Chloroplast transfer RNAs and tRNA genes of wheat

Fractionation (by two-dimensional polyacrylamide gel electrophoresis) of total tRNA from wheat chloroplasts yields about 33 RNA spots. Of these, 30 have been identified by aminoacylation as containing tRNAs specific for 17 amino acids. Hybridization of labeled individual tRNAs to cloned chloroplast DNA fragments has revealed the location of at least nine pairs of tRNA genes in the segments of the inverted repeat, at least twelve tRNA genes in the large single copy region and one tRNA gene in the small single copy region. A comparison of this wheat chloroplast tRNA gene map to that of maize and of other higher plants suggests that gene rearrangements have occurred during evolution, even within cereal chloroplast DNA. These rearrangements have taken place within the inverted repeat, within the large single copy region and between the inverted repeat and the large single copy region.     

Sequences of three transfer RNAs from mosquito mitochondria

The sequences of three transfer RNAs from mosquito cell mitochondria, tRNA_UCG^Arg, tRNA_GUC^Asp, and tRNA_GAU^Ile, determined using a combination of rapid ladder and fingerprinting procedures are reported. These were compared with hamster mitochondrial tRNA_UCG^Arg and tRNA_GUC^Asp determined similarly, and a bovine mitochondrial tRNA_GAU^Ile determined using a somewhat different approach. The primary sequences of the mosquito tRNAs were 35 to 65% homologous to the corresponding mammalian mitochondrial species, and bore little homology to “conventional” (bacterial or eucaryotic cytoplasmic) tRNA. The modification status of the mosquito mitochondrial tRNAs resembled that of mammalian mitochondrial tRNA. The results contribute to the generalization that metazoan mitochondrial tRNA constitutes a distinctive, albeit loosely structured, phylogenetic group.     

Identification of small non-coding RNAs from mitochondria and chloroplasts

Small non-protein-coding RNAs (ncRNAs) have been identified in a wide spectrum of organisms ranging from bacteria to humans. In eukarya, systematic searches for ncRNAs have so far been restricted to the nuclear or cytosolic compartments of cells. Whether or not small stable non-coding RNA species also exist in cell organelles, in addition to tRNAs or ribosomal RNAs, is unknown. We have thus generated cDNA libraries from size-selected mammalian mitochondrial RNA and plant chloroplast RNA and searched for small ncRNA species in these two types of DNA-containing cell organelles. In total, we have identified 18 novel candidates for organellar ncRNAs in these two cellular compartments and confirmed expression of six of them by northern blot analysis or RNase A protection assays. Most candidate ncRNA genes map to intergenic regions of the organellar genomes. As found previously in bacteria, the presumptive ancestors of present-day chloroplasts and mitochondria, we also observed examples of antisense ncRNAs that potentially could target organelle-encoded mRNAs. The structural features of the identified ncRNAs as well as their possible cellular functions are discussed. The absence from our libraries of abundant small RNA species that are not encoded by the organellar genomes suggests that the import of RNAs into cell organelles is of very limited significance or does not occur at all.     

Identification and characterization of receptors for protein import into chloroplasts and mitochondria

An anti-idiotypic antibody approach was used to identify chloroplast and mitochondrial protein component(s) which interact with the corresponding signal sequence. The proteins thus identified can be operationally defined as receptor(s) for import of proteins into chloroplasts and mitochondria. The import receptor(s) was found in "contact sites" between the outer and inner membrane of chloroplast envelope or of mitochondria.     

Arabidopsis thaliana ferrochelatase-I and -II are not imported into Arabidopsis mitochondria

Using in vitro import assays into purified mitochondria and chloroplasts we found that Arabidopsis ferrochelatase-I and ferrochelatase-II were not imported into mitochondria purified from Arabidopsis (or several other plants) but were imported into pea leaf chloroplasts. Other dual targeted proteins could be imported into purified mitochondria from Arabidopsis. As only two ferrochelatase genes are present in the completed Arabidopsis genome, the presence of ferrochelatase activity in plant mitochondria needs to be re-evaluated. Previous reports of Arabidopsis ferrochelatase-I import into pea mitochondria are due to the fact that pea leaf (and root) mitochondria appear to import a variety, but not all chloroplast proteins. Thus pea mitochondria are not a suitable system to either study dual targeting, or to distinguish between isozymes present in mitochondria and chloroplasts.     

Multiple physical forms of excised group II intron RNAs in wheat mitochondria

Plant mitochondrial group II introns do not all possess hallmark ribozymic features such as the bulged adenosine involved in lariat formation. To gain insight into their splicing pathways, we have examined the physical form of excised introns in germinating wheat embryos. Using RT–PCR and cRT–PCR, we observed conventional lariats consistent with a two-step transesterification pathway for introns such as nad2 intron 4, but this was not the case for the cox2 intron or nad1 intron 2. For cox2, we detected full-length linear introns, which possess non-encoded 3′terminaladenosines, as well as heterogeneous circular introns, which lack 3′ nucleotide stretches. These observations are consistent with hydrolytic splicing followed by polyadenylation as well as an in vivo circularization pathway, respectively. The presence of both linear and circular species in vivo is supported by RNase H analysis. Furthermore, the nad1 intron 2, which lacks a bulged nucleotide at the branchpoint position, comprised a mixed population of precisely full-length molecules and circular ones which also include a short, discrete block of non-encoded nucleotides. The presence of these various linear and circular forms of excised intron molecules in plant mitochondria points to multiple novel group II splicing mechanisms in vivo.     

Identification and characterization of respirasomes in potato mitochondria

Plant mitochondria were previously shown to comprise respiratory supercomplexes containing cytochrome c reductase (complex III) and NADH dehydrogenase (complex I) of I(1)III(2) and I(2)III(4) composition. Here we report the discovery of additional supercomplexes in potato (Solanum tuberosum) mitochondria, which are of lower abundance and include cytochrome c oxidase (complex IV). Highly active mitochondria were isolated from potato tubers and stems, solubilized by digitonin, and subsequently analyzed by Blue-native (BN) polyacrylamide gel electrophoresis (PAGE). Visualization of supercomplexes by in-gel activity stains for complex IV revealed five novel supercomplexes of 850, 1,200, 1,850, 2,200, and 3,000 kD in potato tuber mitochondria. These supercomplexes have III(2)IV(1), III(2)IV(2), I(1)III(2)IV(1), I(1)III(2)IV(2), and I(1)III(2)IV(4) compositions as shown by two-dimensional BN/sodium dodecyl sulfate (SDS)-PAGE and BN/BN-PAGE in combination with activity stains for cytochrome c oxidase. Potato stem mitochondria include similar supercomplexes, but complex IV is partially present in a smaller version that lacks the Cox6b protein and possibly other subunits. However, in mitochondria from potato tubers and stems, about 90% of complex IV was present in monomeric form. It was suggested that the I(1)III(2)IV(4) supercomplex represents a basic unit for respiration in mammalian mitochondria termed respirasome. Respirasomes also occur in potato mitochondria but were of low concentrations under all conditions applied. We speculate that respirasomes are more abundant under in vivo conditions.     

Fractional and structural characterization of wheat straw hemicelluloses

Six hemicellulosic fractions were extracted successively from dewaxed wheat straw with sodium hydroxide at increasing strength from 0.25 to 2.00M, and the chemical composition are reported. The structure of the hemicellulosic fraction 2 was investigated using acid hydrolysis, methylation analysis and ¹³C-NMR experiments. The hemicelluloses were confirmed to be a (1→4)-linked β-D-xylan with D-glucopyranosyluronic acid (or 4-O-methyl--D-glucopyranosyluronic acid) group attached at position 2, and L-arabinofuranosyl and D-xylopyranosyl groups attached at position 3. For every 26 D-xylopyranosyl residues in the main chain, there was one uronic acid unit. For 13 such D-xylopyranosyl residues, there was one L-arabinofuranosyl group, and for 18 such D-xylopyranosyl residues, there was one D-xylopyranosyl group.     

A nuclear encoded tRNA of Trypanosoma brucei is imported into mitochondria.

The mitochondrial genome of trypanosomes, unlike that of most other eukaryotes, does not appear to encode any tRNAs. Therefore, mitochondrial tRNAs must be either imported into the organelle or created through a novel mitochondrial process, such as RNA editing. Trypanosomal tRNA(Tyr), whose gene contains an 11-nucleotide intron, is present in both the cytosol and the mitochondrion and is encoded by a single-copy nuclear gene. By site-directed mutagenesis, point mutations were introduced into this tRNA gene, and the mutated gene was reintroduced into the trypanosomal nuclear genome by DNA transfection. Expression of the mutant tRNA led to the accumulation of unspliced tRNA(Tyr) (A. Schneider, K. P. McNally, and N. Agabian, J. Biol. Chem. 268:21868-21874, 1993). Cell fractionation revealed that a significant portion of the unspliced mutant tRNA(Tyr) was recovered in the mitochondrial fraction and was resistant to micrococcal nuclease treatment in the intact organelle. Expression of the nuclear integrated, mutated tRNA gene and recovery of its gene product in the mitochondrial fraction directly demonstrated import. In vitro experiments showed that the unspliced mutant tRNA(Tyr), in contrast to the spliced wild-type form, was no longer a substrate for the cognate aminoacyl synthetase. The presence of uncharged tRNA in the mitochondria demonstrated that aminoacylation was not coupled to import.     

Identification and characterization of endogenous small interfering RNAs from rice

RNA silencing-mediated small interfering RNAs (siRNAs) and microRNAs (miRNAs) have diverse natural roles, ranging from regulation of gene expression and heterochromatin formation to genome defense against transposons and viruses. Unlike miRNAs, endogenous siRNAs are generally not conserved between species; consequently, their identification requires experimental approaches. Thus far, endogenous siRNAs have not been reported from rice, which is a model species for monocotyledonous plants. We identified a large set of putative endogenous siRNAs from root, shoot and inflorescence small RNA cDNA libraries of rice. Most of these siRNAs are from intergenic regions, although a substantial proportion (22%) originates from the introns and exons of protein-coding genes. Northern and RT–PCR analysis revealed that the expression of some of the siRNAs is tissue specific or developmental stage specific. A total of 25 transposons and 21 protein-coding genes were predicted to be cis-targets of some of the siRNAs. Based on sequence homology, we also predicted 111 putative trans-targets for 44 of the siRNAs. Interestingly, ~46% of the predicted trans-targets are transposable elements, which suggests that endogenous siRNAs may play an important role in the suppression of transposon proliferation. Using RNA ligase-mediated-5′ rapid amplification of cDNA end assays, we validated three of the predicted targets and provided evidence for both cis- and trans-silencing of target genes by siRNAs-guided mRNA cleavage.     

Identification and characterization of tumorigenic circular RNAs in cervical cancer

Circular RNAs (circRNAs) are a distinctive family of ncRNAs, and they function as key regulators in the initiation, development and progression of various diseases. However, the regulatory roles of circRNAs in the tumorigenesis of cervical cancer (CC) have not been fully understood. In this study, we identified a set of circRNAs in CC and paired normal tissues, using RNA sequencing data, and found that the cancer and normal tissues could be told apart by those differentially expressed (DE) circRNAs, indicating that circRNA expression profiles in CC were significantly different from those in the normal tissues. Meanwhile, the upregulated genes in CC were enriched in inflammation-related pathways, and the correlation analysis between the DE circRNAs and genes revealed that the abundance of DE circRNAs was positively related to the expression of their host genes. However, the expression of TGFBR2 and KDM4C were found to exhibit a negative correlation with their corresponding circRNAs. Furthermore, we also predicted the interactions between circRNAs and proteins, and constructed a competing endogenous RNA (ceRNA) network. Specifically, hsa_circ_0001495 was predicted to interact with ESRP2, and acted as a sponge by competing for miRNAs with TBL1XR1. Functionally, hsa_circ_0001495 was predicted to regulate epithelial cell proliferation and NOTCH signaling via ESRP2 and TBL1XR1, respectively. We also evaluated the prognostic values of downstream target genes of selected circRNAs, using clinical records of CC patients. In summary, the present study provided some regulatory circRNAs involved in CC tumorigenesis based on bioinformatics approaches, which brought strong evidences for researchers to further explore their biological and clinical values.