Sequence features responsible for intron retention in human

Abstract Background One of the least common types of alternative splicing is the complete retention of an intron in a mature transcript. Intron retention (IR) is believed to be the result of intron, rather than exon, definition associated with failure of the recognition of weak splice sites flanking short introns. Although studies on individual retained introns have been published, few systematic surveys of large amounts of data have been conducted on the mechanisms that lead to IR. Results TTo understand how sequence features are associated with or control IR, and to produce a generalized model that could reveal previously unknown signals that regulate this type of alternative splicing, we partitioned intron retention events observed in human cDNAs into two groups based on the relative abundance of both isoforms and compared relevant features. We found that a higher frequency of IR in human is associated with individual introns that have weaker splice sites, genes with shorter intron lengths, higher expression levels and lower density of both a set of exon splicing silencers (ESSs) and the intronic splicing enhancer GGG. Both groups of retained introns presented events conserved in mouse, in which the retained introns were also short and presented weaker splice sites. Conclusion Although our results confirmed that weaker splice sites are associated with IR, they showed that this feature alone cannot explain a non-negligible fraction of events. Our analysis suggests that cis-regulatory elements are likely to play a crucial role in regulating IR and also reveals 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 IR.

Caracterização funcional e estrutural das proteínas RUV-1 e RUV-2 do fungo Neurospora crassa

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Identical sequence patterns in the ends of exons and introns of human protein-coding genes

Intron splicing is one of the most important steps involved in the maturation process of a pre-mRNA. Although the sequence profiles around the splice sites have been studied extensively, the levels of sequence identity between the exonic sequences preceding the donor sites and the intronic sequences preceding the acceptor sites has not been examined as thoroughly. In this study we investigated identity patterns between the last 15 nucleotides of the exonic sequence preceding the 5' splice site and the intronic sequence preceding the 3' splice site in a set of human protein-coding genes that do not exhibit intron retention. We found that almost 60% of consecutive exons and introns in human protein-coding genes share at least two identical nucleotides at their 3' ends and, on average, the sequence identity length is 2.47 nucleotides. Based on our findings we conclude that the 3' ends of exons and introns tend to have longer identical sequences within a gene than when being taken from different genes. Our results hold even if the pairs are non-consecutive in the transcription order. (C) 2012 Elsevier Ltd. All rights reserved.

A new portal for the analysis of human splicing variants

Understanding alternative splicing is crucial to elucidate the mechanisms behind several biological phenomena, including diseases. The huge amount of expressed sequences available nowadays represents an opportunity and a challenge to catalog and display alternative splicing events (ASEs). Although several groups have faced this challenge with relative success, we still lack a computational tool that uses a simple and straightforward method to retrieve, name and present ASEs. Here we present SPLOOCE, a portal for the analysis of human splicing variants. SPLOOCE uses a method based on regular expressions for retrieval of ASEs. We propose a simple syntax that is able to capture the complexity of ASEs.

Identification of protein-coding and non-coding RNA expression profiles in CD34+ and in stromal cells in refractory anemia with ringed sideroblasts

Abstract Background Myelodysplastic syndromes (MDS) are a group of clonal hematological disorders characterized by ineffective hematopoiesis with morphological evidence of marrow cell dysplasia resulting in peripheral blood cytopenia. Microarray technology has permitted a refined high-throughput mapping of the transcriptional activity in the human genome. Non-coding RNAs (ncRNAs) transcribed from intronic regions of genes are involved in a number of processes related to post-transcriptional control of gene expression, and in the regulation of exon-skipping and intron retention. Characterization of ncRNAs in progenitor cells and stromal cells of MDS patients could be strategic for understanding gene expression regulation in this disease. Methods In this study, gene expression profiles of CD34+ cells of 4 patients with MDS of refractory anemia with ringed sideroblasts (RARS) subgroup and stromal cells of 3 patients with MDS-RARS were compared with healthy individuals using 44 k combined intron-exon oligoarrays, which included probes for exons of protein-coding genes, and for non-coding RNAs transcribed from intronic regions in either the sense or antisense strands. Real-time RT-PCR was performed to confirm the expression levels of selected transcripts. Results In CD34+ cells of MDS-RARS patients, 216 genes were significantly differentially expressed (q-value ≤ 0.01) in comparison to healthy individuals, of which 65 (30%) were non-coding transcripts. In stromal cells of MDS-RARS, 12 genes were significantly differentially expressed (q-value ≤ 0.05) in comparison to healthy individuals, of which 3 (25%) were non-coding transcripts. Conclusions These results demonstrated, for the first time, the differential ncRNA expression profile between MDS-RARS and healthy individuals, in CD34+ cells and stromal cells, suggesting that ncRNAs may play an important role during the development of myelodysplastic syndromes.

Ultraconserved elements in insect genomes: A highly conserved intronic sequence implicated in the control of homothorax mRNA splicing

Recently, we identified a large number of ultraconserved (uc) sequences in noncoding regions of human, mouse, and rat genomes that appear to be essential for vertebrate and amniote ontogeny. Here, we used similar methods to identify ultraconserved genomic regions between the insect species Drosophila melanogaster and Drosophila pseudoobscura, as well as the more distantly related Anopheles gambiae. As with vertebrates, ultraconserved sequences in insects appear to Occur primarily in intergenic and intronic sequences, and at intron-exon junctions. The sequences are significantly associated with genes encoding developmental regulators and transcription factors, but are less frequent and are smaller in size than in vertebrates. The longest identical, nongapped orthologous match between the three genomes was found within the homothorax (hth) gene. This sequence spans an internal exon-intron junction, with the majority located within the intron, and is predicted to form a highly stable stem-loop RNA structure. Real-time quantitative PCR analysis of different hth splice isoforms and Northern blotting showed that the conserved element is associated with a high incidence of intron retention in hth pre-mRNA, suggesting that the conserved intronic element is critically important in the post-transcriptional regulation of hth expression in Diptera.

CCK2 receptor splice variant with intron 4 retention in human gastrointestinal and lung tumours

The wild-type cholecystokinin type 2 (CCK(2)) receptor is expressed in many gastrointestinal and lung tumours. A splice variant of the CCK(2) receptor with retention of intron 4 (CCK(2)Ri4sv) showing constitutive activity associated with increased tumour growth was described in few colorectal, pancreatic and gastric cancers. Given the potential functional and clinical importance of this spliceoform, its occurrence was quantitatively characterized in a broad collection of 81 gastrointestinal and lung tumours, including insulinomas, ileal carcinoids, gastrointestinal stromal tumours (GIST), gastric, colorectal and pancreatic ductal adenocarcinomas, cholangiocellular and hepatocellular carcinomas, small cell lung cancers (SCLC), non-SCLC (nSCLC) and bronchopulmonary carcinoids, as well as 21 samples of corresponding normal tissues. These samples were assessed for transcript expression of total CCK(2) receptor, wild-type CCK(2) receptor and CCK(2)Ri4sv with end-point and real-time RT-PCR, and for total CCK(2) receptor protein expression on the basis of receptor binding with in vitro receptor autoradiography. Wild-type CCK(2) receptor transcripts were found in the vast majority of tumours and normal tissues. CCK(2)Ri4sv mRNA expression was present predominantly in insulinomas (incidence 100%), GIST (100%) and SCLC (67%), but rarely in pancreatic, colorectal and gastric carcinomas and nSCLC. It was not found in wild-type CCK(2) receptor negative tumours or any normal tissues tested. CCK(2)Ri4sv transcript levels in individual tumours were low, ranging from 0.02% to 0.14% of total CCK(2) receptor transcripts. In conclusion, the CCK(2)Ri4sv is a marker of specific gastrointestinal and lung tumours. With its high selectivity for and high incidence in SCLC and GIST, it may represent an attractive clinical target.

Nonsense mutation in the phosphofructokinase muscle subunit gene associated with retention of intron 10 in one of the isolated transcripts in Ashkenazi Jewish patients with Tarui disease.

Mutations in the human phosphofructokinase muscle subunit gene (PFKM) are known to cause myopathy classified as glycogenosis type VII (Tarui disease). Previously described molecular defects include base substitutions altering encoded amino acids or resulting in abnormal splicing. We report a mutation resulting in phosphofructokinase deficiency in three patients from an Ashkenazi Jewish family. Using a reverse transcription PCR assay, PFKM subunit transcripts differing by length were detected in skeletal muscle tissue of all three affected subjects. In the longer transcript, an insertion of 252 nucleotides totally homologous to the structure of the 10th intron of the PFKM gene was found separating exon 10 from exon 11. In addition, two single base transitions were identified by direct sequencing: [exon 6; codon 95; CGA (Arg) to TGA (stop)] and [exon 7; codon 172; ACC (Thr) to ACT (Thr)] in either transcript. Single-stranded conformational polymorphism and restriction enzyme analyses confirmed the presence of these point substitutions in genomic DNA and strongly suggested homozygosity for the pathogenic allele. The nonsense mutation at codon 95 appeared solely responsible for the phenotype in these patients, further expanding genetic heterogeneity of Tarui disease. Transcripts with and without intron 10 arising from identical mutant alleles probably resulted from differential pre-mRNA processing and may represent a novel message from the PFKM gene.

An intronic G run within HIV-1 intron 2 is critical for splicing regulation of vif mRNA

Within target T lymphocytes, human immunodeficiency virus type I (HIV-1) encounters the retroviral restriction factor APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; A3G), which is counteracted by the HIV-1 accessory protein Vif. Vif is encoded by intron-containing viral RNAs that are generated by splicing at 3' splice site (3'ss) A1 but lack splicing at 5'ss D2, which results in the retention of a large downstream intron. Hence, the extents of activation of 3'ss A1 and repression of D2, respectively, determine the levels of vif mRNA and thus the ability to evade A3G-mediated antiviral effects. The use of 3'ss A1 can be enhanced or repressed by splicing regulatory elements that control the recognition of downstream 5'ss D2. Here we show that an intronic G run (G(I2)-1) represses the use of a second 5'ss, termed D2b, that is embedded within intron 2 and, as determined by RNA deep-sequencing analysis, is normally inefficiently used. Mutations of G(I2)-1 and activation of D2b led to the generation of transcripts coding for Gp41 and Rev protein isoforms but primarily led to considerable upregulation of vif mRNA expression. We further demonstrate, however, that higher levels of Vif protein are actually detrimental to viral replication in A3G-expressing T cell lines but not in A3G-deficient cells. These observations suggest that an appropriate ratio of Vif-to-A3G protein levels is required for optimal virus replication and that part of Vif level regulation is effected by the novel G run identified here.

Membership retention within the sporting industry : factors affecting relationship dissolution

Relationship dissolution has been somewhat ignored in the study of relationship marketing paradigm. While there has been an abundance of literature giving broad conceptualizations on how to master the intricacies of relationships, very little has discussed the concept of relationship dissolution. This is especially true of the sporting industry, which does not yet understand the factors that contribute to members relinquishing their membership and severing relationship ties with the club. Team performance was found to be the most powerful predictor of relationship dissolution; however, both satisfaction with the sportscape and emotional bonds had a significant influence on the decision for a member not to renew their membership. Although team performance is mostly out of the hands of sport marketers, greater focus should be given to implementing strategies that enhance the emotional aspects of the club-member relationship while also improving aspects of the service facility.

A holistic approach to the support and engagement of first year students : a retention strategy

Institutions should enact holistic approaches that address students’ personal, social and academic engagement in the early weeks of first year to facilitate retention (Nelson, Kift & Clarke, 2008). This holistic approach is central to the FYE program at Queensland University of Technology (QUT), which was established to maximise learning engagement and hence positively influence the retention of commencing students. The program aims to • engage students in their learning through an intentionally designed and enacted curriculum (Kift, 2008) • facilitate timely access to life and learning support • promote a sense of belonging to the discipline, cohort and profession. The FYE program’s aims are achieved by strategic alliances between academic and professional staff across the institution.

A holistic approach to the support and engagement of first year students : a retention strategy.

Institutions should enact holistic approaches that address students’ personal, social and academic engagement in the early weeks of first year to facilitate retention (Nelson, Kift & Clarke, 2008). This holistic approach is central to the FYE program at Queensland University of Technology (QUT), which was established to maximise learning engagement and hence positively influence the retention of commencing students.