Fully edited and partially edited nad9 transcripts differ in size and both are associated with polysomes in potato mitochondria.
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ABSTRACT:
SUBMITTER: Lu B
PROVIDER: S-EPMC145777 | biostudies-other | 1996 Apr
REPOSITORIES: biostudies-other
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Project description:The pool of partially and completely edited mRNAs present in plant mitochondria could potentially be translated into a mixture of divergent proteins. This possibility was investigated for the nad9 gene in potato by characterization of the mRNA population and the corresponding protein sequence. The deduced amino acid sequence of the nad9 gene product has significant similarity to the nuclear-encoded 30 kDa subunit of the bovine and Neurospora NADH:ubiquinone oxidoreductase (complex I) and to the chloroplast ndhJ gene product. Immunoprecipitation of a 27 kDa in-organello 35S labelled mitochondrial translation product with an antibody directed against the wheat nad9 gene product demonstrates its functional expression in potato and wheat. Comparison of the nad9 genomic DNA and cDNA sequences reveals seven codons to be changed by a C to U RNA-editing. Direct sequencing of RT-PCR products derived from cDNAs of different tissues of potato plants shows the presence of a significant portion of only partially edited nad9 transcripts in the various tissues. Amino acid sequencing of internal peptides of the isolated 27 kDa protein from potato tubers demonstrates homogenous translation products of only completely edited nad9 mRNAs even in the presence of partially edited mRNAs. This result suggests a pretranslational selection between edited and incompletely edited mRNAs in plant mitochondria.
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Project description:Eukaryotic translation initiation factor 3 (eIF3) plays a central role in translation initiation and consists of five core (conserved) subunits present in both budding yeast and higher eukaryotes. Higher eukaryotic eIF3 contains additional (noncore or nonconserved) subunits of poorly defined function, including sub-unit h (eIF3h), which in zebrafish is encoded by two distinct genes (eif3ha and eif3hb). Previously we showed that eif3ha encodes the predominant isoform during zebrafish embryogenesis and that depletion of this factor causes defects in the development of the brain and eyes. To investigate the molecular mechanism governing this regulation, we developed a genome-wide polysome-profiling strategy using stage-matched WT and eif3ha morphant zebrafish embryos. This strategy identified a large set of predominantly neural-associated translationally regulated mRNAs. A striking finding was a cohort of lens-associated crystallin isoform mRNAs lost from the eif3ha morphant polysomes, revealing a mechanism by which lens development is translationally controlled. We show that both UTR sequences of a targeted crystallin transcript are necessary but not sufficient for translational regulation by eif3ha. Therefore, our study reveals the role of a noncore eIF3 subunit in modulating a specific developmental program by regulating translation of defined transcripts and highlights the potential of the zebrafish system to identify translational regulatory mechanisms controlling vertebrate development.
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Project description:We have studied partially edited molecules for the cytochrome-c oxidase subunit III (COIII) transcript from two species of the insect trypanosome Herpetomonas. We found unexpected patterns of editing, in which editing does not proceed strictly 3' to 5', in 24 of 61 partially edited clones. A comparison of the partially edited molecules between the two kinetoplastid species revealed an 8- to 10-nt shift in precisely defined editing boundaries, sites at which editing pauses before binding of the next guide RNA after formation of a stable duplex between a guide RNA and mRNA. This suggests that the region of base pairing between individual guide RNAs and the COIII transcript is not strictly conserved in kinetoplastids, implying gradual evolution of the editing process.
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2008-05-28 | GSE10871 | GEO
Project description:In Cebus apella monkey, as with other mammalian species tested to date, two different forms of haem oxygenase, HO-1 and HO-2, are detected. With the use of cDNA fragment corresponding to HO-1 nucleotides +71 to +833, blot hybridization of RNA revealed the presence of only one HO-1 mRNA of approx. 1.8 kb in both rat and monkey liver, kidney and brain. With the use of a full-length HO-2 DNA probe, blot hybridization of RNA isolated from the same rat organs revealed the presence of two HO-2 homologous transcripts of approx. 1.3 kb and approx. 1.9 kb. The same probe detected only one message of approx. 1.7 kb in monkey organs. The rat 1.3 kb mRNA has been previously shown [Rotenberg & Maines (1990) J. Biol. Chem. 265, 7501-7506] to encode HO-2 (36 kDa). The monkey 1.7 kb mRNA and the rat 1.3 kb mRNA encode proteins with similar molecular masses and immunochemical properties as indicated by Western-immunoblotting analysis. In rat organs the relative abundance of the two mRNAs differed as follows: in the liver the 1.3 kb mRNA was by far the most abundant form; in the brain equal amounts of the two mRNAs were detected, whereas in the kidney the 1.3 kb mRNA was somewhat more abundant. The protein encoded by the 1.8 kb HO-1 mRNA in the monkey did not exhibit immunochemical reactivity with antibody to rat HO-1 in Western blotting and direct e.l.i.s.a. analysis. The data suggest that, at the primary structural level, both HO-1 and HO-2 share extensive base sequence similarity in the rat and the Cebus apella monkey. The HO-1 protein, however, appears to undergo differential post-translational and/or conformational modifications in the two species, whereas the secondary structure of HO-2 protein and antigenic epitopes are conserved among the two mammalian species.
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Project description:Penguins face a major thermal transition when returning to land in a hypothermic state after a foraging trip. Uninsulated appendages (flippers and feet) could provide flexible heat exchange during subsequent rewarming. Here, we tested the hypothesis that peripheral vasodilation could be delayed during this recovery stage. To this end, we designed an experiment to examine patterns of surface rewarming in fully hypothermic (the cloaca and peripheral regions (here; flippers, feet and the breast) < 37 °C) and partially hypothermic (cloaca at normothermia ≥ 37 °C, but periphery at hypothermia) king penguins (Aptenodytes patagonicus) when they rewarmed in the laboratory. Both groups rewarmed during the 21 min observation period, but the temperature changes were larger in fully than in partially hypothermic birds. Moreover, we observed a 5 min delay of peripheral temperature in fully compared to partially hypothermic birds, suggesting that this process was impacted by low internal temperature. To investigate whether our laboratory data were applicable to field conditions, we also recorded surface temperatures of free-ranging penguins after they came ashore to the colony. Initial surface temperatures were lower in these birds compared to in those that rewarmed in the laboratory, and changed less over a comparable period of time on land. This could be explained both by environmental conditions and possible handling-induced thermogenesis in the laboratory. Nevertheless, this study demonstrated that appendage vasodilation is flexibly used during rewarming and that recovery may be influenced by both internal temperature and environmental conditions when penguins transition from sea to land.
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Project description:Menaquinones (MKs) contain both a redox active quinone moiety and a hydrophobic repeating isoprenyl side chain of varying lengths and degrees of saturation. This characteristic structure allows MKs to play a key role in the respiratory electron transport system of some prokaryotes by shuttling electrons and protons between membrane-bound protein complexes, which act as electron acceptors and donors. Hydrophobic MK molecules with partially and fully saturated isoprenyl side chains are found in a wide range of eubacteria and archaea, and the structural variations of the MK analogues are evolutionarily conserved but poorly understood. For example, Mycobacterium tuberculosis, the causative agent of tuberculosis, uses predominantly MK-9(II-H2) (saturated at the second isoprene unit) as its electron carrier and depends on the synthesis of MK-9(II-H2) for survival in host macrophages. Thus, MKs with partially saturated isoprenyl side chains may represent a novel virulence factor. Naturally occurring longer MKs are very hydrophobic, whereas MK analogues that have a truncated (i.e., one to three isoprenes) isoprenyl side chain are less hydrophobic. This improves their solubility in aqueous solutions, allowing rigorous study of their structure and biological activity. We present the synthesis and characterization of two partially saturated MK analogues, MK-2(II-H2) and MK-3(II-H2), and two novel fully saturated MK derivatives, MK-2(I,II-H4) and MK-3(I,II,III-H6).
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Project description:Porcine Reproductive and Respiratory Syndrome (PRRS) is a panzootic infectious disease of pigs, causing major economic losses to the world-wide pig industry. PRRS manifests differently in pigs of all ages but primarily causes late-term abortions and stillbirths in sows and respiratory disease in piglets. The causative agent of the disease is the positive-strand RNA PRRS virus (PRRSV). PRRSV has a narrow host cell tropism, limited to cells of the monocyte/macrophage lineage. CD163 has been described as a fusion receptor for PRRSV, whereby the scavenger receptor cysteine-rich domain 5 (SRCR5) region was shown to be an interaction site for the virus in vitro. CD163 is expressed at high levels on the surface of macrophages, particularly in the respiratory system. Here we describe the application of CRISPR/Cas9 to pig zygotes, resulting in the generation of pigs with a deletion of Exon 7 of the CD163 gene, encoding SRCR5. Deletion of SRCR5 showed no adverse effects in pigs maintained under standard husbandry conditions with normal growth rates and complete blood counts observed. Pulmonary alveolar macrophages (PAMs) and peripheral blood monocytes (PBMCs) were isolated from the animals and assessed in vitro. Both PAMs and macrophages obtained from PBMCs by CSF1 stimulation (PMMs) show the characteristic differentiation and cell surface marker expression of macrophages of the respective origin. Expression and correct folding of the SRCR5 deletion CD163 on the surface of macrophages and biological activity of the protein as hemoglobin-haptoglobin scavenger was confirmed. Challenge of both PAMs and PMMs with PRRSV genotype 1, subtypes 1, 2, and 3 and PMMs with PRRSV genotype 2 showed complete resistance to viral infections assessed by replication. Confocal microscopy revealed the absence of replication structures in the SRCR5 CD163 deletion macrophages, indicating an inhibition of infection prior to gene expression, i.e. at entry/fusion or unpacking stages.
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Project description:Creating accurate maps of A-to-I RNA editing activity is vital to improving our understanding of the biological role of this process and harnessing it as a signal for disease diagnosis. Current RNA sequencing techniques are susceptible to random sampling limitations due to the complexity of the transcriptome and require large amounts of RNA material, specialized instrumentation, and high read counts to accurately interrogate A-to-I editing sites. To address these challenges, we show that Escherichia coli Endonuclease V (eEndoV), an inosine-cleaving enzyme, can be repurposed to bind and isolate A-to-I edited transcripts from cellular RNA. While Mg2+ enables eEndoV to catalyze RNA cleavage, we show that similar levels of Ca2+ instead promote binding of inosine without cleavage and thus enable high affinity capture of inosine in RNA. We leverage this capability to demonstrate EndoVIPER-seq (Endonuclease V inosine precipitation enrichment sequencing) as a facile and effective method to enrich A-to-I edited transcripts prior to RNA-seq, producing significant increases in the coverage and detection of identified editing sites. We envision the use of this approach as a straightforward and cost-effective strategy to improve the epitranscriptomic informational density of RNA samples, facilitating a deeper understanding of the functional roles of A-to-I editing.
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