A possible role of exon-shuffling in the evolution of signal peptides of human proteins

AbstractIt was recently shown that there is a predominance of phase 1 introns near the cleavage site of signal peptides encoded by human genes [Tordai, H. and Patthy, L. (2004) Insertion of spliceosomal introns in proto-splice sites: the case of secretory signal peptides. FEBS Lett. 575, 109–111]. It was suggested that this biased distribution was due to intron insertion at AG∣G proto-splice sites. However, we found that there is no disproportional excess of AG∣G that would support insertion at proto-splice sites. In fact, all nG∣G sites are enriched in the vicinity of the cleavage site. Additional analyses support an alternative scenario in which exon-shuffling is largely responsible for such excess of phase 1 introns

Analysis of genomic sequence features related to alternative splicing events (intron retention) in the human transcriptome

Os genes eucarióticos, em sua maioria, são divididos em exons e introns, requerendo processamento do RNAm para remover as sequências intrônicas e juntar os exons (splicing). As bordas exon/intron são definidas por sítios de splice que normalmente são reconhecidos com alta fidelidade, gerando os mesmos RNAms processados a cada vez. Apesar desse reconhecimento preciso, tem sido observada a junção de exons de maneiras alternativas (splicing alternativo), foco de muitos estudos recentes devido à sua importância em vários processos biológicos. Este processamento alternativo do RNAm pode ser principalmente de três tipos: exclusão de exon, no qual um exon pode ser incluído ou não no RNAm maduro; uso alternativo de sítios de splice, resultando em exons mais longos ou mais curtos e retenção de intron, o tipo menos estudado, no qual uma sequência intrônica é mantida no RNAm maduro. Um dos aspectos cruciais no entendimento de splicing alternativo é conhecer os mecanismos que levam à geração de diferentes transcritos. Coerente com a importância dos sítios de splice no splicing de RNAms, a retenção de intron parece ser causada por falha no reconhecimento daqueles que são sub-ótimos. Como os sítios de splice são reconhecidos aos pares ao se estabelecer uma ponte através de exons ou introns, dependendo de qual é mais curto, uma falha no reconhecimento de um exon ou de um intron leva a diferentes tipos de splicing alternativo (exclusão de exon ou retenção de intron, respectivamente). Desta forma, acredita-se que a ocorrência de retenção de intron esteja também associada a uma falha no reconhecimento de introns curtos. Embora estudos de introns retidos individuais tenham abordado estas questões, poucas análises sistemáticas de grandes quantidades de dados foram conduzidas sobre as características gerais que levam à retenção de intron. Para este fim, realizamos uma análise de bioinformática de sequências do genoma e transcriptoma (RNAm) humanos armazenadas em formato de computador. Para realizar as análises computacionais, desenvolvemos um sistema de anotação de splicing alternativo completo. Particionamos os eventos de retenção de intron identificados em sequências expressas pelo nosso sistema de anotação em dois grupos, com base na abundância relativa das duas isoformas (um grupo de eventos com 50% de transcritos retendo o intron) e comparamos características relevantes. Verificamos que uma maior frequência de retenção de intron em humano está associada a sítios de splice mais fracos, genes com introns mais curtos e maior nível de expressão gênica, e menor densidade de um conjunto de elementos inibitórios exônicos e do promotor de splicing intrônico GGG. Os dois grupos apresentaram eventos conservados em camundongo, nos quais os introns retidos também eram curtos e apresentavam sítios de splice mais fracos. Embora nossos resultados tenham confirmado que sítios de splice mais fracos estão associados à retenção de intron, eles mostram que uma fração não-desprezível dos eventos não pode ser explicada apenas por esta característica. Nossa análise sugere que elementos reguladores em cis provavelmente têm um papel na regulação da retenção de intron e também revelou características previamente desconhecidas que parecem influenciar sua ocorrência. Estes resultados salientam a importância de considerar o compromisso entre estas características na regulação da frequência relativa de retenção de intron.Most eukaryotic genes are split in exons and introns, requiring mRNA processing to remove intervening sequences and join exons (splicing). Exon/intron borders are defined by splice sites that are normally recognized with high fidelity, yielding the same processed mRNA each time. Notwithstanding such precise recognition, alternative joining of exons has been observed (alternative splicing) and is the focus of many recent studies, due to its importance in several biological processes. This alternative mRNA processing can be mainly of three types: exon skipping, whereby an exon may be included or not in the mature mRNA; alternative use of splice sites, resulting in longer or shorter exons and intron retention, the least studied type whereby an intronic sequence is maintained in the mature mRNA. One of the key aspects in understanding alternative splicing is to know the mechanisms that lead to the generation of different transcripts. Coherent with the importance of splice sites in mRNA splicing, intron retention seems to be caused by failure in the recognition of those that are sub-optimal. As splice sites are recognized in pairs by bridging either exons or introns, depending on which is the shortest, failure to recognize an exon or an intron leads to different types of alternative splicing (exon skipping or intron retention, respectively). This way, the occurrence of intron retention is believed to be associated to failure in recognition of short introns also. Although studies on individual retained introns have addressed such issues, few systematic surveys of large amounts of data have been conducted on the general features leading to intron retention. To this end, we performed a bioinformatics analysis of human genome and transcriptome (mRNA) sequences stored in computer format. To perform the computational analyses we developed a complete alternative splicing annotation system. We partitioned intron retention events identified in expressed sequences by our annotation system in two groups based on the relative abundance of both isoforms (one group of events with 50% of transcripts retaining the intron) and compared relevant features. We found that a higher frequency of intron retention in human is associated to weaker splice sites, genes with shorter intron lengths and higher expression level, and lower density of a set of exonic inhibitory elements and the intronic splicing enhancer GGG. Both groups of events presented conserved events in mouse, in which the retained introns were also short and presented weaker splice sites. Although our results confirmed that weaker splice sites are associated to intron retention, they showed that a non-negligible fraction of events can not be explained by this feature alone. Our analysis suggests that cis-regulatory elements are likely to play a crucial role in regulating intron retention and also revealed previously unknown features that seem to influence its occurrence. These results highlight the importance of considering the interplay among these features in the regulation of the relative frequency of intron retention

Detection and evaluation of intron retention events in the human transcriptome

Alternative splicing is a very frequent phenomenon in the human transcriptome. There are four major types of alternative splicing: exon skipping, alternative 3′ splice site, alternative 5′ splice site, and intron retention. Here we present a large-scale analysis of intron retention in a set of 21,106 known human genes. We observed that 14.8% of these genes showed evidence of at least one intron retention event. Most of the events are located within the untranslated regions (UTRs) of human transcripts. For those retained introns interrupting the coding region, the GC content, codon usage, and the frequency of stop codons suggest that these sequences are under selection for coding potential. Furthermore, 26% of the introns within the coding region participate in the coding of a protein domain. A comparison with mouse shows that at least 22% of all informative examples of retained introns in human are also present in the mouse transcriptome. We discuss that the data we present suggest that a significant fraction of the observed events is not spurious and might reflect biological significance. The analyses also allowed us to generate a reliable set of intron retention events that can be used for the identification of splicing regulatory elements

A large-scale study of SNPs in regulatory elements of

The synthesis of a protein in eukaryotic organisms involves the transcription of a molecule of DNA to RNA (pre-mRNA), the excision of introns and fusion of exons by a process named splicing (yielding the mature mRNA molecule) and finally the translation of mRNA to protein. Not all exons of a gene are necessarily included in a mRNA, generating different isoforms of a protein, a phenomenon named alternative splicing. As a result, the mature mRNA may contain different combinations of exons, longer or shorter exons and even incorporate intronic sequences. The recognition of splice sites (exon-intron borders) involves i-specific sequences in the mRNA known as exonic or intronic splicing enhancers/silencers and iimany protein and ribonucleoprotein factors that recognize the sequences described in (i). Mutations that alter regulatory elements in the mRNA (i), thus interfering in the recognition by the factors described in (ii) may significantly alter the inclusion of a given exon [1]. It is known that mutations in a single nucleotide in specific positions of regulatory elements can interfere in the splicing of exons regulated by such sequences [2]. A common form of variation between two genomes of the same organism is the single nucleotide polimorfisms (SNPs) [3]. There are many studies that correlate SNPs to human diseases, since a single nucleotide change may radically affect the biochemical properties of a protein and may change its expression patterns. Our aim is to perform a large scale analysis of the occurrence of human SNPs in regulatory elements of alternative splicing, using a transcriptome database available in our lab. The database allows one to verify the frequency of SNPs in exons, introns and regulatory elements and make correlation with transcripts. OMIM [4] wi..