Functional properties and evolutionary splicing constraints on a composite exonic regulatory element of splicing in CFTR exon 12

In general, splicing regulatory elements are defined as Enhancers or Silencers depending on their positive or negative effect upon exon inclusion. Often, these sequences are usually present separate from each other in exonic/intronic sequences. The Composite Exonic Splicing Regulatory Elements (CERES) represent an extreme physical overlap of enhancer/silencer activity. As a result, when CERES elements are mutated the consequences on the splicing process are difficult to predict. Here, we show that the functional activity of the CERES2 sequence in CFTR exon 12 is regulated by the binding, in very close proximity to each other, of several SR and hnRNP proteins. Moreover, our results show that practically the entire exon 12 sequence context participate in its definition. The consequences of this situation can be observed at the evolutionary level by comparing changes in conservation of different splicing elements in different species. In conclusion, our study highlights how it is increasingly difficult to define many exonic sequences by simply breaking them down in isolated enhancer/silencer or even neutral elements. The real picture is close to one of continuous competition between positive and negative factors where affinity for the target sequences and other dynamic factors decide the inclusion or exclusion of the exon.     

CS-PSeq-Gen: simulating the evolution of protein sequence under constraints

CS-PSeq-Gen is a program derived from PSeq-Gen, designed to perform simulations of the evolution of protein sequences under the constraints of a reconstructed phylogeny. It also provides a basis for the investigation of the correlated evolution of sites. AVAILABILITY: http://condor.urbb.jussieu.fr/CS-PSeq-Gen.html     

Structural constraints and emergence of sequence patterns in protein evolution

The aim of this work was to study the relationship between structure conservation and sequence divergence in protein evolution. To this end, we developed a model of structurally constrained protein evolution (SCPE) in which trial sequences, generated by random mutations at gene level, are selected against departure from a reference three-dimensional structure. Since at the mutational level SCPE is completely unbiased, any emergent sequence pattern will be due exclusively to structural constraints. In this first report, it is shown that SCPE correctly predicts the characteristic hexapeptide motif of the left-handed parallel beta helix (LbetaH) domain of UDP-N-acetylglucosamine acyltransferases (LpxA).     

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.     

Simultaneous assessment of the effects of exonic mutations on RNA splicing and protein functions

To simultaneously assess the effects of exonic mutations on RNA splicing and protein functions, we report here an intron-inclusive cDNA (Intinc) expression system. As a test model, twenty-four mutations in exon 9 of the phenylalanine hydroxylase (PAH) gene were examined in an Intinc expression plasmid composed of the PAH cDNA with the exon 9 flanked by its authentic introns. When the PAH enzyme activities from the Intinc plasmid-transfected cells were compared to those of a standard cDNA expression system, five mutations resulted in significant relative differences in PAH activities attributed to altered exon 9-inclusive mRNA levels. Two of the mutations affected exon recognition probably through splice site modifications and the remaining three affected experimentally verified exon splicing enhancer (ESE) motifs. The Intinc expression system allows not only a better link between mutation genotype to disease phenotype but also contributes to further understanding of molecular mechanisms of deleterious effects of mutations.     

Genome sequence of Staphylococcus aureus strain Newman and comparative analysis of staphylococcal genomes: polymorphism and evolution of two major pathogenicity islands

Strains of Staphylococcus aureus, an important human pathogen, display up to 20% variability in their genome sequence, and most sequence information is available for human clinical isolates that have not been subjected to genetic analysis of virulence attributes. S. aureus strain Newman, which was also isolated from a human infection, displays robust virulence properties in animal models of disease and has already been extensively analyzed for its molecular traits of staphylococcal pathogenesis. We report here the complete genome sequence of S. aureus Newman, which carries four integrated prophages, as well as two large pathogenicity islands. In agreement with the view that S. aureus Newman prophages contribute important properties to pathogenesis, fewer virulence factors are found outside of the prophages than for the highly virulent strain MW2. The absence of drug resistance genes reflects the general antibiotic-susceptible phenotype of S. aureus Newman. Phylogenetic analyses reveal clonal relationships between the staphylococcal strains Newman, COL, NCTC8325, and USA300 and a greater evolutionary distance to strains MRSA252, MW2, MSSA476, N315, Mu50, JH1, JH9, and RF122. However, polymorphism analysis of two large pathogenicity islands distributed among these strains shows that the two islands were acquired independently from the evolutionary pathway of the chromosomal backbones of staphylococcal genomes. Prophages and pathogenicity islands play central roles in S. aureus virulence and evolution.     

Birth-and-death evolution of protein-coding regions and concerted evolution of non-coding regions in the multi-component genomes of nanoviruses

Genomes of the four plant viruses of the genus Nanovirus consist of multiple circular single-stranded DNA components, each of which encodes a single protein. Protein phylogenies supported the hypothesis that faba bean necrotic yellows virus (FBNYV) and milk vetch disease virus (MDV) are sister taxa; that subterranean clover stunt virus (SCSV) branched next; and that banana bunchy top virus (BBTV) is an outgroup to the three other species. The phylogeny of replication (Rep) proteins indicate that this small viral multi-gene family has evolved by a process of duplication and subsequent loss of Rep-encoding genome components, analogous to the "birth-and-death" process of evolution which has been described in eukaryotic multi-gene families. By contrast, repeated recombinational events between components were found to have homogenized the non-coding portions of several components encoding unrelated components. For example, as result of recent recombination a portion of the non-coding region is virtually identical among SCSV components 1, 3, 4, 5, and 7. Thus, there is a process of concerted evolution of non-coding regions of Nanovirus genome components, which raises the possibility that certain non-coding regions are subject to functional constraint.     

Selective constraints, amino acid composition, and the rate of protein evolution

What are the major forces governing protein evolution? A common view is that proteins with strong structural and functional requirements evolve more slowly than proteins with weak constraints, because a stringent negative selection pressure limits the number of substitutions. In contrast, Graur claimed that the substitution rate of a protein is mainly determined by its amino acid composition and the changeabilities of amino acids. In this paper, however, we found that the relative changeabilities of amino acids in mammalian proteins are different for transmembranal and nontransmembranal segments, which have very distinct structural requirements. This indicates that the changeability of a given residue is influenced by the structural and functional context. We also reexamined the relationship between substitution rate and amino acid composition. Indeed, the two kinds of segments exhibit contrasting amino acid compositions: transmembranal regions are made up mainly of hydrophobic residues (a total frequency of approximately 60%) and are very poor in polar amino acids (<5%), whereas nontransmembranal segments have frequencies of 30% and 22%, respectively. Interestingly, we found that within a given integral membrane protein, nontransmembranal segments accumulate, on average, twice as many substitutions as transmembranal regions. However, regression analyses showed that the variability in amino acid frequencies among proteins cannot explain more than 30% of the variability in substitution rate for the transmembranal and nontransmembranal data sets. Furthermore, transmembranal and nontransmembranal segments evolving at the same rate in different proteins have different compositions, and the compositions of slowly evolving and rapidly evolving segments of the same type are similar. From these observations, we conclude that the rate of protein evolution is only weakly affected by amino acid composition but is mostly determined by the strength of functional requirements or selective constraints.     

Nature and evolution of B chromosomes in plants: A non-coding but information-rich part of plant genomes

This work reviews recent advances providing insights on the origin and evolution of B chromosomes (Bs) in representative plant species. Brachyome dichromosomatica has large and micro Bs. Both carry an inactive ribosomal gene cluster. The large Bs contain the B-specific Bd49 family, mainly located at the centromere. Multiple copies are present in the A chromosomes (As) of related species, whereas only a few copies exist in B. dichromosomatica As. The micro Bs share sequences with the As, the large Bs and have the B-specific repeats Bdm29 and Bdm54. It seems that the large and micro Bs are related in origin. It is very unlikely that the Bs originated by simple excision from the As. Rye Bs are composed of sequences predominantly shared with the As. B-specific sequences are located at the heterochromatic end of the long arm. Probably, they originated from the As after many rearrangements, with a tendency for duplication. The E3900 family derives from a Ty3 gypsy retrotransposon, but the D1100 family shows no evidence of genic origin. The overall composition of maize As and Bs is similar suggesting a common origin. Several B-specific sequences have been found, the most studied being pZmBs, which is located at the B centromere. It shows partial homology to the centromere of chromosome 4 and to the knobs. It is not known whether the B centromere derives from centromere 4, or whether both have a more distant common origin. The dynamics of Bs in populations depends on their non-Mendelian mechanisms of transmission, their effects on carrier fitness and on A genes modulating their parasitic properties. Three representative examples are reviewed. The Bs of Allium schoenoprassum are transmitted at a mean lower than Mendelian and adversely affect vigour and fertility. However, there is a differential selection operating in favour of B-containing seedlings. Rye Bs undergo strong drive, which is counteracted by harmful effects on fertility and instabilities at meiosis. Both nondisjunction and meiotic behaviour, and consequently the establishment of B polymorphisms, mainly depend on the Bs themselves. B nondisjunction in maize is controlled by the B, but the As control preferential fertilisation. Considering the non-equilibrium model, the Bs of Allium seem to have been neutralised by the A genome, the As of maize provide defence against B attack, whereas the Bs of rye are only slightly neutralized.     

Comparable contributions of structural-functional constraints and expression level to the rate of protein sequence evolution

Background  

Proteins show a broad range of evolutionary rates. Understanding the factors that are responsible for the characteristic rate of evolution of a given protein arguably is one of the major goals of evolutionary biology. A long-standing general assumption used to be that the evolution rate is, primarily, determined by the specific functional constraints that affect the given protein. These constrains were traditionally thought to depend both on the specific features of the protein's structure and its biological role. The advent of systems biology brought about new types of data, such as expression level and protein-protein interactions, and unexpectedly, a variety of correlations between protein evolution rate and these variables have been observed. The strongest connections by far were repeatedly seen between protein sequence evolution rate and the expression level of the respective gene. It has been hypothesized that this link is due to the selection for the robustness of the protein structure to mistranslation-induced misfolding that is particularly important for highly expressed proteins and is the dominant determinant of the sequence evolution rate.     

Pre-mRNA splicing: awash in a sea of proteins

What's in a spliceosome? More than we ever imagined, according to recent reports employing proteomics techniques to analyze this multi-megadalton machine. As of 1999, around 100 splicing factors were identified (Burge et al., 1999); however, that number has now nearly doubled due primarily to improved purification of spliceosomes coupled with advances in mass spectrometry analyses of complex mixtures. Gratifyingly, most of the previously identified splicing factors were found in the recent mass spec studies. Nonetheless, the number of new proteins emerging with no prior connection to splicing was surprising. Without functional validation, it would be premature to label these proteins as bona fide splicing factors. Yet many were identified multiple times in complexes purified under diverse conditions or from different organisms. Another recurring theme regards the dynamic nature of spliceosomal complexes, which may be even more intricate than previously thought.     

CpG islands: features and distribution in the genomes of vertebrates

We have investigated the distribution of unmethylated CpG islands in vertebrate genomes fractionated according to their base composition. Genomes from warm-blooded vertebrates (man, mouse and chicken) are characterized by abundant CpG islands, whose frequency increases in DNA fractions of increasing % of guanine + cytosine; % G + C (GC), in parallel with the distribution of genes and CpG doublets. Small, yet significant, differences in the distribution of CpG islands were found in the three genomes. In contrast, genomes from cold-blooded vertebrates (two reptiles, one amphibian, and two fishes) were characterized by an extreme scarcity or absence of CpG islands (detected in these experiments as HpaII tiny fragments or HTF). CpG islands associated with homologous genes from cold- and warm-blooded vertebrates were then compared by analyzing CpG frequencies, GC levels, HpaII sites, rare-cutter sites and G/C boxes (GGGGCGGGGC and closely related motifs) in sequences available in gene banks. Small, yet significant, differences were again detected among the CpG islands associated with homologous genes from warm-blooded vertebrates, in that CpG islands associated with mouse or rat genes often showed low CpG and/or GC levels, as well as low numbers of HpaII sites, rare-cutter sites and G/C boxes, compared to homologous human genes; more rarely, CpG islands were just absent. As far as cold-blooded vertebrates were concerned, a number of genes showed CpG islands, which exhibited a much lower frequency of CpG doublets than that found in CpG islands of warm-blooded vertebrates, but still approached the statistically expected frequency; none of the other features of CpG islands associated with genes from warm-blooded vertebrates were present. Other genes did not show any associated CpG islands, unlike their homologues from warm-blooded vertebrates.     

Distribution and evolution of short tandem repeats in closely related bacterial genomes

Simultaneous identification and comparison of perfect and imperfect microsatellites within a genome is a valuable tool both to overcome the lack of a consensus definition of SSRs and to assess repeat history. Detailed analysis of the overall distribution of perfect and imperfect microsatellites in closely related bacterial taxa is expected to give new insight into the evolution of prokaryotic genomes. We have performed a genome-wide analysis of microsatellite distribution in four Escherichia coli and seven Chlamydial strains. Chlamydial strains generally have a higher density of SSRs and show greater intra-group differences of SSR distribution patterns than E. coli genomes. In most investigated genomes the distribution of the total lengths of matching perfect and imperfect trinucleotide repeats are highly similar, with the notable exception of C. muridarum. Closely related strains show more similar repeat distribution patterns than strains separated by a longer divergence time. The discrepancy between the preferred classes of perfect and imperfect repeats in C. muridarum implies accelerated evolution of SSRs in this particular strain. Our results suggest that microsatellites, although considerably less abundant than in eukaryotic genomes, may nevertheless play an important role in the evolution of prokaryotic genomes and several gene families.     

Missense meanderings in sequence space: a biophysical view of protein evolution

Proteins are finicky molecules; they are barely stable and are prone to aggregate, but they must function in a crowded environment that is full of degradative enzymes bent on their destruction. It is no surprise that many common diseases are due to missense mutations that affect protein stability and aggregation. Here we review the literature on biophysics as it relates to molecular evolution, focusing on how protein stability and aggregation affect organismal fitness. We then advance a biophysical model of protein evolution that helps us to understand phenomena that range from the dynamics of molecular adaptation to the clock-like rate of protein evolution.     

Finding the most significant common sequence and structure motifs in a set of RNA sequences.

We present a computational scheme to locally align a collection of RNA sequences using sequence and structure constraints. In addition, the method searches for the resulting alignments with the most significant common motifs, among all possible collections. The first part utilizes a simplified version of the Sankoff algorithm for simultaneous folding and alignment of RNA sequences, but maintains tractability by constructing multi-sequence alignments from pairwise comparisons. The algorithm finds the multiple alignments using a greedy approach and has similarities to both CLUSTAL and CONSENSUS, but the core algorithm assures that the pairwise alignments are optimized for both sequence and structure conservation. The choice of scoring system and the method of progressively constructing the final solution are important considerations that are discussed. Example solutions, and comparisons with other approaches, are provided. The solutions include finding consensus structures identical to published ones.     

Conservation of sequences in related genomes of Apodemus: constraints on the maintenance of satellite DNA sequences.

Satellites from two related species of the Apodemus genus, A. sylvaticus and A. flavicollis, have been analysed with restriction enzymes Taq I, Alu I and Hind III. The restriction maps are closely conserved between species and show a novel feature of two differing internal periodicities within a 375 base pair repeating unit detected by two different restriction enzymes. This places constraints on the introduction of the observed restriction sites according to current models such as unequal crossing-over. The implications of such a conserved sequence and its presence in other species are discussed.     

SWAP pre-mRNA splicing regulators are a novel, ancient protein family sharing a highly conserved sequence motif with the prp21 family of constitutive splicing proteins.

Regulators responsible for the pervasive, nonsex-specific alternative pre-mRNA splicing characteristic of metazoans are almost entirely unknown or uncertain. We describe here a novel family of splicing regulators present throughout metazoans. Specifically, we analyze two nematode (Caenorhabditis elegans) genes. One, CeSWAP, is a cognate of the suppressor-of-white-apricot (DmSWAP) splicing regulator from the arthropod Drosophila. Our results define the ancient, conserved SWAP protein family whose members share a colinearly arrayed series of novel sequence motifs. Further, we describe evidence that the CeSWAP protein autoregulates its levels by feedback control of splicing of its own pre-mRNA analogously to the DmSWAP protein and as expected of a splicing regulator. The second nematode gene, Ceprp21, encodes an abundant nuclear cognate of the constitutive yeast splicing protein, prp21, on the basis of several lines of evidence. Our analysis defines prp21 as a second novel, ancient protein family. One of the motifs conserved in prp21 proteins--designated surp--is shared with SWAP proteins. Several lines of evidence indicate that both new families of surp-containing proteins act at the same (or very similar) step in early prespliceosome assembly. We discuss implications of our results for regulated metazoan pre-mRNA splicing.     

The mechanisms controlling ribosomal protein L1 pre-mRNA splicing are maintained in evolution and rely on conserved intron sequences.

Sequences corresponding to the third intron of the X.laevis L1 ribosomal protein gene were isolated from the second copy of the X.laevis gene and from the single copy of X.tropicalis. Sequence comparison revealed that the three introns share an unusual sequence conservation which spans a region of 110 nucleotides. In addition, they have the same suboptimal 5' splice sites. The three introns show similar features upon oocyte microinjection: they have very low splicing efficiency and undergo the same site specific cleavages which lead to the accumulation of truncated molecules. Computer analysis and RNAse digestions have allowed to assign to the conserved region a specific secondary structure. Mutational analysis has shown that this structure is important for conferring the cleavage phenotype to these three introns. Competition experiments show that the cleavage phenotype can be prevented by coinjection of excess amounts of homologous sequences.