MicroRNAs (miRNAs) are endogenous ~23 nt RNAs which regulate message RNA (mRNA) targets mainly through perfect pairing with their seed region (positions 2-7). Several instances of UTR sequence with an additional nucleotide that might form a bulge within the pairing region, can also be recognized by miRNA as their target (bugle-target). But the prevalence of such imperfect base pairings in human and their roles in the evolution are incompletely understood. We found that human miRNAs with the CG dinucleotides (CG dimer) in their seed region have a significant low mutation rate than their putative binding sites in mRNA targets. Interspecific comparation shows that these miRNAs had very few conservative targets with the perfect seed-pairing, while potentially having a subclass of bulge-targets. Compared with the canonical target (perfect seed-pairing), these bulge-targets had a lower negative correlation with the miRNA expression, and either were down-regulated in the miRNA overexpression experiment or up-regulated in the miRNA knock-down experiment. Our results show that the bulge-targets are widespread in the miRNAs with CG dinucleotide within their seed regions, which could in part explain the rare conserved targets of these miRNAs based on seed rule. Incorporating these bulge-targets, together with conservation information, could more accurately predict the entire targets of these miRNAs.

BACKGROUND: Statistical approaches to genetic sequences have revealed helpful to gain deeper insight into biological and structural functionalities, using ideas coming from information theory and stochastic modelling of symbolic sequences. In particular, previous analyses on CG dinucleotide position along the genome allowed to highlight its epigenetic role in DNA methylation, showing a different distribution tail as compared to other dinucleotides. In this paper we extend the analysis to the whole CG distance distribution over a selected set of higher-order organisms. Then we apply the best fitting probability density function to a large range of organisms (>4400) of different complexity (from bacteria to mammals) and we characterize some emerging global features.
RESULTS: We find that the Gamma distribution is optimal for the selected subset as compared to a group of several distributions, chosen for their physical meaning or because recently used in literature for similar studies. The parameters of this distribution, when applied to our larger set of organisms, allows to highlight some biologically relavant features for the considered organism classes, that can be useful also for classification purposes.
CONCLUSIONS: The quantification of statistical properties of CG dinucleotide positioning along the genome is confirmed as a useful tool to characterize broad classes of organisms, spanning the whole range of biological complexity.

Characterizing the transmitted/founder (T/F) viruses of multi-variant SIV infection may shed new light on the understanding of mucosal transmission. We intrarectally inoculated six Chinese rhesus macaques with a single high dose of SIVmac251 (3.1 × 104 TCID50) and obtained 985 full-length env sequences from multiple tissues at 6 and 10 days post-infection by single genome amplification (SGA). All 6 monkeys were infected with a range of 2 to 8 T/F viruses and the dominant variants from the inoculum were still dominant in different tissues from each monkey. Interestingly, our data showed that a cluster of rare T/F viruses was unequally represented in different tissues. This cluster of rare T/F viruses phylogenetically related to the non-dominant SIV variants in the inoculum and was not detected in any rectum tissues, but could be identified in the descending colon, jejunum, spleen, or plasma. In 2 out of 6 macaques, identical SIVmac251 variants belonging to this cluster were detected simultaneously in descending colon/jejunum and the inoculum. We also demonstrated that the average CG dinucleotide frequency of these rare T/F viruses found in tissues, as well as non-dominant variants in the inoculum, was significantly higher than the dominant T/F viruses in tissues and the inoculum. Collectively, these findings suggest that descending colon/jejunum might be more susceptible than rectum to SIV in the very early phase of infection. And host CG suppression, which was previously shown to inhibit HIV replication in vitro, may also contribute to the bottleneck selection during in vivo transmission.

During mammalian development, some methylated cytosines (5mC) in CG dinucleotides are iteratively oxidized by TET dioxygenases to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). The effect of these cytosine oxidative products on the sequence-specific DNA binding of transcription factors is being actively investigated. Here, we used the electrophoretic mobility shift assay (EMSA) to examine C/EBPα and C/EBPβ homodimers binding to all 25 chemical forms of a CG dinucleotide for two DNA sequences: the canonical C/EBP 8-mer TTGC|GCAA and the chimeric C/EBP|CRE 8-mer TTGC|GTCA. 5hmC in the CG dinucleotide in the C/EBP|CRE motif 8-mer TGAC|GCAA inhibits binding of C/EBPβ but not C/EBPα. Binding was increased by 5mC, 5fC and 5caC. Circular dichroism monitored thermal denaturations for C/EBPβ bound to the C/EBP|CRE motif confirmed the EMSA. The structural differences between C/EBPα and C/EBPβ that may account for the difference in binding 5hmC in the 8-mer TGAC|GCAA are explored.

DNA methylation is responsible for regulating gene expression and cellular differentiation and for maintaining genomic stability during normal human development. Furthermore, it plays a significant role in the regulation of hematopoiesis. In order to elucidate the influence of DNA methylation during B-cell development, genome-wide DNA methylation status of pro-B, pre-BI, pre-BII, and naïve-B-cells isolated from human umbilical cord blood was determined using the methylated CpG island recovery assay followed by next generation sequencing. On average, 182-200 million sequences were generated for each precursor B-cell subset in 10 biological replicates. An overall decrease in methylation was observed during the transition from pro-B to pre-BI, whereas no differential methylation was observed in the pre-BI to pre-BII transition or in the pre-BII to naïve B-cell transition. Most of the methylated regions were located within intergenic and intronic regions not present in a CpG island context. Putative novel enhancers were identified in these regions that were differentially methylated between pro-B and pre-BI cells. The genome-wide methylation profiles are publically available and may be used to gain a better understanding of the involvement of atypical DNA methylation in the pathogenesis of malignancies associated with precursor B-cells.

To date, the main criterion by which long ncRNAs (lncRNAs) are discriminated from mRNAs is based on the capacity of the transcripts to encode a protein. However, it becomes important to identify non-ORF-based sequence characteristics that can be used to parse between ncRNAs and mRNAs. In this study, we first established an extremely selective workflow to define a highly refined database of lncRNAs which was used for comparison with mRNAs. Then using this highly selective collection of lncRNAs, we found the CG dinucleotide frequencies were clearly distinct. In addition, we showed that the bias in CG dinucleotide frequency was conserved in human and mouse genomes. We propose that this sequence feature will serve as a useful classifier in transcript classification pipelines. We also suggest that our refined database of \"bona fide\" lncRNAs will be valuable for the discovery of other sequence characteristics distinct to lncRNAs.

Three oxidative products of 5-methylcytosine (5mC) occur in mammalian genomes. We evaluated if these cytosine modifications in a CG dinucleotide altered DNA binding of four B-HLH homodimers and three heterodimers to the E-Box motif CGCAG|GTG. We examined 25 DNA probes containing all combinations of cytosine in a CG dinucleotide and none changed binding except for carboxylation of cytosine (5caC) in the strand CGCAG|GTG. 5caC enhanced binding of all examined B-HLH homodimers and heterodimers, particularly the Tcf3|Ascl1 heterodimer which increased binding ~10-fold. These results highlight a potential function of the oxidative products of 5mC, changing the DNA binding of sequence-specific transcription factors.