, and confronted the predicted pattern of expression with in vitro translation results. Transactivation functions of the identified MR forms obtained by targeted mutagenesis were evaluated by transient transfection assays with different promoter reporter genes. RESULTS: Two major forms of human MR (hMR), named MRA and MRB, generated by alternative initiation of translation from start methionine 1 and 15 respectively, were predicted. Their expected expressions were confirmed by in vitro transcription/translation studies. Interestingly, each form of MR displayed reduced transactivation capacities with wild type (WT)=A+B>MRA>MRB suggesting that the extreme N-terminal fragment of MR has an effect on the transcriptional properties of the receptor. CONCLUSIONS: Altogether these data indicate that MR is expressed, through alternative translation initiation, as distinct protein variants which possess different functional properties. These MR forms could tightly dictate the modulation of aldosterone responsiveness in various tissues or in pathophysiological situations.

scholarly journals Retapamulin-assisted ribosome profiling reveals the alternative bacterial proteome

2019 ◽  
Author(s):  
Sezen Meydan ◽  
James Marks ◽  
Dorota Klepacki ◽  
Virag Sharma ◽  
Pavel V. Baranov ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Translation Initiation ◽  
Single Gene ◽  
Ribosome Profiling ◽  
Genome Wide ◽  
Translation Start ◽  
Alternative Translation

SUMMARYThe use of alternative translation initiation sites enables production of more than one protein from a single gene, thereby expanding cellular proteome. Although several such examples have been serendipitously found in bacteria, genome-wide mapping of alternative translation start sites has been unattainable. We found that the antibiotic retapamulin specifically arrests initiating ribosomes at start codons of the genes. Retapamulin-enhanced Ribo-seq analysis (Ribo-RET) not only allowed mapping of conventional initiation sites at the beginning of the genes but, strikingly, it also revealed putative internal start sites in a number of Escherichia coli genes. Experiments demonstrated that the internal start codons can be recognized by the ribosomes and direct translation initiation in vitro and in vivo. Proteins, whose synthesis is initiated at an internal in-frame and out-of-frame start sites, can be functionally important and contribute to the ‘alternative’ bacterial proteome. The internal start sites my also play regulatory roles in gene expression.

scholarly journals Eukaryotic Translation Initiation Factors 4G and 4A from Saccharomyces cerevisiae Interact Physically and Functionally

1999 ◽  
Vol 19 (8) ◽  
pp. 5557-5564 ◽  
Author(s):  
Carrie L. Neff ◽  
Alan B. Sachs
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
In Vitro Translation ◽  
Initiation Factor ◽  
Temperature Sensitive ◽  
Eukaryotic Translation ◽  
Initiation Of Translation ◽  
Eukaryotic Translation Initiation

ABSTRACT The initiation of translation in eukaryotes requires several multisubunit complexes, including eukaryotic translation initiation factor 4F (eIF4F). In higher eukaryotes eIF4F is composed of the cap binding protein eIF4E, the adapter protein eIF4G, and the RNA-stimulated ATPase eIF4A. The association of eIF4A withSaccharomyces cerevisiae eIF4F has not yet been demonstrated, and therefore the degree to which eIF4A’s conserved function relies upon this association has remained unclear. Here we report an interaction between yeast eIF4G and eIF4A. Specifically, we found that the growth arrest phenotype associated with three temperature-sensitive alleles of yeast eIF4G2 was suppressed by excess eIF4A and that this suppression was allele specific. In addition, in vitro translation extracts derived from an eIF4G2 mutant strain could be heat inactivated, and this inactivation could be reversed upon the addition of recombinant eIF4A. Finally, in vitro binding between yeast eIF4G and eIF4A was demonstrated, as was diminished binding between mutant eIF4G2 proteins and eIF4A. In total, these data indicate that yeast eIF4G and eIF4A physically associate and that this association performs an essential function.

Structural and Genetic Aspects of Proline-rich Proteins

1987 ◽  
Vol 66 (2) ◽  
pp. 457-461 ◽  
Author(s):  
A. Bennick
Keyword(s):  
General Structure ◽  
Single Gene ◽  
Conclusive Evidence ◽  
In Vitro Translation ◽  
Binding Studies ◽  
Nmr Studies ◽  
Post Translational Modifications ◽  
Single Precursor ◽  
Made In

Considerable advances have been made in the genetics of salivary proline-rich proteins (PRP). The genes for acidic, basic, and glycosylated PRP have been cloned. They code for precursor proteins that all have an acidic N-terminal followed by proline-rich repeat sequences. Structural studies on secreted proteins have demonstrated that not only acidic but also some basic PRPs have this general structure. It is possible that mRNA for different PRP may have originated from a single gene by differential mRNA splicing, but post-translational cleavages of the primary translation product apparently also occur. In vitro translation of salivary gland mRNA results in a single precursor protein for acidic PRP. Such in vitro translated protein can be cleaved by salivary kallikrein, giving rise to two commonly secreted acidic PRPs, and kallikrein or kallikrein-like enzymes may be responsible for other post-translational cleavages of PRPs. Acidic as well as some basic PRPs are phosphorylated. A protein kinase has been demonstrated in salivary glands which phosphorylates the PRPs and other secreted salivary proteins in a cAMP and Ca2+-calmodulinindependent manner. Knowledge of the conformation of PRPs is limited. There is no conclusive evidence of polyproline-like structure in the proline-rich part of PRPs. Ca2+ binding studies on acidic PRPs indicate that there is interaction between the Ca2+ binding N-terminal end and the proline-rich C-terminal part. This interaction is relieved by modification of arginine side-chains. 1H, 32P, and 43Ca NMR studies have further elucidated the conformation of acidic PRPs in solution. Present evidence shows that salivary PRPs constitute a unique superfamily of proteins which pose a number of interesting questions concerning gene structure, pre- and post-translational modifications, and protein conformation.

A cDNA clone for a polyadenylated RNA-binding protein of Xenopus laevis oocytes hybridizes to four developmentally regulated mRNAs

1985 ◽  
Vol 5 (10) ◽  
pp. 2697-2704
Author(s):  
L J Lorenz ◽  
J D Richter
Keyword(s):  
Xenopus Laevis ◽  
Cdna Clone ◽  
Rna Binding ◽  
Binding Protein ◽  
Single Gene ◽  
In Vitro Translation ◽  
Xenopus Laevis Oocytes ◽  
Cdna Insert ◽  
Alternative Processing

Xenopus laevis oocytes contain a unique group of proteins which decrease during oogenesis, bind poly(A) RNA, and possibly play a role in the regulation of translation. A monoclonal antibody generated against one of these proteins was used to screen an expression vector cDNA library. A cDNA clone was isolated and confirmed to code for the binding protein by in vitro translation of hybrid-selected RNA followed by immunoprecipitation. This cDNA, when used in RNA gel blots, hybridized to four transcripts of 2.0, 1.7 (two transcripts of similar size), and 1.2 kilobases. All of the transcripts decreased in amount during oogenesis and were not evident in somatic cells. In addition, the fraction of the transcripts associated with polysomes decreased during oogenesis. Digestion of the cDNA insert with PstI generated two fragments of 220 and 480 base pairs which, when used as probes in an RNA gel blot, hybridized to unique as well as common transcripts. Genomic Southern blots suggested the presence of a single gene, indicating that these transcripts arose by alternative processing.

cDNA cloning and in vitro transcription of the complete brome mosaic virus genome

1984 ◽  
Vol 4 (12) ◽  
pp. 2876-2882 ◽  
Author(s):  
P Ahlquist ◽  
M Janda
Keyword(s):  
Mosaic Virus ◽  
Transcription Initiation ◽  
Direct Sequencing ◽  
In Vitro Transcription ◽  
Brome Mosaic Virus ◽  
Viral Rna ◽  
In Vitro Translation ◽  
In Vitro Production ◽  
Genomic Rnas

Complete cDNA copies of each of the brome mosaic virus genomic RNAs (3.2, 2.8, and 2.1 kilobases in length) were cloned in a novel transcription vector, pPM1, designed to provide exact control of the transcription initiation site. After cleavage at a unique EcoRI site immediately downstream of the inserted cDNA, these clones can be transcribed in vitro by Escherichia coli RNA polymerase to yield complete copies of the brome mosaic virus RNAs. Dideoxy sequencing of 5' transcript cDNA runoff products and direct sequencing of 32P-3'-end-labeled transcripts show that such transcripts initiate at the same 5' position as natural viral RNA and terminate within the EcoRI runoff site after copying the entire viral RNA sequence. When synthesized in the presence of m7GpppG, the transcripts bear the natural capped 5' terminus of brome mosaic virus RNAs. Such transcripts direct the in vitro translation of proteins which coelectrophorese with the translation products of natural brome mosaic virus RNAs. pPM1 should facilitate in vitro production of other viral and nonviral RNAs.

scholarly journals Isolation of cDNA clones for genes exhibiting reduced expression after differentiation of murine teratocarcinoma stem cells.

1984 ◽  
Vol 4 (10) ◽  
pp. 2142-2150 ◽  
Author(s):  
R A Levine ◽  
G J LaRosa ◽  
L J Gudas
Keyword(s):  
Stem Cells ◽  
Retinoic Acid ◽  
Cyclic Amp ◽  
Cdna Library ◽  
In Vitro Transcription ◽  
In Vitro Translation ◽  
Transcriptional Level ◽  
Cdna Clones ◽  
Dibutyryl Cyclic Amp

In the absence of retinoic acid, PSA-G teratocarcinoma stem cells spontaneously differentiate at a moderate frequency into fibroblast-like cells. In the presence of retinoic acid and dibutyryl cyclic AMP, PSA-G stem cells differentiate into parietal endoderm cells. We prepared a cDNA library from undifferentiated PSA-G teratocarcinoma stem cells; this cDNA library was then screened for gene sequences which exhibit a reduction in expression during the differentiation of these stem cells. From ca. 1,000 clones screened, eight independent sequences were isolated. The level of expression of these cloned genes decreases by 3.0-fold to more than 10-fold after differentiation of PSA-G cells into fibroblast-like cells. After treatment of either PSA-G or F9 teratocarcinoma cells with retinoic acid and dibutyryl cyclic AMP for 72 h, the expression of seven genes is inhibited by two- to fourfold. This decrease of clone-specific transcripts can be detected within 12 h after the addition of retinoic acid. Hybridization-selection and in vitro translation experiments identified the proteins encoded by three of the cloned genes: pST 6-23 codes for a 89,000-dalton protein, pST 7-105 codes for a 41,000-dalton protein, and pST 9-31 codes for a 34,000-dalton protein. The 89,000-dalton protein encoded by pST 6-23 is a heat shock protein. In vitro transcription experiments demonstrate that the retinoic acid-mediated decrease in pST 6-135- and pST 1-68-specific RNA occurs at the transcriptional level and that dibutyryl cyclic AMP acts posttranscriptionally to further depress the levels of these RNAs.

scholarly journals Translation initiation factor 4A from Saccharomyces cerevisiae: analysis of residues conserved in the D-E-A-D family of RNA helicases.

1991 ◽  
Vol 11 (7) ◽  
pp. 3463-3471 ◽  
Author(s):  
S R Schmid ◽  
P Linder
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Clear Correlation ◽  
Temperature Sensitive ◽  
Rna Helicases ◽  
Initiation Of Translation

The eukaryotic translation initiation factor 4A (eIF-4A) possesses an in vitro helicase activity that allows the unwinding of double-stranded RNA. This activity is dependent on ATP hydrolysis and the presence of another translation initiation factor, eIF-4B. These two initiation factors are thought to unwind mRNA secondary structures in preparation for ribosome binding and initiation of translation. To further characterize the function of eIF-4A in cellular translation and its interaction with other elements of the translation machinery, we have isolated mutations in the TIF1 and TIF2 genes encoding eIF-4A in Saccharomyces cerevisiae. We show that three highly conserved domains of the D-E-A-D protein family, encoding eIF-4A and other RNA helicases, are essential for protein function. Only in rare cases could we make a conservative substitution without affecting cell growth. The mutants show a clear correlation between their growth and in vivo translation rates. One mutation that results in a temperature-sensitive phenotype reveals an immediate decrease in translation activity following a shift to the nonpermissive temperature. These in vivo results confirm previous in vitro data demonstrating an absolute dependence of translation on the TIF1 and TIF2 gene products.

scholarly journals Implications of the Use of Eukaryotic Translation Initiation Factor 5A (eIF5A) for Prognosis and Treatment of Hepatocellular Carcinoma

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Felix H. Shek ◽  
Sarwat Fatima ◽  
Nikki P. Lee
Keyword(s):  
Hepatocellular Carcinoma ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation Initiation Factor ◽  
Eukaryotic Translation ◽  
Initiation Of Translation ◽  
Eukaryotic Translation Initiation ◽  
Hcc Cells

Hepatocellular carcinoma (HCC) is a primary liver malignancy and accounts for most of the total liver cancer cases. Lack of treatment options and late diagnosis contribute to high mortality rate of HCC. In eukaryotes, translation of messenger RNA (mRNA) to protein is a key process in protein biosynthesis in which initiation of translation involves interaction of different eukaryotic translation initiation factors (eIFs), ribosome subunits and mRNAs. Eukaryotic translation initiation factor 5A (eIF5A) is one of the eIFs involved in translation initiation and eIF5A2, one of its isoforms, is upregulated in various cancers including HCC as a result of chromosomal instability, where it resides. In HCC, eIF5A2 expression is associated with adverse prognosis such as presence of tumor metastasis and venous infiltration. Based on eIF5A2 functional studies, suppressing eIF5A2 expression by short interfering RNA alleviates the tumorigenic properties of HCC cellsin vitrowhile ectopic expression of eIF5A2 enhances the aggressiveness of HCC cellsin vivoandin vitroby inducing epithelial-mesenchymal transition. In conclusion, eIF5A2 is a potential prognostic marker as well as a therapeutic target for HCC.

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