Targeting of oleosins to the oil bodies of oilseed rape (Brassica napus L.)

Oleosins of Brassica napus L. (oilseed rape) synthesized by in-vitro translation were found to be very efficiently targeted to microsomal membranes but only poorly translocated to oil bodies or emulsified oil. The use of other bilayer membranes as controls showed that this interaction was specific. The rate of oleosin synthesis in the presence of microsomes was enhanced about threefold, indicative of the involvement of the signal-recognition particle in the targeting process. There is no evidence for the cleavage of the protein during targeting and the protein sequence reveals no consensus cleavage site for the signal peptide. Protection experiments using Proteinase K revealed that about 6 kDa of the protein is exposed on the cytoplasmic side of the ER but the remainder is protected. Carbonate (pH 11) washing of microsomal membranes after in-vitro translation confirmed that oleosins have a domain which remains inserted in the ER rather than the protein being transported completely into the lumen of the ER. These results indicate that oleosins are transported via the ER prior to their accumulation on oil bodies.     

Changes in DNA strand breaks in non parenchymal cells following hepatocyte regeneration in CCl4-induced rat liver injury

DNA strand breaks (nicks) in non-parenchymal cells (NPCs) in CCl₄-induced acute or chronic liver injury in rats were detected using an in situ nick translation method; their dynamic changes were analysed in relation to the proliferation pattern of hepatocytes and NPCs, as revealed by bromodeoxyuridine (BrdU)-up-take. In acute injury, hepatocyte proliferation started before centrilobular necrosis had occurred, whereas BrdU-labeled sinusoidal NPCs markedly increased only after centrilobular necrosis was apparent. DNA breakages in NPCs paralleled the proliferation pattern of these cells, suggesting that nicks are physiological, and reflect proliferation and activated gene expression. In chronic injury, liver cirrhosis developed after 9 weeks, but BrdU-labeling of hepatocytes was almost the same level as that in untreated liver. The number of BrdU-labeled NPCs showed only a slight increase, while those with DNA breakages were much more frequent in the cirrhotic stage, suggesting a significant role for NPCs in the fibrotic process. These results indicate that DNA strand breaks in NPCs act as a marker for activation states such as proliferation, differentiation and/or activated gene expression.     

Identification of potential proteins translated from circular RNA splice variants

Circular RNAs (circRNAs) are covalently closed RNA molecules generated from precursor RNAs by the head-to-tail backsplicing of exons. Hundreds of studies demonstrated that circRNAs are ubiquitously expressed and regulate cellular events by modulating microRNA (miRNA) and RNA-binding protein (RBP) activities. A few circRNAs are also known to translate into functional polypeptides regulating cellular physiology. All these functions primarily depend on the full-length sequence of the circRNAs. CircRNA backsplice junction sequence is the key to identifying circRNAs and their full-length mature sequence. However, some multi-exonic circRNAs exist in different isoforms sharing identical backsplice junction sequences and are termed circRNA splice variants. Here, we analyzed the previously published HeLa cell RNA-seq datasets to identify circRNA splice variants using the de novo module of the CIRCexplorer2 circRNA annotation pipeline. A subset of circRNAs with splice variants was validated by the circRNA-rolling circle amplification (circRNA-RCA) method. Interestingly, several validated circRNAs were predicted to translate into proteins by the riboCIRC database. Furthermore, polyribosome fractionation followed by quantitative PCR confirmed the association of a subset of circRNAs with polyribosome supporting their protein-coding potential. Finally, bioinformatics analysis of proteins derived from splice variants of circCORO1C and circASPH suggested altered protein sequences and structures that could affect their physiological functions. Together, our study identified novel circRNA splice variants and their potential translation into protein isoforms which may regulate various physiological processes.     

The sequence surrounding the translation initiation codon of the pea plastocyanin gene increases translational efficiency of a reporter gene

The 5-upstream region of the pea plastocyanin gene (petE) directed 5–10-fold higher levels of -glucuronidase (GUS) activity than the cauliflower mosaic virus 35S promoter in transgenic tobacco plants, although the levels of GUS mRNA were similar. The sequence (AAAAAUGG) around the translation initiation codon of petE enhanced translation of the GUS mRNA 10-fold compared to translation from the GUS translation initiation codon in transgenic tobacco plants and transfected protoplasts.     

Generation of anNco I restriction site for translational fusions using PCR-mediated,site-directed mutagenesis

A polymerase chain reaction-based method of site-directed mutagenesis was used to introduce anNco I restriction site on the translation start site of a tomato peroxidase gene. This quick and efficient method utilized two overlapping synthetic oligonucleotide primers containing the requisite base pair changes on the ATG translation start site and two flanking primers in PCR. The resulting DNA amplified fragments were fused together byNco I digestion at the mutated ends followed by a T4 ligation reaction. A rapid alternative method utilizing the overlapping fragments and the flanking primers in PCR can also be used for ligating the two fragments. Cloning and sequencing of the PCR-amplified fragments provided additional evidence for the presence of the site-specific mutations. Unique restriction sites upstream and downstream of the site-specific mutation allows for the easy transfer of this mutated region into the wild type peroxidase gene.     

Poly(A)-containing RNA in early embryogenesis of sea urchins

The content, biosynthesis and template activity of poly(A)+ RNA in the early stages of sea urchin development have been studied. The amount of poly(A)+ RNA reaches a maximum at the middle blastula stage in polyribosomes and at the 8-blastomere stage in the cytoplasm. Poly(A)+ RNA synthesis becomes noticeable at the 64-blastomere stage and the spectrum of newly synthesized molecules is different from that at the middle blastula stage. The products of translation in vitro of poly(A)+ RNA at all the stages studied show insignificant differences and contain a major group of polypeptides of molecular mass 10-20 kDa.     

Synthesis of subunit III of CF0 by thylakoid-bound polysomes from pea chloroplasts

Summary Wahsed thylakoid membranes from pea chloroplasts incorporate label from (³⁵S)-methionine into protein when supplemented with S-30 soluble factors from E. coli. One of the products associated with the thylakoids is soluble in butanol, precipitated by ether and has an apparent molecular mss of 8200D on urea-lithium dodecyl sulphate (LDS) polyacrylamide gels. In addition, the protein covalently binds dicyclohexylcarbo-diimide (DCCD) which causes it to migrate as two slower forms on gels. Based on these criteria we establish that the proteolipid or subunit III of CF₀ (the intrinsic sector of the ATPase complex) is synthesized by the thylakoid bound polysomes.     

Post-translational processing of 7S and 11S components of soybean storage proteins

The synthesis and processing of the major storage proteins in soybean cotyledons was studied both in vivo and in vitro. The and subunits of 7S as well as the 11S proteins are synthesized as higher molecular weight-precursors on membrane-bound polysomes. The initial translation products of the 7S are proteolytically cleaved during translation suggesting the removal of a signal peptide as evidenced by the presence of 2 and 2 peptides immunoreactive with 7S antibody in the in vitro chain completion products of the membrane-bound polysomes. This is followed or accompanied by cotranslational glycosylation, which increases their size equivalent to that of initially-synthesized precursors. In vivo pulse-labelled 7S and products are of slightly higher molecular weights than the immunoprecipitable chain-completion products, indicating further post-translational modifications. A slow post-translational processing during a period of 1.5 to 16 h yields the final 7S and glycoproteins.Acidic and basic subunits of the 11S protein appear to be synthesized from common large molecular weight (60K-59K) precursors. Antibodies to the 11S acidic component recognize both acidic and basic domains in the precursor while those raised against basic subunits appear to be specific for that region only. The processing of the 11S precursor is also very slow and occurs post-translationally. This slow rate of processing, coupled with a temporal difference in the synthesis of 7S and 11S components, suggests a highly coordinated mechanism for synthesis and packaging of these proteins into protein bodies during seed development.