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Results for “Translation reinitiation”

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11 recordsLinked to original sources
PubMed2024

Elucidating the clinical and genetic spectrum of inositol polyphosphate phosphatase INPP4A-related neurodevelopmental disorder.

PURPOSE: Biallelic INPP4A variants have recently been associated with severe neurodevelopmental disease in single-case reports. Here, we expand and elucidate the clinical-genetic spectrum and provide a pathomechanistic explanation for genotype-phenotype correlations. METHODS: Clinical and genomic investigations of 30 individuals were undertaken alongside molecular and in silico modelling and translation reinitiation studies. RESULTS: We characterize a clinically variable disorder with cardinal features, including global developmental delay, severe-profound intellectual disability, microcephaly, limb weakness, cerebellar signs, and short stature. A more severe presentation associated with biallelic INPP4A variants downstream of exon 4 has additional features of (ponto)cerebellar hypoplasia, reduced cerebral volume, peripheral spasticity, contractures, intractable seizures, and cortical visual impairment. Our studies identify the likely pathomechanism of this genotype-phenotype correlation entailing translational reinitiation in exon 4 resulting in an N-terminal truncated INPP4A protein retaining partial functionality, associated with less severe disease. We also identified identical reinitiation site conservation in Inpp4a-/- mouse models displaying similar genotype-phenotype correlation. Additionally, we show fibroblasts from a single affected individual exhibit disrupted endocytic trafficking pathways, indicating the potential biological basis of the condition. CONCLUSION: Our studies comprehensively characterize INPP4A-related neurodevelopmental disorder and suggest genotype-specific clinical assessment guidelines. We propose that the potential mechanistic basis of observed genotype-phenotype correlations entails exon 4 translation reinitiation.

Humans↗
PubMed1977

5'-Terminal nucleotide sequence of Escherichia coli lactose repressor mRNA: features of translational initiation and reinitiation sites.

In a sequence of 214 nucleotides at the 5' terminus of the I gene mRNA, which codes for the lactose repressor protein of Escherichia coli, (i) an untranslated leader sequence of 28 residues precedes the repressor coding region; (ii) a GUG initiates synthesis of the wild-type repressor; (iii) GUG and AUG are the functional initiators for the synthesis of restart polypeptides activated by early I gene amber mutations, confirming previous assignments for these residues based on protein sequencing data; and (iv) sequences complementary to 16S ribosomal RNA provide stronger potential mRNA.16S rRNA interaction at the wild-type initiation site than at the restart sites. When I mRNA is used to direct the formation of initiation complexes in vitro, ribosomes bind only to the wild-type initiator region.A striking feature of the I mRNA sequence is the presence of a number of in-phase GUGs that have not been observed to serve as initiation signals in vivo in the nonsense mutant strains examined. The selective use of potential initiator triplets in the I mRNA leads to the following conclusions. First, when presented with several neighboring initiator triplets at the wild-type initiator region, ribosomes select the one preceded by the strongest appropriately positioned complementarity to the 16S 3' end. Second, ribosomes do not restart after termination simply by moving to the next available initiator codon. Third, the formation of stable secondary structures predicted for the untranslated I mRNA beyond chain-terminating nonsense mutations may prevent ribosome access to some potential reinitiation sites.

Bacterial Proteins↗
PubMed1977

Encephalomyocarditis virus RNA. II. Polyadenylic acid requirement for efficient translation.

Differentially polyadenylated subpopulatons of encephalomyocarditis (EMC) viral RNA were isolated by affinity chromatography on oligodeoxythymidylic acid-cellulose. Translation of these RNA fractions in several in vitro protein-synthesizing systems, isolated from Ehrlich ascites tumor cells, demonstrated that poly(A)+EMC viral RNA was translated two to three times more efficiently than poly(A)-EMC viral RNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the polypetides synthesized by the in vitro system in response to the different RNAs showed no detectable differences in the size or relative amount- of the translational products. mRNA saturation curves indicated that the in vitro systems were stimulated maximally by equivalent amounts of RNA, wheter it be poly(A)-or poly(A)+ EMC viral RNA. Time course experiments showed that the differences in translatability were more pronounced late in the reaction when reinitiation was required, and that by eliminating reinitiation with high salt the apparent effect of poly(A) on translation was diminished. Together, these results suggest that poly(A) may be required for efficient initiation and reinitiation of protein synthesis in the cell-free systems. This interpretation is discussed relative to earlier data.

Animals↗
PubMed1978

Diminished sensitivity of re-initiation of translation to inhibition by cap analogues in reticulocyte lysates.

In a nuclease-treated reticuloyte lysate reinitiation of protein biosynthesis was less inhibited by 7-methylguanosine 5'-monophosphate (m7GMP), whereas primary-initiation could be totally blocked by cap analogues. We suggest that a cap binding factor binds to mRNA as a first step in initiation of protein synthesis and that this factor remains bound during the subsequent reinitiation cycles. Primarily because of the greater relative importance of the reinitiation process, globin synthesis in the untreated lysate was less inhibited by m7GMP.

Animals↗
PubMed1979

Genetic evidence for the creation of a reinitiation site by mutation inside the yeast ura 2 gene.

The ura 2 gene of yeast codes for two enzymatic activities which are translated from a unique messenger RNA in the order carbamoyl-phosphate synthetase (CPSase), aspartate transcarbamylase (ATCase) (Lacroute, 1968; Denis-Duphil and Kaplan, 1976). Nonsense mutations in the CPSPase region cause a complete loss in ATCase activity by a total polar effect, characteristic of eukaryotic mRNA translation, and due to the unique site of protein initiation present on each messenger (Shaffer et al., 1969). A triple nonsense mutant in the CPSase has been constructed by recombination and ATCase+ revertants have been selected from it. Among seventeen revertants obtained, three had a deletion covering the three nonsense mutations relieving thus the polar effect (Fink and Styles, 1974) but fourteen others examined had retained all the CPSase DNA including the three nonsense mutations; this can be explained in the present state of knowledge only by the creation by mutation of reinitiation site either for transcription or for translation in the region of the ura 2 gene distal to the last nonsense mutation.

Aspartate Carbamoyltransferase↗
PubMedDate not supplied

[Initiation in a polyribosome-dependent protein-synthesizing cell-free system from Saccharomyces (author's transl)].

A method to prepare polyribosomes from yeasts by using the french-press is described. The highest yield of polyribosomes was derived from late log-phase cells. These polyribosomes, incubated in a cell-free system, were able to reinitiate protein synthesis, which was shown by inhibiting aminoacid incorporation by aurintricarboxylic acid, edeine and sodiumfluoride. We developed the translational system in order to look for the optimal ion-conditions of a DNA-dependent protein-synthesizing system. We found out that at the optimal MgCl2-concentration (6 mM) protein synthesis was strongly inhibited by Mangan ions which are required for transcription in yeast. If protein-synthesis was carried out with 2 mM and 3 mM MgCl2 maximal aminoacid incorporation was observed at 2 mM and 1.5 mM MnCl2.

Aurintricarboxylic Acid↗
PubMed1978

Formylation of initiator tRNA methionine in procaryotic protein synthesis: in vivo polarity in lactose operon expression.

Eucaryotic and procaryotic organisms differ in two aspects of their translation machinery: polycistronic messengers are expressed as a sequence of individual proteins only in procaryotes, and the initiation of protein synthesis proceeds with an initiator tRNA which is found to be modified (formylated) in procaryotes and not in eucaryotes. In the present study, we show that formylation is required in vivo for the coordinate expression of the Escherichia coli lactose operon. Our experiments are consistent with a translation mechanism using dissociated ribosomes at the 5' end of the mRNA in a reaction that is only weakly dependent on formylation at this initiation step; the ribosomes then travel along the messenger and can reinitiate after the intracistronic barrier without dissociation. This latter initiation step is strongly dependent on the level of formylation: a low level of the formyl group, obtained by the antifolic agent trimethoprim, induces a strong polarity in the expression of the lactose operon. There exist mutant strains in which this polarity is much less apparent than in the wild type. We show here that such is the case of rpsL mutants. Ribosomes mutated in the S12 protein (rpsL) are found to be much more easily dissociated than the wild type. This might explain why the expression of the lactose operon on rpsL strains remains coordinated when the intracellular level of formylation is decreased.

Acetyltransferases↗
PubMed1979

Effect of blocking protein synthesis at nonpermissive temperatures on temperature-sensitive deoxyribonucleic acid mutants of Escherichia coli.

When protein synthesis was blocked in temperature-sensitive deoxyribonucleic acid synthesis mutants of Escherichia coli at nonpermissive temperatures, it reduced the amount of apparent subsequent chain elongation to approximately half that observed in the mutants either at nonpermissive temperatures alone or when protein synthesis was blocked at the permissive temperature. Blocking protein synthesis at the nonpermissive temperatures for periods of 40 min caused the loss of ability to reinitiate deoxyribonucleic acid synthesis at the permissive temperature.

Amino Acids↗
PubMed1978

The effects of tiamulin, a semisynthetic pleuromutilin derivative, on bacterial polypeptide chain initiation.

Tiamulin, a water-soluble and highly effective semisynthetic derivative of pleuromutilin leads to the formation of physiologically inactive polypeptide chain initiation complexes which readily decompose and do not enter the phase of peptide chain elongation. Once elongation has begun it continues even in the presence of tiamulin as has been shown by measuring the formation of N-acetylphenylalanine-poly(phenylalanine). The formation of abortive initiation complexes was observed regardless of whether AcPhe-tRNA of fMet-tRNA was used as an initiator or whether artificial messengers or a natural messenger, like R17 bacteriophage RNA, was used. When this drug was acting on whole cells, it led to the disappearance of polysomes. The only structures which could be detected were of the monosome size. Therefore, polysomes seem to elongate the polypeptide chains in whole cells in the presence of this antibiotic, but since effective reinitiation is blocked, the polysome pool of the cell soon becomes depleted.

Anti-Bacterial Agents↗
PubMed1975

Estrogenic inhibition of the hepatic synthesis of alpha2u globulin in the rat.

The hepatic synthesis of the androgen-dependent urinary protein in the rat, called alpha2u globulin, is strongly inhibited by estrogens. In mature male rats, treatment with estradiol-17beta (0.5 mug/g body weight) completely inhibits alpha2u synthesis within 6-7 days. Following withdrawal of estrogen treatment alpha 2u synthesis is not reinitiated for approximately 20 days. Parabiotic joining of estrogen-suppressed male rats with their normal littermates within this lag period fails to change the preparabiotic pattern of alpha2u synthesis in the respective partners. Besides estradiol-17beta, other estrane derivatives such as estrone, estriol and estradiol-17alpha were also found to inhibit the synthesis of alpha2u globulin. All of the above estrane derivatives which inhibit alpha2u synthesis are also found to inhibit the uptake of 5alpha-dihydrotestosterone by the hepatic cytosol androgen binding protein of the mature male rat. Unlike cycloheximide, a known translational inhibitor, estradiol-17beta does not inhibit alpha 2u synthesis in the perfused rat liver.

Alpha-Globulins↗