Divergence of conserved non-coding sequences: rate estimates and relative rate tests

In many eukaryotic genomes only a small fraction of the DNA codes for proteins, but the non-protein coding DNA harbors important genetic elements directing the development and the physiology of the organisms, like promoters, enhancers, insulators, and micro-RNA genes. The molecular evolution of these genetic elements is difficult to study because their functional significance is hard to deduce from sequence information alone. Here we propose an approach to the study of the rate of evolution of functional non-coding sequences at a macro-evolutionary scale. We identify functionally important non-coding sequences as Conserved Non-Coding Nucleotide (CNCN) sequences from the comparison of two outgroup species. The CNCN sequences so identified are then compared to their homologous sequences in a pair of ingroup species, and we monitor the degree of modification these sequences suffered in the two ingroup lineages. We propose a method to test for rate differences in the modification of CNCN sequences among the two ingroup lineages, as well as a method to estimate their rate of modification. We apply this method to the full sequences of the HoxA clusters from six gnathostome species: a shark, Heterodontus francisci; a basal ray finned fish, Polypterus senegalus; the amphibian, Xenopus tropicalis; as well as three mammalian species, human, rat and mouse. The results show that the evolutionary rate of CNCN sequences is not distinguishable among the three mammalian lineages, while the Xenopus lineage has a significantly increased rate of evolution. Furthermore the estimates of the rate parameters suggest that in the stem lineage of mammals the rate of CNCN sequence evolution was more than twice the rate observed within the placental amniotes clade, suggesting a high rate of evolution of cis-regulatory elements during the origin of amniotes and mammals. We conclude that the proposed methods can be used for testing hypotheses about the rate and pattern of evolution of putative cis-regulatory elements.     

Fugu and human sequence comparison identifies novel human genes and conserved non-coding sequences

The compact genome of the pufferfish, Fugu rubripes, has been proposed as a 'reference' genome to aid in annotating and analysing the human genome. We have annotated and compared 85 kb of Fugu sequence containing 17 genes with its homologous loci in the human draft genome and identified three 'novel' human genes that were missed or incompletely predicted by the previous gene prediction methods. Two of the novel genes contain zinc finger domains and are designated ZNF366 and ZNF367. They map to human chromosomes 5q13.2 and 9q22.32, respectively. The third novel gene, designated C9orf21, maps to chromosome 9q22.32. This gene is unique to vertebrates, and the protein encoded by it does not contain any known domains. We could not find human homologs for two Fugu genes, a novel chemokine gene and a kinase gene. These genes are either specific to teleosts or lost in the human lineage. The Fugu-human comparison identified several conserved non-coding sequences in the promoter and intronic regions. These sequences, conserved during 450 million years of vertebrate evolution, are likely to be involved in gene regulation. The 85 kb Fugu locus is dispersed over four human loci, occupying about 1.5 Mb. Contiguity is conserved in the human genome between six out of 16 Fugu gene pairs. These contiguous chromosomal segments should share a common evolutionary history dating back to the common ancestor of mammals and teleosts. We propose contiguity as strong evidence to identify orthologous genes in distant organisms. This study confirms the utility of the Fugu as a supplementary tool to uncover and confirm novel genes and putative gene regulatory regions in the human genome.     

Chromosome conformation capture uncovers potential genome-wide interactions between human conserved non-coding sequences

Comparative analyses of various mammalian genomes have identified numerous conserved non-coding (CNC) DNA elements that display striking conservation among species, suggesting that they have maintained specific functions throughout evolution. CNC function remains poorly understood, although recent studies have identified a role in gene regulation. We hypothesized that the identification of genomic loci that interact physically with CNCs would provide information on their functions. We have used circular chromosome conformation capture (4C) to characterize interactions of 10 CNCs from human chromosome 21 in K562 cells. The data provide evidence that CNCs are capable of interacting with loci that are enriched for CNCs. The number of trans interactions varies among CNCs; some show interactions with many loci, while others interact with few. Some of the tested CNCs are capable of driving the expression of a reporter gene in the mouse embryo, and associate with the oligodendrocyte genes OLIG1 and OLIG2. Our results underscore the power of chromosome conformation capture for the identification of targets of functional DNA elements and raise the possibility that CNCs exert their functions by physical association with defined genomic regions enriched in CNCs. These CNC-CNC interactions may in part explain their stringent conservation as a group of regulatory sequences.     

Striking nucleotide frequency pattern at the borders of highly conserved vertebrate non-coding sequences

In a recent study, 1373 highly conserved non-coding elements (CNEs) were detected by aligning the human and Takifugu rubripes (Fugu) genomes. The remarkable degree of sequence conservation in CNEs compared with their surroundings suggested comparing the base composition within CNEs with their 5' and 3' flanking regions. The analysis reveals a novel, sharp and distinct signal of nucleotide frequency bias precisely at the border between CNEs and flanking regions.     

Cis-regulation and conserved non-coding elements in amphioxus

The origin of the vertebrates was a major event in the evolution of morphological diversity and the genetic mechanisms responsible for this diversity, once purely theoretical, can now be approached experimentally in the genome era. With a prototypical chordate genome, vertebrate-like development and simple morphology, amphioxus provides the appropriate model for investigating the origin of the vertebrates. Comparative genomics is revealing that both conservation and divergence of genes and cis-regulatory elements involved in developmental regulatory networks are required to shape different animal body plans. This article reviews the cis-regulatory studies performed in amphioxus, the discovery of conserved non-coding elements (CNEs) across the metazoans and the examination of amphioxus CNEs. Emerging ideas on the evolution of CNEs after large-scale genome duplication events and the state of cephalochordate genomics are also discussed.     

Plant non-coding RNAs and epigenetics

正Epigenetics plays a large role in various aspects of plant biology,including development,response to biotic and abiotic stresses,silencing of transposable elements,and maintenance of genome stability.In plants,epigenetic regulation involves histone and DNA modifications and noncoding RNAs including small RNAs(s RNAs)and long noncoding RNAs(lnc RNAs).This issue of Science China Life Sciences includes five review articles and eight research     

Unexpected conserved non-coding DNA blocks in mammals

The significance of non-coding DNA is a longstanding riddle in the study of molecular evolution. Using a comparative genomics approach, Dermitzakis and colleagues have recently shown that at least some non-coding sequence, frequently ignored as meaningless noise, might bear the signature of natural selection. If functional, it could mark a turning point in the way we think about the evolution of the genome.     

Uricase protein sequences: conserved during vertebrate evolution but absent in humans

Uricase is a peroxisomal liver enzyme that catalyzes the oxidation of uric acid to allantoin during purine catabolism. It is present in vertebrates in most species of fish, amphibians, and mammals but its enzymatic activity is absent in hominoids. We have used Western blot analysis in a comparative study to establish a homology among uricases from different species of vertebrates. Using antibodies against denatured rat liver uricase, we have been able to detect for the first time cross-reactivity with the uricase of species ranging in the evolutionary scale from fish to primates (macaque). Our results suggest that these uricases have a common evolutionary origin. Our conclusion is also supported by the fact that uricase from different species exhibits identical tissue, subcellular localization, and similarity of molecular weights. This study was extended to include human liver samples. Using the same approach but with a more sensitive detection system (alkaline phosphatase instead of peroxidase), we did not detect polypeptide species related to rat uricase in human fetal or adult liver samples, which indicates that during hominoid evolution, the mutational event responsible for the loss of uricase activity in humans precluded formation of a translatable uricase mRNA.     

The boundary of macaque rDNA is constituted by low-copy sequences conserved during evolution

In Macaca mulatta, the single rDNA array is flanked by a patchwork of sequences including subregions of human Yp11.2, 4q35.2, and 10p15.3. This composite DNA region is characterized by unique or low-copy sequences, resembling a potentially transcribed region. The analysis of Cercopithecus aethiops, Presbytis cristata, and Hylobates lar suggests that this complex sequence organization could be shared by Old World monkey and lesser ape species. After the lesser apes/great apes divergence, the unique or nonduplicated DNA region underwent amplification and spreading, preferentially marking the p arm of acrocentric chromosomes bearing the rDNA. The molecular analysis of human acrocentric chromosomes revealed some extent of remodeling of the rDNA boundary: near the human NOR, a large 4q35.2 duplication partially resembles that found in MMU; conversely, infrequently represented Yp11.2 sequences totally differed from those of the macaque, and 10p15.3 sequences were lacking. Thus, although evolutionary events modified the sequence organization of the MMU rDNA boundary, its overall sequence feature and the preferential location in vicinity to the NOR have been conserved.     

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).     

The most frequent short sequences in non-coding DNA

The purpose of this work is to determine the most frequent short sequences in non-coding DNA. They may play a role in maintaining the structure and function of eukaryotic chromosomes. We present a simple method for the detection and analysis of such sequences in several genomes, including Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster and Homo sapiens. We also study two chromosomes of man and mouse with a length similar to the whole genomes of the other species. We provide a list of the most common sequences of 9–14 bases in each genome. As expected, they are present in human Alu sequences. Our programs may also give a graph and a list of their position in the genome. Detection of clusters is also possible. In most cases, these sequences contain few alternating regions. Their intrinsic structure and their influence on nucleosome formation are not known. In particular, we have found new features of short sequences in C. elegans, which are distributed in heterogeneous clusters. They appear as punctuation marks in the chromosomes. Such clusters are not found in either A. thaliana or D. melanogaster. We discuss the possibility that they play a role in centromere function and homolog recognition in meiosis.     

Evolutionary divergence and limits of conserved non-coding sequence detection in plant genomes

The discovery of regulatory motifs embedded in upstream regions of plants is a particularly challenging bioinformatics task. Previous studies have shown that motifs in plants are short compared with those found in vertebrates. Furthermore, plant genomes have undergone several diversification mechanisms such as genome duplication events which impact the evolution of regulatory motifs. In this article, a systematic phylogenomic comparison of upstream regions is conducted to further identify features of the plant regulatory genomes, the component of genomes regulating gene expression, to enable future de novo discoveries. The findings highlight differences in upstream region properties between major plant groups and the effects of divergence times and duplication events. First, clear differences in upstream region evolution can be detected between monocots and dicots, thus suggesting that a separation of these groups should be made when searching for novel regulatory motifs, particularly since universal motifs such as the TATA box are rare. Second, investigating the decay rate of significantly aligned regions suggests that a divergence time of ~100 mya sets a limit for reliable conserved non-coding sequence (CNS) detection. Insights presented here will set a framework to help identify embedded motifs of functional relevance by understanding the limits of bioinformatics detection for CNSs.