RNA methyltransferases utilize two cysteine residues in the formation of 5-methylcytosine

Proteins that have sequence homology with known RNA m(5)C methyltransferases contain two conserved cysteines, each of which lies within a sequence that bears similarity to a methyltransferase active site. Other enzymes that transfer a methyl group to carbon 5 of a pyrimidine nucleotide, such as the bacterial DNA m(5)C methyltransferases, utilize their single conserved cysteine residue to form a covalent Michael adduct with carbon 6 of the pyrimidine ring during catalysis. We present a model for the utilization of two cysteines in catalysis by RNA m(5)C methyltransferases. It is proposed that one thiol acts in a classical fashion by forming a covalent link to carbon 6 of the pyrimidine base, while the other cysteine assists breakdown of the covalent adduct. Therefore, alteration of the assisting cysteine is anticipated to stabilize the covalent enzyme-RNA intermediate. The model was conceived as a possible explanation for the effects of mutations that change the conserved cysteines in Nop2p, an apparent RNA m(5)C methyltransferase that is essential for ribosome assembly and yeast viability. Evidence for the predicted accumulation of protein-RNA complexes following mutation of the assisting cysteine has been obtained with Nop2p and a known tRNA m(5)C methyltransferase called Ncl1p (Trm4).     

Reconstructing the coding and non-coding RNA regulatory networks of miRNAs and mRNAs in breast cancer

microRNAs (miRNAs) are a class of small non-coding RNAs that deregulate and/or decrease the expression of target messenger RNAs (mRNAs), which specifically contribute to complex diseases. In our study, we reanalyzed an integrated data to promote classification performance by rebuilding miRNA–mRNA modules, in which a group of deregulated miRNAs cooperatively regulated a group of significant mRNAs. In five-fold cross validation, the multiple processes flow considered the biological and statistical significant correlations. First, of statistical significant miRNAs, 6 were identified as core miRNAs. Second, in the 13 significant pathways enriched by gene set enrichment analysis (GSEA), 705 deregulated mRNAs were found. Based on the union of predicted sets and correlation sets, 6 modules were built. Finally, after verified by test sets, three indexes, including area under the ROC curve (AUC), Accuracy and Matthews correlation coefficients (MCCs), indicated only 4 modules (miR-106b-CIT-KPNA2-miR-93, miR-106b-POLQ-miR-93, miR-107-BTRC-UBR3-miR-16 and miR-200c-miR-16-EIF2B5-miR-15b) had discriminated ability and their classification performance were prior to that of the single molecules. By applying this flow to different subtypes, Module 1 was the consistent module across subtypes, but some different modules were still specific to each subtype. Taken together, this method gives new insight to building modules related to complex diseases and simultaneously can give a supplement to explain the mechanism of breast cancer (BC).     

The 5-methylcytosine content of DNA from human tumors

The over-all 5-methylcytosine (m5C) content of DNA from normal tissues varies considerably in a tissue-specific manner. By high-performance liquid chromatography, we have examined the m5C contents of enzymatic digests of DNA from 103 human tumors including benign, primary malignant and secondary malignant neoplasms. The diversity and large number of these tumor samples allowed us to compare the range of DNA methylation levels from neoplastic tissues to that of normal tissues from humans. Most of the metastatic neoplasms had significantly lower genomic m5C contents than did most of the benign neoplasms or normal tissues. The percentage of primary malignancies with hypomethylated DNA was intermediate between those of metastases and benign neoplasms. These findings might reflect an involvement of extensive demethylation of DNA in tumor progression. Such demethylation could be a source of the continually generated cellular diversity associated with cancer.     

Widespread occurrence of organelle genome-encoded 5S rRNAs including permuted molecules

5S Ribosomal RNA (5S rRNA) is a universal component of ribosomes, and the corresponding gene is easily identified in archaeal, bacterial and nuclear genome sequences. However, organelle gene homologs (rrn5) appear to be absent from most mitochondrial and several chloroplast genomes. Here, we re-examine the distribution of organelle rrn5 by building mitochondrion- and plastid-specific covariance models (CMs) with which we screened organelle genome sequences. We not only recover all organelle rrn5 genes annotated in GenBank records, but also identify more than 50 previously unrecognized homologs in mitochondrial genomes of various stramenopiles, red algae, cryptomonads, malawimonads and apusozoans, and surprisingly, in the apicoplast (highly derived plastid) genomes of the coccidian pathogens Toxoplasma gondii and Eimeria tenella. Comparative modeling of RNA secondary structure reveals that mitochondrial 5S rRNAs from brown algae adopt a permuted triskelion shape that has not been seen elsewhere. Expression of the newly predicted rrn5 genes is confirmed experimentally in 10 instances, based on our own and published RNA-Seq data. This study establishes that particularly mitochondrial 5S rRNA has a much broader taxonomic distribution and a much larger structural variability than previously thought. The newly developed CMs will be made available via the Rfam database and the MFannot organelle genome annotator.     

The 5-methylcytosine content of highly repeated sequences in human DNA.

Previously, we found much tissue- or cell-specificity in the levels of 5-methylcytosine (m5C) in the total human genome as well as in DNA fractions resolved by reassociation kinetics. We now report that there were even greater differences in the m5C content of the highly repeated, tandem EcoRI family of DNA sequences from different human organs or cell populations. The ratio of m5C levels in this DNA fraction from brain, placenta, and sperm was 2.0:1.2:1.0. At a HhaI site in this repeat family, sperm DNA was 5-10 fold less methylated than somatic DNAs. In contrast, the highly repeated Alu family, which is approximately 5% of the genome, had almost the same high m5C content in brain and placenta despite marked tissue-specific differences in m5C levels of the single copy sequences with which these repeats are interspersed. These data show that very different degrees of change in methylation levels of various highly repeated DNA sequences accompany differentiation.     

ADAR-mediated RNA editing in non-coding RNA sequences

Adenosine to inosine (A-to-I) RNA editing is the most abundant editing event in animals. It converts adenosine to inosine in double-stranded RNA regions through the action of the adenosine deaminase acting on RNA (ADAR) proteins. Editing of pre-mRNA coding regions can alter the protein codon and increase functional diversity. However, most of the A-to-I editing sites occur in the non-coding regions of pre-mRNA or mRNA and non-coding RNAs. Untranslated regions (UTRs) and introns are located in pre-mRNA non-coding regions, thus A-to-I editing can influence gene expression by nuclear retention, degradation, alternative splicing, and translation regulation. Non-coding RNAs such as microRNA (miRNA), small interfering RNA (siRNA) and long non-coding RNA (lncRNA) are related to pre-mRNA splicing, translation, and gene regulation. A-to-I editing could therefore affect the stability, biogenesis, and target recognition of non-coding RNAs. Finally, it may influence the function of non-coding RNAs, resulting in regulation of gene expression. This review focuses on the function of ADAR-mediated RNA editing on mRNA non-coding regions (UTRs and introns) and non-coding RNAs (miRNA, siRNA, and lncRNA).     

Molecular evolution of coding and non-coding regions in Plasmodium

Recurrence analysis provides a useful tool for the characterisation of oligonucleotide usage along genomic tracts. While coding regions are characterised by a low-recurrence regimen (except in the case of intragenic repeats) introns and intergenic regions exhibit a high density of recurring oligos, and appear to be correlated from the point of view of oligonucleotide preference. By comparing homologous loci in Plasmodium falciparum and P. berghei, it can be seen that introns and intergenic regions, though exhibiting very low sequence similarity, do not drift without constraints, but maintain a consistent use of the same oligos in the two species.     

Structural architecture of the human long non-coding RNA, steroid receptor RNA activator

While functional roles of several long non-coding RNAs (lncRNAs) have been determined, the molecular mechanisms are not well understood. Here, we report the first experimentally derived secondary structure of a human lncRNA, the steroid receptor RNA activator (SRA), 0.87 kB in size. The SRA RNA is a non-coding RNA that coactivates several human sex hormone receptors and is strongly associated with breast cancer. Coding isoforms of SRA are also expressed to produce proteins, making the SRA gene a unique bifunctional system. Our experimental findings (SHAPE, in-line, DMS and RNase V1 probing) reveal that this lncRNA has a complex structural organization, consisting of four domains, with a variety of secondary structure elements. We examine the coevolution of the SRA gene at the RNA structure and protein structure levels using comparative sequence analysis across vertebrates. Rapid evolutionary stabilization of RNA structure, combined with frame-disrupting mutations in conserved regions, suggests that evolutionary pressure preserves the RNA structural core rather than its translational product. We perform similar experiments on alternatively spliced SRA isoforms to assess their structural features.