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Background: Amelogenesis imperfecta (AI) is a developmental enamel defect affecting the structure of enamel, esthetic appearance, and the tooth masticatory function. Gene mutations are reported to be relevant to AI. However, the mechanism underlying AI caused by different mutations is still unclear. This study aimed to reveal the molecular pathogenesis in AI families with 2 novel pre-mRNA splicing mutations. Methods: Two Chinese families with AI were recruited. Whole-exome sequencing and Sanger sequencing were performed to identify mutations in candidate genes. Minigene splicing assays were performed to analyze the mutation effects on mRNA splicing alteration. Furthermore, three-dimensional structures of mutant proteins were predicted by AlphaFold2 to evaluate the detrimental effect. Results: The affected enamel in family 1 was thin, rough, and stained, which was diagnosed as hypoplastic-hypomature AI. Genomic analysis revealed a novel splicing mutation (NM_001142.2: c.570 + 1G > A) in the intron 6 of amelogenin (AMELX) gene in family 1, resulting in a partial intron 6 retention effect. The proband in family 2 exhibited a typical hypoplastic AI, and the splicing mutation (NM_031889.2: c.123 + 4 A > G) in the intron 4 of enamelin (ENAM) gene was observed in the proband and her father. This mutation led to exon 4 skipping. The predicted structures showed that there were obvious differences in the mutation proteins compared with wild type, leading to impaired function of mutant proteins. Conclusions: In this study, we identified two new splicing mutations in AMELX and ENAM genes, which cause hypoplastic-hypomature and hypoplastic AI, respectively. These results expand the spectrum of genes causing AI and broaden our understanding of molecular genetic pathology of enamel formation. [ABSTRACT FROM AUTHOR]

Background: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by hamartomas in multiple organs associated with germline mutations in TSC1 and TSC2, including exonic, intronic, or mosaic mutations. Gastrointestinal (GI) tract Lymphangioleiomyomatosis (LAM) is an extremely rare manifestation of TSC, with few reported cases. Herein, we aimed to determine the driver mutation, pathogenesis, and relationship of germline and somatic mutations of LAM through whole-genome sequencing (WGS) of the tumor and blood samples and whole transcriptome sequencing (WTS) analysis. Case presentation: A nine-year-old girl with a full-blown TSC presented with abdominal masses detected during a routine check-up. Resected intestinal masses were diagnosed as LAM by thorough pathological examination. Interestingly, the LAM presented a somatic TSC2 gene mutation in exon 24 (p.R905W, c.C2713T), and the patient had intron retention by a novel germline mutation in the intron region of TSC2 (chr16:2126489, C > G). Conclusion: Our case suggests that intron retention by a single nucleotide intronic mutation of TSC2 is sufficient to develop severe manifestations of TSC, but the development of LAM requires an additional somatic oncogenic mutation of TSC2. [ABSTRACT FROM AUTHOR]

Background: Despite the growing interest in the female side of copulatory interactions, the roles played by differential expression and alternative splicing mechanisms of pre-RNA on tissues outside of the reproductive tract have remained largely unknown. Here we addressed these questions in the context of con- vs heterospecific matings between Drosophila mojavensis and its sister species, D. arizonae. We analyzed transcriptional responses in female heads using an integrated investigation of genome-wide patterns of gene expression, including differential expression (DE), alternative splicing (AS) and intron retention (IR). Results: Our results indicated that early transcriptional responses were largely congruent between con- and heterospecific matings but are substantially perturbed over time. Conspecific matings induced functional pathways related to amino acid balance previously associated with the brain's physiology and female postmating behavior. Heterospecific matings often failed to activate regulation of some of these genes and induced expression of additional genes when compared with those of conspecifically-mated females. These mechanisms showed functional specializations with DE genes mostly linked to pathways of proteolysis and nutrient homeostasis, while AS genes were more related to photoreception and muscle assembly pathways. IR seems to play a more general role in DE regulation during the female postmating response. Conclusions: We provide evidence showing that AS genes substantially perturbed by heterospecific matings in female heads evolve at slower evolutionary rates than the genome background. However, DE genes evolve at evolutionary rates similar, or even higher, than those of male reproductive genes, which highlights their potential role in sexual selection and the evolution of reproductive barriers. [ABSTRACT FROM AUTHOR]

Background: Intron retention (IR) has been traditionally overlooked as 'noise' and received negligible attention in the field of gene expression analysis. In recent years, IR has become an emerging field for interrogating transcriptomes because it has been recognized to carry out important biological functions such as gene expression regulation and it has been found to be associated with complex diseases such as cancers. However, methods for detecting IR today are limited. Thus, there is a need to develop novel methods to improve IR detection. Results: Here we present iREAD (intron REtention Analysis and Detector), a tool to detect IR events genome-wide from high-throughput RNA-seq data. The command line interface for iREAD is implemented in Python. iREAD takes as input a BAM file, representing the transcriptome, and a text file containing the intron coordinates of a genome. It then 1) counts all reads that overlap intron regions, 2) detects IR events by analyzing the features of reads such as depth and distribution patterns, and 3) outputs a list of retained introns into a tab-delimited text file. iREAD provides significant added value in detecting IR compared with output from IRFinder with a higher AUC on all datasets tested. Both methods showed low false positive rates and high false negative rates in different regimes, indicating that use together is generally beneficial. The output from iREAD can be directly used for further exploratory analysis such as differential intron expression and functional enrichment. The software is freely available at https://github.com/genemine/iread. Conclusion: Being complementary to existing tools, iREAD provides a new and generic tool to interrogate poly-A enriched transcriptomic data of intron regions. Intron retention analysis provides a complementary approach for understanding transcriptome. [ABSTRACT FROM AUTHOR]

Background: In eukaryotes, most genes code for multiple transcript isoforms that are generated through the complex and tightly regulated process of RNA splicing. Despite arising from identical precursor transcripts, alternatively spliced RNAs can have dramatically different functions. Transcriptome complexity is elevated further by the production of circular RNAs (circRNAs), another class of mature RNA that results from the splicing of a downstream splice donor to an upstream splice acceptor. While there has been a rapid expansion of circRNA catalogs in the last few years through the utilization of next generation sequencing approaches, our understanding of the mechanisms and regulation of circular RNA biogenesis, the impact that circRNA generation has on parental transcript processing, and the functions carried out by circular RNAs remains limited. Results: Here, we present a visualization and analysis tool, SpliceV, that rapidly plots all relevant forward- and back-splice data, with exon and single nucleotide level coverage information from RNA-seq experiments in a publication quality format. SpliceV also integrates analysis features that assist investigations into splicing regulation and transcript functions through the display of predicted RNA binding protein sites and the configuration of repetitive elements along the primary transcript. Conclusions: SpliceV is an easy-to-use splicing visualization tool, compatible with both Python 2.7 and 3+, and distributed under the GNU Public License. The source code is freely available for download at https://github.com/flemingtonlab/SpliceV and can be installed from PyPI using pip. [ABSTRACT FROM AUTHOR]

Background: Intron retention (IR), the most prevalent alternative splicing form in plants, plays a critical role in gene expression during plant development and stress response. However, the molecular mechanisms underlying IR regulation remain largely unknown. Results: Knockdown of SDG725, a histone H3 lysine 36 (H3K36)-specific methyltransferase in rice, leads to alterations of IR in more than 4700 genes. Surprisingly, IR events are globally increased at the 5′ region but decreased at the 3′ region of the gene body in the SDG725-knockdown mutant. Chromatin immunoprecipitation sequencing analyses reveal that SDG725 depletion results in a genome-wide increase of the H3K36 mono-methylation (H3K36me1) but, unexpectedly, promoter-proximal shifts of H3K36 di- and tri-methylation (H3K36me2 and H3K36me3). Consistent with the results in animals, the levels of H3K36me1/me2/me3 in rice positively correlate with gene expression levels, whereas shifts of H3K36me2/me3 coincide with position-specific alterations of IR. We find that either H3K36me2 or H3K36me3 alone contributes to the positional change of IR caused by SDG725 knockdown, although IR shift is more significant when both H3K36me2 and H3K36me3 modifications are simultaneously shifted. Conclusions: Our results revealed that SDG725 modulates IR in a position-specific manner, indicating that H3K36 methylation plays a role in RNA splicing, probably by marking the retained introns in plants. [ABSTRACT FROM AUTHOR]

Background: In-depth study of the intron retention levels of transcripts provide insights on the mechanisms regulating pre-mRNA splicing efficiency. Additionally, detailed analysis of retained introns can link these introns to post-transcriptional regulation or identify aberrant splicing events in human diseases. Results: We present IntEREst, Intron–Exon Retention Estimator, an R package that supports rigorous analysis of non-annotated intron retention events (in addition to the ones annotated by RefSeq or similar databases), and support intra-sample in addition to inter-sample comparisons. It accepts binary sequence alignment/map (.bam) files as input and determines genome-wide estimates of intron retention or exon-exon junction levels. Moreover, it includes functions for comparing subsets of user-defined introns (e.g. U12-type vs U2-type) and its plotting functions allow visualization of the distribution of the retention levels of the introns. Statistical methods are adapted from the DESeq2, edgeR and DEXSeq R packages to extract the significantly more or less retained introns. Analyses can be performed either sequentially (on single core) or in parallel (on multiple cores). We used IntEREst to investigate the U12- and U2-type intron retention in human and plant RNAseq dataset with defects in the U12-dependent spliceosome due to mutations in the ZRSR2 component of this spliceosome. Additionally, we compared the retained introns discovered by IntEREst with that of other methods and studies. Conclusion: IntEREst is an R package for Intron retention and exon-exon junction levels analysis of RNA-seq data. Both the human and plant analyses show that the U12-type introns are retained at higher level compared to the U2-type introns already in the control samples, but the retention is exacerbated in patient or plant samples carrying a mutated ZRSR2 gene. Intron retention events caused by ZRSR2 mutation that we discovered using IntEREst (DESeq2 based function) show considerable overlap with the retained introns discovered by other methods (e.g. IRFinder and edgeR based function of IntEREst). Our results indicate that increase in both the number of biological replicates and the depth of sequencing library promote the discovery of retained introns, but the effect of library size gradually decreases with more than 35 million reads mapped to the introns. [ABSTRACT FROM AUTHOR]

Background: Intron retention (IR) is the most prevalent form of alternative splicing in plants. IR, like other forms of alternative splicing, has an important role in increasing gene product diversity and regulating transcript functionality. Splicing is known to occur co-transcriptionally and is influenced by the speed of transcription which in turn, is affected by chromatin structure. It follows that chromatin structure may have an important role in the regulation of splicing, and there is preliminary evidence in metazoans to suggest that this is indeed the case; however, nothing is known about the role of chromatin structure in regulating IR in plants. DNase I-seq is a useful experimental tool for genome-wide interrogation of chromatin accessibility, providing information on regions of chromatin with very high likelihood of cleavage by the enzyme DNase I, known as DNase I Hypersensitive Sites (DHSs). While it is well-established that promoter regions are highly accessible and are over-represented with DHSs, not much is known about DHSs in the bodies of genes, and their relationship to splicing in general, and IR in particular. Results: In this study we use publicly available DNase I-seq data in arabidopsis and rice to investigate the relationship between IR and chromatin structure. We find that IR events are highly enriched in DHSs in both species. This implies that chromatin is more open in retained introns, which is consistent with a kinetic model of the process whereby higher speeds of transcription in those regions give less time for the spliceosomal machinery to recognize and splice out those introns co-transcriptionally. The more open chromatin in IR can also be the result of regulation mediated by DNA-binding proteins. To test this, we performed an exhaustive search for footprints left by DNA-binding proteins that are associated with IR. We identified several hundred short sequence elements that exhibit footprints in their DNase I-seq coverage, the telltale sign for binding events of a regulatory protein, protecting its binding site from cleavage by DNase I. A highly significant fraction of those sequence elements are conserved between arabidopsis and rice, a strong indication of their functional importance. Conclusions: In this study we have established an association between IR and chromatin accessibility, and presented a mechanistic hypothesis that explains the observed association from the perspective of the co-transcriptional nature of splicing. Furthermore, we identified conserved sequence elements for DNA-binding proteins that affect splicing. [ABSTRACT FROM AUTHOR]

Background: The DMD gene encoding dystrophin is mutated in Duchenne muscular dystrophy, a fatal progressive muscle wasting disease. DMD has also been shown to act as a tumor suppressor gene. Rhabdomyosarcoma (RMS) is a mesodermal sarcoma that shares characteristics of skeletal muscle precursors. Products of the DMD gene in RMS have not yet been fully clarified. Here, DMD products were analyzed in CRL-2061 cells established from alveolar RMS. Methods: The 14-kb long DMD cDNA was PCR amplified as 20 separated fragments, as were nine short intron regions. Dystrophin was analyzed by Western blotting using an antibody against the C-terminal region of dystrophin. Results: Sixteen of the 20 DMD cDNA fragments could be amplified from CRL-2061 cells as muscle cDNA. Three fragments included aberrant gene products, including one in which exon 71 was omitted and one each with retention of introns 40 and 58. In one fragment, extending from exon 70 to 79, no normally spliced product was obtained. Rather, six alternatively spliced products were identified, including a new product deleting exon 73, with the most abundant product showing deletion of exon 78. Although dystrophin expression was expected in CRL-2061 cells, western blotting of cell lysates showed no evidence of dystrophin, suggesting that translation of full-length DMD mRNA was inhibited by intron retention that generated a premature stop codon. Intron specific PCR amplification of nine short introns, showed retention of introns 40, 58, and 70, which constituted about 60, 25 and 9%, respectively, of the total PCR amplified products. The most abundant DMD transcript contained two abnormalities, intron 40 retention and exon 78 skipping. Conclusions: Intron-specific PCR amplification showed that DMD transcripts contained high levels of introns 40, 58 and 70. Retention of these introns may have been responsible for the lack of dystrophin expression by CRL-2061 cells, thereby abolishing the tumor suppressor activity of dystrophin. [ABSTRACT FROM AUTHOR]

Background: Alternative splicing (AS) regulation is extensive and shapes the functional complexity of higher organisms. However, the contribution of alternative splicing to fungal biology is not well studied. Results: This study provides sequences of the transcriptomes of the plant wilt pathogen Verticillium dahliae, using two different strains and multiple methods for cDNA library preparations. We identified alternatively spliced mRNA isoforms in over a half of the multi-exonic fungal genes. Over one-thousand isoforms involve TopHat novel splice junction; multiple types of combinatory alternative splicing patterns were identified. We showed that one Verticillium gene could use four different 5' splice sites and two different 3' donor sites to produce up to five mature mRNAs, representing one of the most sophisticated alternative splicing model in eukaryotes other than animals. Hundreds of novel intron types involving a pair of new splice sites were identified in the V. dahliae genome. All the types of AS events were validated by using RT-PCR. Functional enrichment analysis showed that AS genes are involved in most known biological functions and enriched in ATP biosynthesis, sexual/asexual reproduction, morphogenesis, signal transduction etc., predicting that the AS regulation modulates mRNA isoform output and shapes the V. dahliae proteome plasticity of the pathogen in response to the environmental and developmental changes. Conclusions: These findings demonstrate the comprehensive alternative splicing mechanisms in a fungal plant pathogen, which argues the importance of this fungus in developing complicate genome regulation strategies in eukaryotes. [ABSTRACT FROM AUTHOR]

Background: Alternative splicing is crucial for proteome diversity and functional complexity in higher organisms. However, the alternative splicing landscape in fungi is still elusive. Results: The transcriptome of the filamentous fungus Trichoderma longibrachiatum was deep sequenced using Illumina Solexa technology. A total of 14305 splice junctions were discovered. Analyses of alternative splicing events revealed that the number of all alternative splicing events (10034), intron retentions (IR, 9369), alternative 5' splice sites (A5SS, 167), and alternative 3' splice sites (A3SS, 302) is 7.3, 7.4, 5.1, and 5.9-fold higher, respectively, than those observed in the fungus Aspergillus oryzae using Illumina Solexa technology. This unexpectedly high ratio of alternative splicing suggests that alternative splicing is important to the transcriptome diversity of T. longibrachiatum. Alternatively spliced introns had longer lengths, higher GC contents, and lower splice site scores than constitutive introns. Further analysis demonstrated that the isoform relative frequencies were correlated with the splice site scores of the isoforms. Moreover, comparative transcriptomics determined that most enzymes related to glycolysis and the citrate cycle and glyoxylate cycle as well as a few carbohydrate-active enzymes are transcriptionally regulated. Conclusions: This study, consisting of a comprehensive analysis of the alternative splicing landscape in the filamentous fungus T. longibrachiatum, revealed an unexpectedly high ratio of alternative splicing events and provided new insights into transcriptome diversity in fungi. [ABSTRACT FROM AUTHOR]

Background: Global concern over the rapid expansion of the Asian tiger mosquito, Aedes albopictus, and its vector competence has highlighted an urgent need to improve currently available population control methods, like the Sterile Insect Technique. Knowledge of the sex determination cascade is a prerequisite for the development of early-stage sexing systems. To this end, we have characterised the putative sex determination gene, Nix, in this species. In Aedes species the chromosome complement consists of three pairs of chromosomes. The sex determination alleles are linked to the smallest homomorphic chromosome. Results: We identified the male-specific chromosome 1 of Ae. albopictus that carries the putative male-determining gene Nix. We have also characterised the complete genomic sequence of the Nix gene which is composed of two exons and a short intron. The gene displays different levels of intron retention during development. Comparison of DNA sequences covering most of the Nix gene from individuals across the species range revealed no polymorphism. Conclusions: Our characterisation of the Nix gene in Ae. albopictus represents an initial step in the analysis of the sex determination cascade in this species. We found evidence of intron retention (IR) in Nix. IR might play a role in regulating the expression of Nix during development. Our results provide the basis for the development of new genetic control strategies. [ABSTRACT FROM AUTHOR]

Background: The DMD gene encoding dystrophin is mutated in Duchenne muscular dystrophy, a fatal progressive muscle wasting disease. DMD has also been shown to act as a tumor suppressor gene. Rhabdomyosarcoma (RMS) is a mesodermal sarcoma that shares characteristics of skeletal muscle precursors. Products of the DMD gene in RMS have not yet been fully clarified. Here, DMD products were analyzed in CRL-2061 cells established from alveolar RMS., Methods: The 14-kb long DMD cDNA was PCR amplified as 20 separated fragments, as were nine short intron regions. Dystrophin was analyzed by Western blotting using an antibody against the C-terminal region of dystrophin., Results: Sixteen of the 20 DMD cDNA fragments could be amplified from CRL-2061 cells as muscle cDNA. Three fragments included aberrant gene products, including one in which exon 71 was omitted and one each with retention of introns 40 and 58. In one fragment, extending from exon 70 to 79, no normally spliced product was obtained. Rather, six alternatively spliced products were identified, including a new product deleting exon 73, with the most abundant product showing deletion of exon 78. Although dystrophin expression was expected in CRL-2061 cells, western blotting of cell lysates showed no evidence of dystrophin, suggesting that translation of full-length DMD mRNA was inhibited by intron retention that generated a premature stop codon. Intron specific PCR amplification of nine short introns, showed retention of introns 40, 58, and 70, which constituted about 60, 25 and 9%, respectively, of the total PCR amplified products. The most abundant DMD transcript contained two abnormalities, intron 40 retention and exon 78 skipping., Conclusions: Intron-specific PCR amplification showed that DMD transcripts contained high levels of introns 40, 58 and 70. Retention of these introns may have been responsible for the lack of dystrophin expression by CRL-2061 cells, thereby abolishing the tumor suppressor activity of dystrophin.

Background: Alternative splicing (AS) regulation is extensive and shapes the functional complexity of higher organisms. However, the contribution of alternative splicing to fungal biology is not well studied., Results: This study provides sequences of the transcriptomes of the plant wilt pathogen Verticillium dahliae, using two different strains and multiple methods for cDNA library preparations. We identified alternatively spliced mRNA isoforms in over a half of the multi-exonic fungal genes. Over one-thousand isoforms involve TopHat novel splice junction; multiple types of combinatory alternative splicing patterns were identified. We showed that one Verticillium gene could use four different 5' splice sites and two different 3' donor sites to produce up to five mature mRNAs, representing one of the most sophisticated alternative splicing model in eukaryotes other than animals. Hundreds of novel intron types involving a pair of new splice sites were identified in the V. dahliae genome. All the types of AS events were validated by using RT-PCR. Functional enrichment analysis showed that AS genes are involved in most known biological functions and enriched in ATP biosynthesis, sexual/asexual reproduction, morphogenesis, signal transduction etc., predicting that the AS regulation modulates mRNA isoform output and shapes the V. dahliae proteome plasticity of the pathogen in response to the environmental and developmental changes., Conclusions: These findings demonstrate the comprehensive alternative splicing mechanisms in a fungal plant pathogen, which argues the importance of this fungus in developing complicate genome regulation strategies in eukaryotes.

Intron retention (IR) occurs when an intron is transcribed into pre-mRNA and remains in the final mRNA. We have developed a program and database called IRFinder to accurately detect IR from mRNA sequencing data. Analysis of 2573 samples showed that IR occurs in all tissues analyzed, affects over 80% of all coding genes and is associated with cell differentiation and the cell cycle. Frequently retained introns are enriched for specific RNA binding protein sites and are often retained in clusters in the same gene. IR is associated with lower protein levels and intron-retaining transcripts that escape nonsense-mediated decay are not actively translated. Electronic supplementary material The online version of this article (doi:10.1186/s13059-017-1184-4) contains supplementary material, which is available to authorized users.