One parameter to describe the mechanism of splice sites competition

The choice of a splice site is not only related to its own intrinsic strength, but also is influenced by its flanking competitors. Splice site competition is an important mechanism for splice site prediction, especially, it is a new insight for alternative splice site prediction. In this paper, the position weight matrix scoring function is used to represent splice site strength, and the mechanism of splice site competition is described by only one parameter: scoring function subtraction. While applying on the alternative splice site prediction, based on the only one parameter, 68.22% of donor sites and 70.86% of acceptor sites are correctly classified into alternative and constitutive. The prediction abilities are approximately equal to the recent method which is based on the mechanism of splice site competition. The results reveal that the scoring function subtraction is the best parameter to describe the mechanism of splice sites competition.     

Intrinsic differences between authentic and cryptic 5' splice sites

Cryptic splice sites are used only when use of a natural splice site is disrupted by mutation. To determine the features that distinguish authentic from cryptic 5′ splice sites (5′ss), we systematically analyzed a set of 76 cryptic 5′ss derived from 46 human genes. These cryptic 5′ss have a similar frequency distribution in exons and introns, and are usually located close to the authentic 5′ss. Statistical analysis of the strengths of the 5′ss using the Shapiro and Senapathy matrix revealed that authentic 5′ss have significantly higher score values than cryptic 5′ss, which in turn have higher values than the mutant ones. β-Globin provides an interesting exception to this rule, so we chose it for detailed experimental analysis in vitro. We found that the sequences of the β-globin authentic and cryptic 5′ss, but not their surrounding context, determine the correct 5′ss choice, although their respective scores do not reflect this functional difference. Our analysis provides a statistical basis to explain the competitive advantage of authentic over cryptic 5′ss in most cases, and should facilitate the development of tools to reliably predict the effect of disease-associated 5′ss-disrupting mutations at the mRNA level.     

Cryptic splice sites and split genes

We describe a new program called cryptic splice finder (CSF) that can reliably identify cryptic splice sites (css), so providing a useful tool to help investigate splicing mutations in genetic disease. We report that many css are not entirely dormant and are often already active at low levels in normal genes prior to their enhancement in genetic disease. We also report a fascinating correlation between the positions of css and introns, whereby css within the exons of one species frequently match the exact position of introns in equivalent genes from another species. These results strongly indicate that many introns were inserted into css during evolution and they also imply that the splicing information that lies outside some introns can be independently recognized by the splicing machinery and was in place prior to intron insertion. This indicates that non-intronic splicing information had a key role in shaping the split structure of eukaryote genes.     

Identification of alternative 5'/3' splice sites based on the mechanism of splice site competition

Alternative splicing plays an important role in regulating gene expression. Currently, most efficient methods use expressed sequence tags or microarray analysis for large-scale detection of alternative splicing. However, it is difficult to detect all alternative splice events with them because of their inherent limitations. Previous computational methods for alternative splicing prediction could only predict particular kinds of alternative splice events. Thus, it would be highly desirable to predict alternative 5'/3' splice sites with various splicing levels using genomic sequences alone. Here, we introduce the competition mechanism of splice sites selection into alternative splice site prediction. This approach allows us to predict not only rarely used but also frequently used alternative splice sites. On a dataset extracted from the AltSplice database, our method correctly classified approximately 70% of the splice sites into alternative and constitutive, as well as approximately 80% of the locations of real competitors for alternative splice sites. It outperforms a method which only considers features extracted from the splice sites themselves. Furthermore, this approach can also predict the changes in activation level arising from mutations in flanking cryptic splice sites of a given splice site. Our approach might be useful for studying alternative splicing in both computational and molecular biology.     

A method for identifying splice sites and translation start sites in human genomic sequences

We describe a new method for identifying the sequences that signal the start of translation, and the boundaries between exons and introns (donor and acceptor sites) in human mRNA. According to the mandatory keyword, ORGANISM, and feature key, CDS, a large set of standard data for each signal site was extracted from the ASCII flat file, gbpri.seq, in the GenBank release 108.0. This was used to generate the scoring matrices, which summarize the sequence information for each signal site. The scoring matrices take into account the independent nucleotide frequencies between adjacent bases in each position within the signal site regions, and the relative weight on each nucleotide in proportion to their probabilities in the known signal sites. Using a scoring scheme that is based on the nucleotide scoring matrices, the method has great sensitivity and specificity when used to locate signals in uncharacterized human genomic DNA. These matrices are especially effective at distinguishing true and false sites.     

Latent splice sites and stop codons revisited

The accuracy of the data we reported in an RNA Letter to the Editor earlier this year on the possible relationship between stop codons and splicing is questioned by Miriami et al. (this issue). We reply here that we see no inaccuracy in our data presentation and offer a possible explanation for their interpretation.     

Characterization and prediction of alternative splice sites

Human alternative isoform, cryptic, skipped, and constitutive splice sites from the ALTEXTRON database were analysed regarding splice site strength, composition, GC content, position and binding site strength of polypyrimidine tract and branch site. Several features were identified which distinguish alternative isoform and cryptic splice sites, but not skipped splice sites from constitutive ones. These include splice site strength, introns GC content, U2AF35 binding site score, and oligonucleotide frequencies. For the predictive classification of splice sites, pattern recognition models for different splicing factor binding sites and oligonucleotide frequency models (OFMs) were combined using backpropagation networks. 67.45% of acceptor sites and 71.23% of donor sites are correctly classified by networks trained for classification of constitutive and alternative isoform/cryptic splice sites. A web-application for the prediction of alternative splice sites is available at http://es.embnet.org/~mwang/assp.html .     

Adenosine discriminates between the caffeine and adenine nucleotide sites on the sheep cardiac sarcoplasmic reticulum calcium-release channel

Calcium-release channels of sheep cardiac sarcoplasmic reticulum were incorporated into phosphatidylethanolamine bilayers and single channel currents were recorded under voltage-clamp conditions. The effect of adenosine on single channel conductance and gating was investigated, as were the interactions between adenosine and caffeine and adenosine and ,-methylene ATP.Addition of adenosine (0.5–5 mm) to the cytosolic but not the luminal side of the membrane increased the open probability of single calcium-activated calcium-release channels by increasing the frequency and duration of open events, yielding an EC₅₀ of 0.75 mm at 10 m activating Ca²⁺.Addition of 1 mm caffeine potentiated the effects of adenosine at 10 or 100 m-activating cytosolic calcium, but had no effect on the inability of adenosine to activate the channel at 80 pmcalcium, suggesting discrete sites of action on the calcium-release channel for adenosine and caffeine. In contrast, addition of 100 m ,-methylene-ATP decreased single channel open probability in the presence of adenosine, suggesting that these compounds act on the same site on the channel.Activation of single channel opening by adenosine, or by adenosine together with caffeine, had no effect on single channel conductance or the Ca²⁺/Tris⁺ permeability ratio. Channels activated by adenosine were characteristically modified by ryanodine and blocked by m ruthenium red or mm magnesium.These results show that adenosine activates the sheep cardiac sarcoplasmic reticulum Ca²⁺-release channel by increasing the frequency and duration of open events in a Ca²⁺-dependent manner. The receptor site on the channel for adenosine is distinct from that for caffeine but probably the same as that for adenine nucleotides.This work was supported by the British Heart Foundation.     

Coevolution of genomic intron number and splice sites

Spliceosomal intron numbers and boundary sequences vary dramatically in eukaryotes. We found a striking correspondence between low intron number and strong sequence conservation of 5' splice sites (5'ss) across eukaryotic genomes. The phylogenetic pattern suggests that ancestral 5'ss were relatively weakly conserved, but that some lineages independently underwent both major intron loss and 5'ss strengthening. It seems that eukaryotic ancestors had relatively large intron numbers and 'weak' 5'ss, a pattern associated with frequent alternative splicing in modern organisms.     

Density discriminates between thermophilic and mesophilic proteins

Despite an intense interest and a remarkable number of studies on the subject, the relationships between thermostability and (primary, secondary and tertiary) structure of proteins are still not fully understood. Here, comparing the protein density – defined by the ratio between the residue number and protein excluded volume – for a set of thermophilic/mesophilic pairs, we provide evidence that this property is connected to the optimal growth temperature. In particular, our results indicate that thermophilic proteins have – in general – a lower density with respect to the mesophilic counterparts, being such a correlation more pronounced for optimal growth temperature differences greater than 40°C. The effect of the protein thermostability changes on the molecular shape is also presented.     

The 5' and 3' splice sites come together via a three dimensional diffusion mechanism.

We present evidence that the splice sites in mammalian pre-mRNAs are brought together via a three dimensional diffusion mechanism. We tested two mechanisms for splice site pairing: a lateral diffusion ('scanning') model and the currently favored three dimensional diffusion ('jumping') model. Two lines of evidence that distinguish between these two models are presented. The first utilized bipartite splicing substrates tethered by double-stranded RNA stems predicted to provide either a moderate or severe block to splice site pairing via a scanning mechanism. Splice site pairing via a jumping mechanism was expected to be unaffected or affected minimally. The second approach utilized a flexible poly(ethylene glycol) moiety within the intron. This insertion was predicted to reduce scanning efficiency but not the efficiency of a three dimensional diffusion mechanism. The best explanation for the data with the bipartite RNAs is that splice site pairing occurs through three dimensional diffusion. Kinetic analysis of the poly(ethylene glycol) containing substrate showed that neither the lag phase nor the initial rates of mRNA production and spliceosome assembly were affected by this insertion. Therefore, both experimental approaches supported the three dimensional diffusion model of splice site pairing.     

Homologies between different procaryotic DNA-binding regulatory proteins and between their sites of action.

Comparison of the amino acid sequences of 13 procaryotic regulatory proteins, including the products of genes crp (catabolite activator protein; CAP), lacI, galR , lexA, lysR, araC, trpR, and tnpR of Escherichia coli, of genes cI, cII and cro of phage lambda, cro of phage 434, and c2 of phage P22, has revealed two regions of homology. The sites of action of these proteins also share common features in their DNA sequence. Taking into account the models proposed for the lambda repressors, cro and cI, and for CAP, a general type of DNA-protein interaction is suggested.     

tau Exon 10 expression involves a bipartite intron 10 regulatory sequence and weak 5' and 3' splice sites

tau mutations that deregulate alternative exon 10 (E10) splicing cause frontotemporal dementia with parkinsonism chromosome 17-type by several mechanisms. Previously we showed that E10 splicing involved exon splicing enhancer sequences at the 5' and 3' ends of E10, an exon splicing silencer, a weak 5' splice site, and an intron splicing silencer (ISS) within intron 10 (I10). Here, we identify additional regulatory sequences in I10 using both non-neuronal and neuronal cells. The ISS sequence extends from I10 nucleotides 11-18, which is sufficient to inhibit use of a weakened 5' splice site of a heterologous exon. Furthermore, ISS function is location-independent but requires proximity to a weak 5' splice site. Thus, the ISS functions as a linear sequence. A new cis-acting element, the intron splicing modulator (ISM), was identified immediately downstream of the ISS at I10 positions 19-26. The ISM and ISS form a bipartite regulatory element, within which the ISM functions when the ISS is present, mitigating E10 repression by the ISS. Additionally, the 3' splice site of E10 is weak and requires exon splicing enhancer elements for efficient E10 inclusion. Thus far, tau FTDP-17 splicing mutations affect six predicted cis-regulatory sequences.     

A novel taxonomic marker that discriminates between morphologically complex actinomycetes

In the era when large whole genome bacterial datasets are generated routinely, rapid and accurate molecular systematics is becoming increasingly important. However, 16S ribosomal RNA sequencing does not always offer sufficient resolution to discriminate between closely related genera. The SsgA-like proteins are developmental regulatory proteins in sporulating actinomycetes, whereby SsgB actively recruits FtsZ during sporulation-specific cell division. Here, we present a novel method to classify actinomycetes, based on the extraordinary way the SsgA and SsgB proteins are conserved. The almost complete conservation of the SsgB amino acid (aa) sequence between members of the same genus and its high divergence between even closely related genera provides high-quality data for the classification of morphologically complex actinomycetes. Our analysis validates Kitasatospora as a sister genus to Streptomyces in the family Streptomycetaceae and suggests that Micromonospora, Salinispora and Verrucosispora may represent different clades of the same genus. It is also apparent that the aa sequence of SsgA is an accurate determinant for the ability of streptomycetes to produce submerged spores, dividing the phylogenetic tree of streptomycetes into liquid-culture sporulation and no liquid-culture sporulation branches. A new phylogenetic tree of industrially relevant actinomycetes is presented and compared with that based on 16S rRNA sequences.