Intron Positions Research Articles

Long-term follow-up evaluation of an acatalasemia boy with severe periodontitis

BackgroundAcatalasemia is a rare genetic catalase deficiency that is inherited as an autosomal recessive trait. Although usually asymptomatic, a syndrome of oral ulcerations and gangrene may be present (Takahara's disease). In this report, we presented the diagnosis and 15-y periodontal treatments of an acatalasemia patient with Takahara's disease in China. MethodsTo confirm the diagnosis of acatalasemia, intron 4 of the catalase gene was amplified and sequenced. Erythrocyte catalase activity was measured by ultraviolet spectrophotometer. Besides, periodontal treatments and 15y follow-up were performed. ResultsDirect sequencing showed a clear splicing mutation of guanine to adenine substitution at the fifth position of intron 4 in the patient. Erythrocyte catalase activity of the patient (5.2MU/l, 4.6%) was 10% lower than the normal range (113.3±16.5MU/l). After 15-y treatments, the periodontal pocket depth ≥4mm and clinical attachment loss reduced to 30% and 3.7±1.2mm. ConclusionsBased on these findings, a diagnosis of acatalasemia was established. And the periodontal therapies have achieved a stable periodontal status.

Shared Gene Structures and Clusters of Mutually Exclusive Spliced Exons within the Metazoan Muscle Myosin Heavy Chain Genes

Multicellular animals possess two to three different types of muscle tissues. Striated muscles have considerable ultrastructural similarity and contain a core set of proteins including the muscle myosin heavy chain (Mhc) protein. The ATPase activity of this myosin motor protein largely dictates muscle performance at the molecular level. Two different solutions to adjusting myosin properties to different muscle subtypes have been identified so far: Vertebrates and nematodes contain many independent differentially expressed Mhc genes while arthropods have single Mhc genes with clusters of mutually exclusive spliced exons (MXEs). The availability of hundreds of metazoan genomes now allowed us to study whether the ancient bilateria already contained MXEs, how MXE complexity subsequently evolved, and whether additional scenarios to control contractile properties in different muscles could be proposed, By reconstructing the Mhc genes from 116 metazoans we showed that all intron positions within the motor domain coding regions are conserved in all bilateria analysed. The last common ancestor of the bilateria already contained a cluster of MXEs coding for part of the loop-2 actin-binding sequence. Subsequently the protostomes and later the arthropods gained many further clusters while MXEs got completely lost independently in several branches (vertebrates and nematodes) and species (for example the annelid Helobdella robusta and the salmon louse Lepeophtheirus salmonis). Several bilateria have been found to encode multiple Mhc genes that might all or in part contain clusters of MXEs. Notable examples are a cluster of six tandemly arrayed Mhc genes, of which two contain MXEs, in the owl limpet Lottia gigantea and four Mhc genes with three encoding MXEs in the predatory mite Metaseiulus occidentalis. Our analysis showed that similar solutions to provide different myosin isoforms (multiple genes or clusters of MXEs or both) have independently been developed several times within bilaterian evolution.

Intron evolution in a phylogenetic perspective: Divergent trends in the two copies of the duplicated def gene in Impatiens L. (Balsaminaceae)

AbstractThe history of MADS box genes is well‐known in angiosperms. While duplication events and gene losses occur frequently, gene structure and intron positions are very conserved. We investigated all six introns in a duplicated MADS box gene (deficiens, def) in selected Impatiens taxa, thereby assessing intron features. For the first time, our study provides a comparison of molecular changes in all introns of a gene from a phylogenetic perspective. Interestingly, a uniform pattern of molecular evolution in the introns of each copy was not observed, but intron length increases, decreases, and size retention can be found in each copy. A tendency to accumulate long autapomorphic indels is also present, thus, a longer intron length does not reflect a higher number of parsimony‐informative characters. Substitution rates vary between introns of each gene copy. While four of the six introns of def1 exhibit a change in their substitution rate, five of the six def2 introns maintain their rates throughout the genus albeit at different levels. In MADS box genes several regulatory sequences are found residing in introns. Thus, presence of putative regulatory motifs was investigated. Most of them are not conserved in position and usually present in only one of the gene copies. In addition, the potential for phylogenetic reconstruction of introns in both def copies is shortly discussed.

Characterization of the GPI-anchored lipid transfer proteins in the moss Physcomitrella patens

The non-specific lipid transfer proteins (nsLTPs) are characterized by a compact structure with a central hydrophobic cavity very suitable for binding hydrophobic ligands, such as lipids. The nsLTPs are encoded by large gene families in all land plant lineages, but seem to be absent from green algae. The nsLTPs are classified to different types based on molecular weight, sequence similarity, intron position or spacing between the cysteine residues. The Type G nsLTPs (LTPGs) have a GPI-anchor in the C-terminal region which may attach the protein to the exterior side of the plasma membrane. Here, we present the first characterization of nsLTPs from an early diverged plant, the moss Physcomitrella patens. Moss LTPGs were heterologously produced and purified from Pichia pastoris. The purified moss LTPGs were found to be extremely heat stable and showed a binding preference for unsaturated fatty acids. Structural modeling implied that high alanine content could be important for the heat stability. Lipid profiling revealed that cutin monomers, such as C16 and C18 mono- and di-hydroxylated fatty acids, could be identified in P. patens. Expression of a moss LTPG-YFP fusion revealed localization to the plasma membrane. The expressions of many of the moss LTPGs were found to be upregulated during drought and cold treatments.

Recruitment and diversification of an ecdysozoan family of neuropeptide hormones for black widow spider venom expression

Venoms have attracted enormous attention because of their potent physiological effects and dynamic evolution, including the convergent recruitment of homologous genes for venom expression. Here we provide novel evidence for the recruitment of genes from the Crustacean Hyperglycemic Hormone (CHH) and arthropod Ion Transport Peptide (ITP) superfamily for venom expression in black widow spiders. We characterized latrodectin peptides from venom gland cDNAs from the Western black widow spider (Latrodectus hesperus), the brown widow (Latrodectus geometricus) and cupboard spider (Steatoda grossa). Phylogenetic analyses of these sequences with homologs from other spider, scorpion and wasp venom cDNAs, as well as CHH/ITP neuropeptides, show latrodectins as derived members of the CHH/ITP superfamily. These analyses suggest that CHH/ITP homologs are more widespread in spider venoms, and were recruited for venom expression in two additional arthropod lineages. We also found that the latrodectin 2 gene and nearly all CHH/ITP genes include a phase 2 intron in the same position, supporting latrodectin's placement within the CHH/ITP superfamily. Evolutionary analyses of latrodectins suggest episodes of positive selection along some sequence lineages, and positive and purifying selection on specific codons, supporting its functional importance in widow venom. We consider how this improved understanding of latrodectin evolution informs functional hypotheses regarding its role in black widow venom as well as its potential convergent recruitment for venom expression across arthropods.

Distribution of Conventional and Nonconventional Introns in Tubulin (α and β) Genes of Euglenids

The nuclear genomes of euglenids contain three types of introns: conventional spliceosomal introns, nonconventional introns for which a splicing mechanism is unknown (variable noncanonical borders, RNA secondary structure bringing together intron ends), and so-called intermediate introns, which combine features of conventional and nonconventional introns. Analysis of two genes, tubA and tubB, from 20 species of euglenids reveals contrasting distribution patterns of conventional and nonconventional introns—positions of conventional introns are conserved, whereas those of the nonconventional ones are unique to individual species or small groups of closely related taxa. Moreover, in the group of phototrophic euglenids, 11 events of conventional intron loss versus 15 events of nonconventional intron gain were identified. A comparison of all nonconventional intron sequences highlighted the most conserved elements in their sequence and secondary structure. Our results led us to put forward two hypotheses. 1) The first one posits that mutational changes in intron sequence could lead to a change in their excision mechanism—intermediate introns would then be a transitional form between the conventional and nonconventional introns. 2) The second hypothesis concerns the origin of nonconventional introns—because of the presence of inverted repeats near their ends, insertion of MITE-like transposon elements is proposed as a possible source of new introns.

Alternative 5’ Untranslated Regions Are Involved in Expression Regulation of Human Heme Oxygenase-1

The single nucleotide polymorphism rs2071746 and a (GT)n microsatellite within the human gene encoding heme oxygenase-1 (HMOX1) are associated with incidence or outcome in a variety of diseases. Most of these associations involve either release of heme or oxidative stress. Both polymorphisms are localized in the promoter region, but previously reported correlations with heme oxygenase-1 expression remain not coherent. This ambiguity suggests a more complex organization of the 5’ gene region which we sought to investigate more fully.We evaluated the 5‘ end of HMOX1 and found a novel first exon 1a placing the two previously reported polymorphisms in intronic or exonic positions within the 5’ untranslated region respectively. Expression of exon 1a can be induced in HepG2 hepatoma cells by hemin and is a repressor of heme oxygenase-1 translation as shown by luciferase reporter assays. Moreover, minigene approaches revealed that the quantitative outcome of alternative splicing within the 5’ untranslated region is affected by the (GT)n microsatellite.This data supporting an extended HMOX1 gene model and provide further insights into expression regulation of heme oxygenase-1. Alternative splicing within the HMOX1 5' untranslated region contributes to translational regulation and is a mechanistic feature involved in the interplay between genetic variations, heme oxygenase-1 expression and disease outcome.

Mutation analysis and prenatal diagnosis of FBN1 gene mutations for four patients with Marfan syndrome

To screen for mutations of fibrillin-1 (FBN1) gene in 4 patients with Marfan syndrome in order to provide prenatal diagnosis and genetic counseling. Potential mutations of the FBN1 gene in the probands were detected with PCR and DNA sequencing. Subsequently, genomic DNA was extracted from amniotic fluid sampled between 18 to 20 weeks gestation. The mutations were confirmed with denaturing high-performance liquid chromatography - robust microsatellite instability (DHPLC-MSI) analysis with maternal DNA as reference. The products were further analyzed by direct sequencing and BLAST search of NCBI database. An IVS46+1G>A substitution was identified in patient A at +1 position of intron 46 of the FBN1 gene. Two novel missense mutations were respectively discovered at positions +4453 of intron 35 in patient B (Cys1485Gly) and position +2585 of intron 21 in patient C (Cys862Tyr). In patient D, a novel deletion (c.3536 delA) was found at position +3536 of intron 28. In all of the 4 cases, the same mutations have been identified in the fetuses. FBN1 gene analysis can provide accurate diagnosis of Marfan syndrome, which can facilitate both prenatal diagnosis and genetic counseling.

Cloning, expression and phylogenetic analysis of a divergent laccase multigene family in Auricularia auricula-judae

Laccases (p-diphenol: oxygen oxidoreductase; EC 1.10.3.2) are multi-copper oxidases encoded by gene family in white rot fungi. Auricularia auricula-judae is one kind of white rot fungi with a soft, jelly-like texture and an ear-like shape. In the present study, seven laccase genes containing the signature sequences L1–L4 were isolated from A. auricula-judae strain Au916 on the basis of the mycelium-derived transcriptome. In the basidiomycetes, the predicted substrate binding loops of the A. auricula-judae laccases were found to be uncommon. Phylogenetic analysis showed that the laccases of the Auricularia were nested into the ascomycete laccases, indicating that the laccase genes from Auricularia are distinctly different in function from other basidiomycetes. Among the seven laccases, the intron positions and cluster distributions in the NJ tree varied from each other and the expression patterns of seven genes estimated by qRT-PCR were also discrepant. The lcc3 gene was highly expressed not only in the free-living mycelium but also in substrate mycelium, furthermore, the lcc5 gene was mostly expressed during the fruiting body formation and maturation indicating that lcc5 might play a major role during the sexual reproduction stage.

The Arabidopsis thaliana MHX gene includes an intronic element that boosts translation when localized in a 5' UTR intron.

The mechanisms that underlie the ability of some introns to increase gene expression, a phenomenon called intron-mediated enhancement (IME), are not fully understood. It is also not known why introns localized in the 5′-untranslated region (5′ UTR) are considerably longer than downstream eukaryotic introns. It was hypothesized that this extra length results from the presence of some functional intronic elements. However, deletion analyses studies carried out thus far were unable to identify specific intronic regions necessary for IME. Using deletion analysis and a gain-of-function approach, an internal element that considerably increases translational efficiency, without affecting splicing, was identified in the 5′ UTR intron of the Arabidopsis thaliana MHX gene. Moreover, the ability of this element to enhance translation was diminished by a minor downstream shift in the position of introns containing it from the 5′ UTR into the coding sequence. These data suggest that some of the extra length of 5′ UTR introns results from the presence of elements that enhance translation, and, moreover, from the ability of 5′ UTR introns to provide preferable platforms for such elements over downstream introns. The impact of the identified intronic element on translational efficiency was augmented upon removal of neighbouring intronic elements. Interference between different intronic elements had not been reported thus far. This interference may support the bioinformatics-based idea that some of the extra sequence of 5′ UTR introns is also necessary for separating different functional intronic elements.

Introns Regulate Gene Expression in Cryptococcus neoformans in a Pab2p Dependent Pathway

Most Cryptococccus neoformans genes are interrupted by introns, and alternative splicing occurs very often. In this study, we examined the influence of introns on C. neoformans gene expression. For most tested genes, elimination of introns greatly reduces mRNA accumulation. Strikingly, the number and the position of introns modulate the gene expression level in a cumulative manner. A screen for mutant strains able to express functionally an intronless allele revealed that the nuclear poly(A) binding protein Pab2 modulates intron-dependent regulation of gene expression in C. neoformans. PAB2 deletion partially restored accumulation of intronless mRNA. In addition, our results demonstrated that the essential nucleases Rrp44p and Xrn2p are implicated in the degradation of mRNA transcribed from an intronless allele in C. neoformans. Double mutant constructions and over-expression experiments suggested that Pab2p and Xrn2p could act in the same pathway whereas Rrp44p appears to act independently. Finally, deletion of the RRP6 or the CID14 gene, encoding the nuclear exosome nuclease and the TRAMP complex associated poly(A) polymerase, respectively, has no effect on intronless allele expression.

Coexpression patterns indicate that GPI-anchored non-specific lipid transfer proteins are involved in accumulation of cuticular wax, suberin and sporopollenin

The non-specific lipid transfer proteins (nsLTP) are unique to land plants. The nsLTPs are characterized by a compact structure with a central hydrophobic cavity and can be classified to different types based on sequence similarity, intron position or spacing between the cysteine residues. The type G nsLTPs (LTPGs) have a GPI-anchor in the C-terminal region which attaches the protein to the exterior side of the plasma membrane. The function of these proteins, which are encoded by large gene families, has not been systematically investigated so far. In this study we have explored microarray data to investigate the expression pattern of the LTPGs in Arabidopsis and rice. We identified that the LTPG genes in each plant can be arranged in three expression modules with significant coexpression within the modules. According to expression patterns and module sizes, the Arabidopsis module AtI is functionally equivalent to the rice module OsI, AtII corresponds to OsII and AtIII is functionally comparable to OsIII. Starting from modules AtI, AtII and AtIII we generated extended networks with Arabidopsis genes coexpressed with the modules. Gene ontology analyses of the obtained networks suggest roles for LTPGs in the synthesis or deposition of cuticular waxes, suberin and sporopollenin. The AtI-module is primarily involved with cuticular wax, the AtII-module with suberin and the AtIII-module with sporopollenin. Further transcript analysis revealed that several transcript forms exist for several of the LTPG genes in both Arabidopsis and rice. The data suggests that the GPI-anchor attachment and localization of LTPGs may be controlled to some extent by alternative splicing.

Cloning and characterization of two allelic glyceraldehyde-3-phosphate dehydrogenase genes in Auricularia auricula-judae

Two allelic variants of the gpd gene, Gpd(a) and Gpd(b), were isolated based on a putative glyceraldehyde-3-phosphate dehydrogenase encoding sequence from the transcriptome of Auricularia auricula-judae strain Au916. The two alleles were found to have a 73 bp length discrepancy and 39 SNP variations. Both of the genomic DNA sequences of two alleles were interrupted by five introns, and encoded a same 340 aa protein. Intron positions analysis showed that the first intron was absent, but the last unique intron was gained in A. auricula-judae. Allele-specific expression analysis showed that the Gpd(a) and Gpd(b) were expressed with no significant difference in dikaryotic mycelia of A. auricula-judae. To the best of our knowledge, this is the first report about the detection of two allelic gpd genes in A. auricula-judae, as well as the application of allele-specific primers in gene expression analysis for this edible fungus.

GSA‐1/ARG1 protects root gravitropism in Arabidopsis under ammonium stress

Gravitropism plays a critical role in plant growth and development, plant stability and acclimation to changes in water and nutrient availability. Ammonium (NH4(+)) is well known to have profound effects on root growth, but its impacts on gravitropism are poorly understood. To determine which genes are essential for the maintenance of root gravitropism under NH4(+) stress, we isolated and identified an NH4 (+)-sensitive mutant, gsa-1 (gravitropism sensitive to ammonium-1), in Arabidopsis thaliana, using an agar plate root reorientation assay. We found that, under NH4(+) stress, gsa-1 displayed increased loss of root gravitropism. Gene cloning and sequencing revealed that gsa-1 contains a G to C transversion mutation at the highly conserved 5'-GT splice position of intron 10 of ARG1 (ALTERED RESPONSE TO GRAVITY1), known to participate in the transduction of the root gravity signal. Genetic complement tests established the locus of GSA-1/ARG1 and its role in resistance to NH4 (+) inhibition on root gravitropism. GSA-1/ARG1 is required for normal AUX1 expression and basipetal auxin transport in root apices. In addition, PIN-FORMED2 (PIN2) is proposed as a target in the reduction of root gravitropism under NH4(+) stress, a response which can be antagonized by the GSA-1/ARG1-dependent pathway. These results suggest that GSA-1/ARG1 protects root gravitropism in Arabidopsis thaliana under ammonium stress.

Reversion of the Arabidopsis rpn12a-1 exon-trap mutation by an intragenic suppressor that weakens the chimeric 5’ splice site

Background: In the Arabidopsis 26S proteasome mutant rpn12a-1, an exon-trap T-DNA is inserted 531 base pairs downstream of the RPN12a STOP codon. We have previously shown that this insertion activates a STOP codon-associated latent 5' splice site that competes with the polyadenylation signal during processing of the pre-mRNA. As a result of this dual input from splicing and polyadenylation in the rpn12a-1 mutant, two RPN12a transcripts are produced and they encode the wild-type RPN12a and a chimeric RPN12a-NPTII protein. Both proteins form complexes with other proteasome subunits leading to the formation of wild-type and mutant proteasome versions. The net result of this heterogeneity of proteasome particles is a reduction of total cellular proteasome activity. One of the consequences of reduced proteasomal activity is decreased sensitivity to the major plant hormone cytokinin. Methods: We performed ethyl methanesulfonate mutagenesis of rpn12a-1 and isolated revertants with wild-type cytokinin sensitivity. Results: We describe the isolation and analyses of suppressor of rpn12a-1 ( sor1). The sor1 mutation is intragenic and located at the fifth position of the chimeric intron. This mutation weakens the activated 5' splice site associated with the STOP codon and tilts the processing of the RPN12a mRNA back towards polyadenylation. Conclusions: These results validate our earlier interpretation of the unusual nature of the rpn12a-1 mutation. Furthermore, the data show that optimal 26S proteasome activity requires RPN12a accumulation beyond a critical threshold. Finally, this finding reinforces our previous conclusion that proteasome function is critical for the cytokinin-dependent regulation of plant growth.

Identification of an intronic regulatory mutation at the buffalo αS1-casein gene that triggers the skipping of exon 6

The characterization of casein polymorphism is an essential step in order to understand the genetic basis of milk quality in dairy ruminants. In this work, we report the identification of a regulatory mutation at the buffalo αs1-casein (CSN1S1) gene that alters the normal processing of the primary transcript. Sequencing of CSN1S1 cDNA from individuals harbouring this new variant revealed that its most distinctive feature is the loss of exon 6 that encodes eight amino acids between positions 35-42 of mature protein. In an effort to map the causal mutation, we sequenced a genomic region spanning exons 5-7 of the buffalo CSN1S1 gene. This experiment allowed us to establish that exon 6-skipping is produced by a G to C substitution at the first position of intron 6 that inactivates the donor splice site. This mutation can be typed by PCR-RFLP by using either TaaI or Bpu10I diagnostic restriction enzymes, and it has a frequency of 0.18 in Romanian buffaloes. This exon skipping phenomenon is the first one described in buffalo CSN1S1 locus.

Abstract 4087: Androgen receptor splice variants in mouse prostate cancer cell lines: altered distribution in lines derived from castration-resistant tumors relative to those primary tumors.

Abstract Prostate cancer is sensitive to androgen deprivation at the primary androgen-dependent growth stage (ADCa). However, it invariably acquires resistance to androgen withdrawal leading to the recurrence of the castration-resistant prostate cancer (CRPC). Androgen receptor (AR) activity that is necessary for the normal prostate development remains active through both phases of the disease. Even at castrate levels of androgen AR remains functional, possibly through several non-mutually exclusive mechanisms including expression of constitutively active AR variants (ARVs). The full-length of AR (ARFL) is a 110 KDa nuclear hormone receptor composed of an N-terminal domain (NTD, exon1), DNA-binding domain (DBD, exon 2 and 3), a hinge region (exon 4), and the C-terminal ligand binding domain (LBD, exon 4-8). To date up to 15 different ARVs lacking LBD have been identified in human prostate cancer cell lines or specimens. It is thought that the altered biological properties of these ARVs may be, in part, correlated to the development of castration resistance. ARVs remain understudied in mouse prostate cancer cell lines; detection of only two ARVs was reported in a mouse cell line. A better definition of ARVs in distinct phases of this cancer in an appropriate mouse model should be valuable for insights into the role of ARVs in therapy and disease progression. To this end, by using cell lines established from the conditional Pten deletion mouse model of prostate adenocarcinoma, we detected low molecular weight AR protein species (∼50 kDa - 80 kDa) besides the presence of the 110 kDa full-length. We performed 3’RACE and subsequent Sanger sequencing on cell lines derived from both ADCa (E-series) and CRPC stages (cE-series) and identified three different ARVs which we named as mARV-abc. Structurally, mARVa is analogous to the previously reported mAR-V4 and the human ARV567es which only lacks LBD domain retaining exon 1-4 followed by the inclusion of a cryptic exon 4 from intron 4 at the 3’-terminal sequence. More interestingly, we identified two novel ARV isoforms (mARV-b and -c) which merely contains the NTD encoded by exon 1 before the addition of specific sequences from different positions of intron 1. Although these mARVs occur in all the murine cell lines tested, their proportion appears to vary with respect to the stage of the disease from which the cell lines originated. In cE-series established from a CRPC tumor, novel ARVb and ARVc are more abundant than that in E-series from an ADCa tumor; in parallel, ARVa displays an opposite expression profile. These new findings together with the results of ongoing experiments, including the single cell expression analysis and on their biological activities of the splice variants will be discussed. Citation Format: Mengmeng Liang, Helty A. Adisetiyo, Xiuqing Li, Ren Liu, Parkash S. Gill, Pradip Roy-Burman. Androgen receptor splice variants in mouse prostate cancer cell lines: altered distribution in lines derived from castration-resistant tumors relative to those primary tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4087. doi:10.1158/1538-7445.AM2013-4087

GenePainter: a fast tool for aligning gene structures of eukaryotic protein families, visualizing the alignments and mapping gene structures onto protein structures

BackgroundAll sequenced eukaryotic genomes have been shown to possess at least a few introns. This includes those unicellular organisms, which were previously suspected to be intron-less. Therefore, gene splicing must have been present at least in the last common ancestor of the eukaryotes. To explain the evolution of introns, basically two mutually exclusive concepts have been developed. The introns-early hypothesis says that already the very first protein-coding genes contained introns while the introns-late concept asserts that eukaryotic genes gained introns only after the emergence of the eukaryotic lineage. A very important aspect in this respect is the conservation of intron positions within homologous genes of different taxa.ResultsGenePainter is a standalone application for mapping gene structure information onto protein multiple sequence alignments. Based on the multiple sequence alignments the gene structures are aligned down to single nucleotides. GenePainter accounts for variable lengths in exons and introns, respects split codons at intron junctions and is able to handle sequencing and assembly errors, which are possible reasons for frame-shifts in exons and gaps in genome assemblies. Thus, even gene structures of considerably divergent proteins can properly be compared, as it is needed in phylogenetic analyses. Conserved intron positions can also be mapped to user-provided protein structures. For their visualization GenePainter provides scripts for the molecular graphics system PyMol.ConclusionsGenePainter is a tool to analyse gene structure conservation providing various visualization options. A stable version of GenePainter for all operating systems as well as documentation and example data are available at http://www.motorprotein.de/genepainter.html.

Reversion of the Arabidopsis rpn12a-1 exon-trap mutation by an intragenic suppressor that weakens the chimeric 5’ splice site

Background: In the Arabidopsis 26S proteasome mutant rpn12a-1 , an exon-trap T-DNA is inserted 531 base pairs downstream of the RPN12a STOP codon. We have previously shown that this insertion activates a STOP codon-associated latent 5' splice site that competes with the polyadenylation signal during processing of the pre-mRNA. As a result of this dual input from splicing and polyadenylation in the rpn12a-1 mutant, two RPN12a transcripts are produced and they encode the wild-type RPN12a and a chimeric RPN12a-NPTII protein. Both proteins form complexes with other proteasome subunits leading to the formation of wild-type and mutant proteasome versions. The net result of this heterogeneity of proteasome particles is a reduction of total cellular proteasome activity. One of the consequences of reduced proteasomal activity is decreased sensitivity to the major plant hormone cytokinin. Methods: We performed ethyl methanesulfonate mutagenesis of rpn12a-1 and isolated revertants with wild-type cytokinin sensitivity. Results: We describe the isolation and analyses of suppressor of rpn12a-1 ( sor1 ). The sor1 mutation is intragenic and located at the fifth position of the chimeric intron. This mutation weakens the activated 5' splice site associated with the STOP codon and tilts the processing of the RPN12a mRNA back towards polyadenylation. Conclusions: These results validate our earlier interpretation of the unusual nature of the rpn12a-1 mutation. Furthermore, the data show that optimal 26S proteasome activity requires RPN12a accumulation beyond a critical threshold. Finally, this finding reinforces our previous conclusion that proteasome function is critical for the cytokinin-dependent regulation of plant growth.

Convergent intron gains in hymenopteran elongation factor-1α

The eukaryotic translation elongation factor-1α gene (eEF1A) has been used extensively in higher level phylogenetics of insects and other groups, despite being present in two or more copies in several taxa. Orthology assessment has relied heavily on the position of introns, but the basic assumption of low rates of intron loss and absence of convergent intron gains has not been tested thoroughly. Here, we study the evolution of eEF1A based on a broad sample of taxa in the insect order Hymenoptera. The gene is universally present in two copies – F1 and F2 – both of which apparently originated before the emergence of the order. An elevated ratio of non-synonymous versus synonymous substitutions and differences in rates of amino acid replacements between the copies suggest that they evolve independently, and phylogenetic methods clearly cluster the copies separately. The F2 copy appears to be ancient; it is orthologous with the copy known as F1 in Diptera, and is likely present in most insect orders. The hymenopteran F1 copy, which may or may not be unique to this order, apparently originated through retroposition and was originally intron free. During the evolution of the Hymenoptera, it has successively accumulated introns, at least three of which have appeared at the same position as introns in the F2 copy or in eEF1A copies in other insects. The sites of convergent intron gain are characterized by highly conserved nucleotides that strongly resemble specific intron-associated sequence motifs, so-called proto-splice sites. The significant rate of convergent intron gain renders intron–exon structure unreliable as an indicator of orthology in eEF1A, and probably also in other protein-coding genes.