Modes Of Alternative Splicing Research Articles

DeepRetention: A Deep Learning Approach for Intron Retention Detection

As the least understood mode of alternative splicing, Intron Retention (IR) is emerging as an interesting area and has attracted more and more attention in the field of gene regulation and disease studies. Existing methods detect IR exclusively based on one or a few predefined metrics describing local or summarized characteristics of retained introns. These metrics are not able to describe the pattern of sequencing depth of intronic reads, which is an intuitive and informative characteristic of retained introns. We hypothesize that incorporating the distribution pattern of intronic reads will improve the accuracy of IR detection. Here we present DeepRetention, a novel approach for IR detection by modeling the pattern of sequencing depth of introns. Due to the lack of a gold standard dataset of IR, we first compare DeepRetention with two state-of-the-art methods, i.e. iREAD and IRFinder, on simulated RNA-seq datasets with retained introns. The results show that DeepRetention outperforms these two methods. Next, DeepRetention performs well when it is applied to third-generation long-read RNA-seq data, while IRFinder and iREAD are not applicable to detecting IR from the third-generation sequencing data. Further, we show that IRs predicted by DeepRetention are biologically meaningful on an RNA-seq dataset from Alzheimer's Disease (AD) samples. The differential IRs are found to be significantly associated with AD based on statistical evaluation of an AD-specific functional gene network. The parent genes of differential IRs are enriched in AD-related functions. In summary, DeepRetention detects IR from a new angle of view, providing a valuable tool for IR analysis.

A comparison of computational approaches for intron retention detection

Intron Retention (IR) is an alternative splicing mode through which introns are retained in mature RNAs rather than being spliced in most cases. IR has been gaining increasing attention in recent years because of its recognized association with gene expression regulation and complex diseases. Continuous efforts have been dedicated to the development of IR detection methods. These methods differ in their metrics to quantify retention propensity, performance to detect IR events, functional enrichment of detected IRs, and computational speed. A systematic experimental comparison would be valuable to the selection and use of existing methods. In this work, we conduct an experimental comparison of existing IR detection methods. Considering the unavailability of a gold standard dataset of intron retention, we compare the IR detection performance on simulation datasets. Then, we compare the IR detection results with real RNA-Seq data. We also describe the use of differential analysis methods to identify disease-associated IRs and compare differential IRs along with their Gene Ontology enrichment, which is illustrated on an Alzheimer's disease RNA-Seq dataset. We discuss key principles and features of existing approaches and outline their differences. This systematic analysis provides helpful guidance for interrogating transcriptomic data from the point of view of IR.

CRISPR/Cas9 induces exon skipping that facilitates development of fragrant rice.

Splicing of precursor-mRNAs (pre-mRNAs) is a critical biological process of gene expression. Normally, pre-mRNAs are processed by spliceosomes to produce mature mRNAs by removing introns at 5'-(donor) and 3'-(acceptor) splice sites based on the canonical GU-AG rule (Reddy et al., 2013). Mutation at either the intron donor or acceptor sites should cause mRNA mis-splicing. Recently, an exon skipping method has been developed to generate loss of gene function in mammalian cells using base editors to mutate nucleotides at the acceptor sites (Gapinske et al., 2018).

A chromosome-scale assembly of the black gram (Vigna mungo) genome.

Black gram (Vigna mungo) is an important short duration grain legume crop. Black gram seeds provide an inexpensive source of dietary protein. Here, we applied the 10X Genomics linked-read technology to obtain a de novo whole genome assembly of V.mungo cultivated variety Chai Nat 80 (CN80). The preliminary assembly contained 12,228 contigs and had an N50 length of 5.2Mb. Subsequent scaffolding using the long-range Chicago and HiC techniques yielded the first high-quality, chromosome-level assembly of 499Mb comprising 11 pseudomolecules. Comparative genomics analyses based on sequence information from single-copy orthologous genes revealed that black gram and mungbean (Vignaradiata) diverged about 2.7 million years ago . The transversion rate (4DTv) analysis in V.mungo revealed no evidence supporting a recent genome-wide duplication event observed in the tetraploid créole bean (Vigna reflexo-pilosa). The proportion of repetitive elements in the black gram genome is slightly lower than the numbers reported for related Vigna species. The majority of long terminal repeat retrotransposons appeared to integrate into the genome within the last five million years. We also examined alternative splicing events in V.mungo using full-length transcript sequences. While intron retention was the most prevalent mode of alternative splicing in several plant species, alternative 3' acceptor site selection represented the majority of events in black gram. Our high-quality genome assembly along with the genomic variation information from the germplasm provides valuable resources for accelerating the development of elite varieties through marker-assisted breeding and for future comparative genomics and phylogenetic studies in legume species.

Intron Retention as a Mode for RNA-Seq Data Analysis

Intron retention (IR) is an alternative splicing mode whereby introns, rather than being spliced out as usual, are retained in mature mRNAs. It was previously considered a consequence of mis-splicing and received very limited attention. Only recently has IR become of interest for transcriptomic data analysis owing to its recognized roles in gene expression regulation and associations with complex diseases. In this article, we first review the function of IR in regulating gene expression in a number of biological processes, such as neuron differentiation and activation of CD4+ T cells. Next, we briefly review its association with diseases, such as Alzheimer's disease and cancers. Then, we describe state-of-the-art methods for IR detection, including RNA-seq analysis tools IRFinder and iREAD, highlighting their underlying principles and discussing their advantages and limitations. Finally, we discuss the challenges for IR detection and potential ways in which IR detection methods could be improved.

Selective release of circRNAs in platelet-derived extracellular vesicles

ABSTRACTCircular RNAs (circRNAs) are a novel class of noncoding RNAs present in all eukaryotic cells investigated so far and generated by a special mode of alternative splicing of pre-mRNAs. Thereby, single exons, or multiple adjacent and spliced exons, are released in a circular form. CircRNAs are cell-type specifically expressed, are unusually stable, and can be found in various body fluids such as blood and saliva. Here we analysed circRNAs and the corresponding linear splice isoforms from human platelets, where circRNAs are particularly abundant, compared with other hematopoietic cell types. In addition, we isolated extracellular vesicles from purified and in vitro activated human platelets, using density-gradient centrifugation, followed by RNA-seq analysis for circRNA detection. We could demonstrate that circRNAs are packaged and released within both types of vesicles (microvesicles and exosomes) derived from platelets. Interestingly, we observed a selective release of circRNAs into the vesicles, suggesting a specific sorting mechanism. In sum, circRNAs represent yet another class of extracellular RNAs that circulate in the body and may be involved in signalling pathways. Since platelets are essential for central physiological processes such as haemostasis, wound healing, inflammation and cancer metastasis, these findings should greatly extend the potential of circRNAs as prognostic and diagnostic biomarkers.

Characterization of an insect heterodimeric voltage-gated sodium channel with unique alternative splicing mode.

Recent discovery of the heterodimeric voltage-gated sodium channels (Nav) in two aphid species, Acyrthosiphon pisum and Myzus persicae, aroused interest in exploring whether this kind of channel is conserved for aphids. Herewith, we aim to provide evidence for the conservation of heterodimeric Navs in aphids and investigate whether they have unique splicing patterns. We found that the only identifiable Nav from Toxoptera citricida consisted of two subunits, forming a heterodimeric Nav, which carried an atypical "DENS" ion selectivity filter and a conventional "MFM" inactivation gate, confirming the heterodimeric Navs' conservation within aphids. These unique heterodimeric channels may form a new Nav subfamily, specific to aphids. A more ancient member of four-domain Nav homolog was well preserved in T. citricida, carrying a typical "DEEA" and "MFL" motif. The presence of "DENS" in mammalian Naxs and "DEKT" in a fungus Nav suggested that the heterodimeric Navs may still preserve Na+ permeability. Sequencing 46 clones from nymphs and adults exposed unique splicing patterns for this heterodimeric Nav from T. citricida, revealing 7 alternatively spliced exons, evidencing that exon 5 was no longer unique to Bombyx mori, and exon k/l was semi-mutually exclusive. Two previously undescribed optional exons and a SNP site seemingly unique to aphids were identified. In conclusion, the dimeric Navs might form a new aphids-specific heterodimeric Nav subfamily. This dimeric Nav from T. citricida was characterized with distinguishable alternative splicing modes, exemplified by the discovery of two novel alternative exons and unique usage patterns of alternative exons.

Functional SNPs of INCENP Affect Semen Quality by Alternative Splicing Mode and Binding Affinity with the Target Bta-miR-378 in Chinese Holstein Bulls

Inner centromere protein (INCENP) plays an important role in mitosis and meiosis as the main member of chromosomal passenger protein complex (CPC). To investigate the functional markers of the INCENP gene associated with semen quality, the single nucleotide polymorphisms (SNPs) g.19970 A>G and g.34078 T>G were identified and analyzed. The new splice variant INCENP-TV is characterized by the deletion of exon 12. The g.19970 A>G in the exonic splicing enhancer (ESE) motif region results in an aberrant splice variant by constructing two minigene expression vectors using the pSPL3 exon capturing vector and transfecting vectors into MLTC-1 cells. INCENP-TV was more highly expressed than INCENP-reference in adult bull testes. The g.34078 T>G located in the binding region of bta-miR-378 could affect the expression of INCENP, which was verified by luciferase assay. To analyze comprehensively the correlation of SNPs with sperm quality, haplotype combinations constructed by g.19970 A>G and g.34078 T>G, as well as g.-692 C>T and g.-556 G>T reported in our previous studies, were analyzed. The bulls with H1H12 and H2H2 exhibited a higher ejaculate volume than those with H2H10 and H9H12, respectively (P < 0.05). Bulls with H11H11 and H2H10 exhibited higher initial sperm motility than those with H2H2 (P < 0.05). The expression levels of INCENP in bulls with H1H12 and H11H11 were significantly higher than those in bulls with H9H12 (P < 0.05), as determined by qRT-PCR. Findings suggest that g.19970 A>G and g.34078 T>G in INCENP both of which appear to change the molecular and biological characteristics of the mRNA transcribed from the locus may serve as a biomarkers of male bovine fertility by affecting alternative splicing mode and binding affinity with the target bta-miR-378.

The Sequence Characteristics and Expression Models Reveal Superoxide Dismutase Involved in Cold Response and Fruiting Body Development in Volvariella volvacea.

As the first defence for cells to counteract the toxicity of active oxygen, superoxide dismutase (SOD) plays an important role in the response of living organisms to stress and cell differentiation. One extracellular Cu-ZnSOD (ecCu-ZnSOD), and two MnSODs, were identified based on the Volvariella volvacea genome sequence. All three genes have complicated alternative splicing modes during transcription; only when the fourth intron is retained can the Vv_Cu-Znsod1 gene be translated into a protein sequence with SOD functional domains. The expression levels of the three sod genes in the pilei are higher than in the stipe. The Vv_Cu-Znsod1 and the Vv_Mnsod2 are co-expressed in different developmental stages of the fruiting body, with the highest level of expression in the pilei of the egg stage, and they show a significant, positive correlation with the efficiency of karyogamy, indicating the potential role of these two genes during karyogamy. The expression of the ecCu-Znsod and two Vv_Mnsod genes showed a significant up-regulated when treated by cold stress for one hour; however, the lack of the intracellular Cu-ZnSOD encoding gene (icCu-Znsod) and the special locus of the ecCu-Znsod gene initiation codon suggested a possible reason for the autolysis phenomenon of V. volvacea in cold conditions.

Global analysis of physical and functional RNA targets of hnRNP L reveals distinct sequence and epigenetic features of repressed and enhanced exons.

HnRNP L is a ubiquitous splicing-regulatory protein that is critical for the development and function of mammalian T cells. Previous work has identified a few targets of hnRNP L-dependent alternative splicing in T cells and has described transcriptome-wide association of hnRNP L with RNA. However, a comprehensive analysis of the impact of hnRNP L on mRNA expression remains lacking. Here we use next-generation sequencing to identify transcriptome changes upon depletion of hnRNP L in a model T-cell line. We demonstrate that hnRNP L primarily regulates cassette-type alternative splicing, with minimal impact of hnRNP L depletion on transcript abundance, intron retention, or other modes of alternative splicing. Strikingly, we find that binding of hnRNP L within or flanking an exon largely correlates with exon repression by hnRNP L. In contrast, exons that are enhanced by hnRNP L generally lack proximal hnRNP L binding. Notably, these hnRNP L-enhanced exons share sequence and context features that correlate with poor nucleosome positioning, suggesting that hnRNP may enhance inclusion of a subset of exons via a cotranscriptional or epigenetic mechanism. Our data demonstrate that hnRNP L controls inclusion of a broad spectrum of alternative cassette exons in T cells and suggest both direct RNA regulation as well as indirect mechanisms sensitive to the epigenetic landscape.

Deep developmental transcriptome sequencing uncovers numerous new genes and enhances gene annotation in the sponge Amphimedon queenslandica.

BackgroundThe demosponge Amphimedon queenslandica is amongst the few early-branching metazoans with an assembled and annotated draft genome, making it an important species in the study of the origin and early evolution of animals. Current gene models in this species are largely based on in silico predictions and low coverage expressed sequence tag (EST) evidence.ResultsAmphimedon queenslandica protein-coding gene models are improved using deep RNA-Seq data from four developmental stages and CEL-Seq data from 82 developmental samples. Over 86% of previously predicted genes are retained in the new gene models, although 24% have additional exons; there is also a marked increase in the total number of annotated 3’ and 5’ untranslated regions (UTRs). Importantly, these new developmental transcriptome data reveal numerous previously unannotated protein-coding genes in the Amphimedon genome, increasing the total gene number by 25%, from 30,060 to 40,122. In general, Amphimedon genes have introns that are markedly smaller than those in other animals and most of the alternatively spliced genes in Amphimedon undergo intron-retention; exon-skipping is the least common mode of alternative splicing. Finally, in addition to canonical polyadenylation signal sequences, Amphimedon genes are enriched in a number of unique AT-rich motifs in their 3’ UTRs.ConclusionsThe inclusion of developmental transcriptome data has substantially improved the structure and composition of protein-coding gene models in Amphimedon queenslandica, providing a more accurate and comprehensive set of genes for functional and comparative studies. These improvements reveal the Amphimedon genome is comprised of a remarkably high number of tightly packed genes. These genes have small introns and there is pervasive intron retention amongst alternatively spliced transcripts. These aspects of the sponge genome are more similar unicellular opisthokont genomes than to other animal genomes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1588-z) contains supplementary material, which is available to authorized users.

Exon Circularization Requires Canonical Splice Signals

Circular RNAs (circRNAs), an abundant class of noncoding RNAs in higher eukaryotes, are generated from pre-mRNAs by circularization of adjacent exons. Using a set of 15 circRNAs, we demonstrated their cell-type-specific expression and circular versus linear processing in mammalian cells. Northern blot analysis combined with RNase H cleavage conclusively proved a circular configuration for two examples, LPAR1 and HIPK3. To address the circularization mechanism, we analyzed the sequence requirements using minigenes derived from natural circRNAs. Both canonical splice sites are required for circularization, although they vary in flexibility and potential use of cryptic sites. Surprisingly, we found that no specific circRNA exon sequence is necessary and that potential flanking intron structures can modulate circularization efficiency. In combination with splice inhibitor assays, our results argue that the canonical spliceosomal machinery functions in circRNA biogenesis, constituting an alternative splicing mode.

Characterization of the Tomato ARF Gene Family Uncovers a Multi-Levels Post-Transcriptional Regulation Including Alternative Splicing

BackgroundThe phytohormone auxin is involved in a wide range of developmental processes and auxin signaling is known to modulate the expression of target genes via two types of transcriptional regulators, namely, Aux/IAA and Auxin Response Factors (ARF). ARFs play a major role in transcriptional activation or repression through direct binding to the promoter of auxin-responsive genes. The present study aims at gaining better insight on distinctive structural and functional features among ARF proteins.ResultsBuilding on the most updated tomato (Solanum lycopersicon) reference genome sequence, a comprehensive set of ARF genes was identified, extending the total number of family members to 22. Upon correction of structural annotation inconsistencies, renaming the tomato ARF family members provided a consensus nomenclature for all ARF genes across plant species. In silico search predicted the presence of putative target site for small interfering RNAs within twelve Sl-ARFs while sequence analysis of the 5′-leader sequences revealed the presence of potential small uORF regulatory elements. Functional characterization carried out by transactivation assay partitioned tomato ARFs into repressors and activators of auxin-dependent gene transcription. Expression studies identified tomato ARFs potentially involved in the fruit set process. Genome-wide expression profiling using RNA-seq revealed that at least one third of the gene family members display alternative splicing mode of regulation during the flower to fruit transition. Moreover, the regulation of several tomato ARF genes by both ethylene and auxin, suggests their potential contribution to the convergence mechanism between the signaling pathways of these two hormones.ConclusionAll together, the data bring new insight on the complexity of the expression control of Sl-ARF genes at the transcriptional and post-transcriptional levels supporting the hypothesis that these transcriptional mediators might represent one of the main components that enable auxin to regulate a wide range of physiological processes in a highly specific and coordinated manner.

NOX2β: A Novel Splice Variant of NOX2 That Regulates NADPH Oxidase Activity in Macrophages

Nox2 oxidase is one isoform in a family of seven NADPH oxidases that generate reactive oxygen species (ROS) and thereby contribute to physiological and pathological processes including host defense, redox signaling and oxidative tissue damage. While alternative mRNA splicing has been shown to influence the activity of several Nox-family proteins, functionally relevant splice variants of Nox2 have not previously been identified. We immunoscreened several mouse tissues and cells for the presence of truncated Nox2 proteins and identified a 30 kDa protein in lung, spleen and macrophages. RT-PCR analysis of mRNA from primary and immortalised (RAW264.7) mouse macrophages, and from human alveolar macrophages, identified a truncated Nox2 transcript which, upon sequence analysis, was found to be a product of the ‘exon skipping’ mode of alternative splicing, lacking exons 4–10 of the Nox2 gene. The predicted protein is comparable in size to that identified by immunoscreening and contains two transmembrane helices and an extended cytosolic C-terminus with binding sites for NADPH and the Nox organiser protein p47phox. Importantly, selective siRNA-mediated knockdown of the transcript reduced expression of the 30 kDa protein in macrophages, and suppressed phorbol ester-stimulated ROS production by 50%. We thus provide the first evidence that Nox2 undergoes alternative mRNA splicing to yield a 30 kDa protein – herein termed Nox2β – that regulates NADPH oxidase activity in macrophages from mice and humans. The discovery of Nox2β paves the way for future examination of its role in physiological and pathological processes.

Identification of glutinous maize landraces and inbred lines with altered transcription of waxy gene

Waxy maize has little or very low content of amylose (<5 %) in grain starch and carries null mutations of the waxy (Wx) gene. With important uses as fresh maize or as an ingredient in food, textile, adhesive, and paper industries, two types of wx allele, wx-D7 and wx-D10, had been identified. In the present study, 10 accessions carrying neither wx-D7 nor wx-D10 allele were identified from a collection of 325 waxy maize accessions. The Wx locus of these 10 accessions was sequenced, but no potential causative mutation was detected. Further sequencing of the full-length complementary DNAs revealed that 6 of the 10 accessions had alternative splicing patterns while the other 4 had wild-type Wx transcripts. Among the six accessions, one generated transcripts identical to the wx-D7 allele, two had the same transcripts as the wx-D10 allele, another two generated transcripts with deletion of the 10th and the 11th exon, yet the other one produced transcripts of various lengths due to deletions from part of the 2nd exon to part of the 12th exon. The wx alleles with the above alternative splicing modes are referred to as wx-tD7, wx-tD10, wx-tD10-11, and wx-tD2-12, respectively. Real-time quantitative reverse-transcription polymerase chain reaction analysis of two waxy accessions that produced Wx transcripts showed significantly decreased expression, having only 47.3 and 3.6 % transcription level compared with B73. Sequence analysis of deletions in the transcripts with comparison with wild one showed short direct repeats at deletion endpoints, similar to reported signatures of DNA deletions. The waxy accessions present unique wx alleles for waxy maize breeding as well as for transcriptional regulation studies in plants.

Characterization and comparison of intron structure and alternative splicing between Medicago truncatula, Populus trichocarpa, Arabidopsis and rice

Alignment of transcripts and genome sequences yielded a set of alternatively spliced transcripts in four angiosperm genomes: three dicotyledon species Medicago truncatula (Medicago), Populus trichocarpa (poplar) and Arabidopsis thaliana (Arabidopsis), and the monocotyledon Oryzae sativa (rice). Intron retention was the predominant mode of alternative splicing (AS) in each species, consistent with previous reports for Arabidopsis and rice. We analyzed the structure of 5'-splice junctions and observed commonalities between species. There was dependency of base composition between sites flanking the 5'-splice junction, with the potential to create a subset of splice sites that interact more weakly or strongly than average with U1 snRNA. Such altered nucleotide composition was correlated with splicing fidelity in all four species. For Medicago, poplar and Arabidopsis, but not in rice, alternative splicing was most prevalent for introns with decreased UA content, consistent with lower UA content for monocot introns and potentially reflecting evolved differences in splicing mechanisms. Similarly, the occurrence of AS between transcript Gene Ontology categories was positively correlated between Arabidopsis and Medicago, with no correlation between dicots and rice. Analysis of within-species paralogs and between-species reciprocal best-hit homologs yielded rare cases of potentially conserved AS events. Reverse transcriptase PCR and amplicon sequencing were used to confirm a subset of the in silico-predicted AS events within Medicago, as well as to characterize conserved AS events between Medicago and Arabidopsis.

Selection against tandem splice sites affecting structured protein regions

BackgroundAlternative selection of splice sites in tandem donors and acceptors is a major mode of alternative splicing. Here, we analyzed whether in-frame tandem sites leading to subtle mRNA insertions/deletions of 3, 6, or 9 nucleotides are under natural selection.ResultsWe found multiple lines of evidence that the human protein coding sequences are under selection against such in-frame tandem splice events, indicating that these events are often deleterious. The strength of selection is not homogeneous within the coding sequence as protein regions that fold into a fixed 3D structure (intrinsically ordered) are under stronger selection, especially against sites with a strong minor splice site. Investigating structures of functional protein domains, we found that tandem acceptors are preferentially located at the domain surface and outside structural elements such as helices and sheets. Using three-species comparisons, we estimate that more than half of all mutations that create NAGNAG acceptors in the coding region have been eliminated by selection.ConclusionWe estimate that ~2,400 introns are under selection against possessing a tandem site.

Characteristics and regulatory elements defining constitutive splicing and different modes of alternative splicing in human and mouse

Alternative splicing is a major contributor to genomic complexity, disease, and development. Previous studies have captured some of the characteristics that distinguish alternative splicing from constitutive splicing. However, most published work only focuses on skipped exons and/or a single species. Here we take advantage of the highly curated data in the MAASE database (see related paper in this issue) to analyze features that characterize different modes of splicing. Our analysis confirms previous observations about alternative splicing, including weaker splicing signals at alternative splice sites, higher sequence conservation surrounding orthologous alternative exons, shorter exon length, and more frequent reading frame maintenance in skipped exons. In addition, our study reveals potentially novel regulatory principles underlying distinct modes of alternative splicing and a role of a specific class of repeat elements (transposons) in the origin/evolution of alternative exons. These features suggest diverse regulatory mechanisms and evolutionary paths for different modes of alternative splicing.

Vascular endothelial growth factor in salivary pleomorphic adenomas: one of the reasons for their poorly vascularized stroma

To better understand the poorly vascularized background of the stroma of pleomorphic adenomas, we attempted to determine the expression of molecules related to blood vessels and hypoxic conditions in pleomorphic adenoma. Surgical specimens and tumor cells in primary culture of salivary pleomorphic adenomas were used for immunohistochemistry for CD31, vascular endothelial growth factor (VEGF) and its receptors Flk-1 and Flt-1, as well as for hypoxia markers, such as hypoxia-inducible factor-1alpha (HIF-1alpha) and lactate dehydrogenase-1 (LDH). At the same time, alternative splicing modes of the VEGF gene and expression levels of the HIF-1alpha gene were analyzed in surgical specimens by means of reverse-transcription polymerase chain reaction (RT-PCR) and direct sequencing of the PCR products. In addition to co-immunolocalization with CD31+ vascular endothelial cells, VEGF and its receptors were demonstrated in normal duct epithelial and myoepithelial cells as well as in tumor cells in ductal structures and in myxochondroid stromata. Immunolocalizations for HIF-1alpha and LDH were confirmed in the VEGF-positive area. Immunofluorescence signals for VEGF and others were confirmed in pleomorphic adenoma cells in culture. RT-PCR results showed that there were at least four splicing modes of the VEGF gene, among which VEGF(121) was most enhanced, and higher HIF-1alpha levels in pleomorphic adenomas. The results suggest that pleomorphic adenoma cells produce VEGF in several functional forms for their own proliferation or differentiation, and that the VEGF expression is controlled by hypoxic circumstances of poorly vascularized pleomorphic adenomas.

Molecular analysis of PHYA in wild-type and phytochrome A-deficient mutants of tomato.

Tomato (Lycopersicon esculentum Mill., recently redesignated Solanum lycopersicum L.), an agronomically important crop plant, has been adopted as a model species complementary to Arabidopsis in which to characterize the phytochrome family. Here we describe the cloning and molecular characterization of the gene encoding the apoprotein of phytochrome A in wild-type tomato and in the far-red-light-insensitive (fri1 and fri2) tomato mutants. The physical organization of this gene is similar to that of other angiosperm phytochromes with the four exons of the coding region interrupted by three introns. The pool of transcripts is heterogeneous due to multiple transcription start sites and to three modes of alternative splicing of the 5' leader. The leader in each alternative transcript carries multiple upstream open reading frames of considerable length. At the genomic level, both fri mutants share an identical base substitution which changes a consensus AG/ to TG/ at the 3' end of the intron between exons 1 and 2. This mutation leads to aberrant processing of the resultant pre-mRNA. While most mature transcripts retain the mutated intron, both cryptic splicing and exon skipping were also detected. Cryptic splicing occurred both upstream and downstream from the wild-type splice site. These observations are consistent with the hypothesis that exon definition in splicing of plant pre-mRNAs plays a secondary role to that of intron definition. Analysis of the frequency with which potentially functional phytochrome A apoproteins might be produced indicates that both fri1 and fri2 have less than 1% of the wild-type phytochrome A level.