Intron Positions Research Articles

Genome-wide identification and characterization of the 14\u20133-3 family in Vitis vinifera L. during berry development and cold- and heat-stress response

BackgroundThe 14–3-3 family of ubiquitous proteins in eukaryotes plays important roles in the regulation of various plant biological processes. However, less information is known about this family in grape fruit.ResultsTo investigate the characteristics and functions of 14–3-3 in grape, a total of 11 14–3-3 proteins were identified. Phylogenetic analysis of 14–3-3 proteins in grape (VviGRFs) with homologous proteins in Arabidopsis showed that these proteins were classified into two groups, namely, epsilon and non-epsilon groups. Epsilon group members commonly contained more introns and motifs than non-epsilon group, and some intron positions were found to be conserved between Vitis and Arabidopsis 14–3-3 genes. RNA-seq and qRT-PCR results indicated that VviGRF genes may be involved in the regulation of grape development and berry ripening. Moreover, six VviGRFs exhibited significantly up- or down-regulated expression in response to cold and heat stresses, thereby revealing their potential roles in the regulation of abiotic stress responses.ConclusionsThis work provides fundamental knowledge for further studies about the biological roles of VviGRFs in grape development and abiotic stress response. The present result will also be beneficial for understanding their molecular mechanisms and improving grape agricultural traits in the future.

Use of modified U1 small nuclear RNA for rescue from exon 7 skipping caused by 5′-splice site mutation of human cathepsin A gene

Cathepsin A (CTSA) is a multifunctional lysosomal enzyme, and its hereditary defect causes an autosomal recessive disorder called galactosialidosis. In a certain number of galactosialidosis patients, a base substitution from adenine to guanine is observed at the +3 position of the 7th intron (IVS7 +3a>g) of the CTSA gene. With this mutation, a splicing error occurs; and consequently mRNA lacking the 7th exon is produced. This skipping of exon 7 causes a frame shift of the transcripts, resulting in a non-functional CTSA protein and hence galactosialidosis. This mutation seems to make the interaction between the 5′-splice site of intron 7 of pre-mRNA and U1 small nuclear RNA (U1 snRNA) much weaker. In the present study, to produce properly spliced mRNA from the CTSA gene harboring this IVS7 +3a>g mutation, we examined the possible usefulness of modified U1 snRNA that could interact with the mutated 5′-splice site. Toward this goal, we first prepared a model system using a mutant CTSA mini gene plasmid for delivery into HeLa cells. Then, we examined the effectiveness of modified U1 snRNA on the formation of properly spliced mRNA from this mutant CTSA mini gene. As a result, we succeeded in obtaining improved formation of properly spliced CTSA mRNA. Our results suggest the usefulness of modified U1 snRNA for rescue from exon 7 skipping caused by the IVS7 +3a>g mutation of the CTSA gene.

Combining RNA-seq data and homology-based gene prediction for plants, animals and fungi

BackgroundGenome annotation is of key importance in many research questions. The identification of protein-coding genes is often based on transcriptome sequencing data, ab-initio or homology-based prediction. Recently, it was demonstrated that intron position conservation improves homology-based gene prediction, and that experimental data improves ab-initio gene prediction.ResultsHere, we present an extension of the gene prediction program GeMoMa that utilizes amino acid sequence conservation, intron position conservation and optionally RNA-seq data for homology-based gene prediction. We show on published benchmark data for plants, animals and fungi that GeMoMa performs better than the gene prediction programs BRAKER1, MAKER2, and CodingQuarry, and purely RNA-seq-based pipelines for transcript identification. In addition, we demonstrate that using multiple reference organisms may help to further improve the performance of GeMoMa. Finally, we apply GeMoMa to four nematode species and to the recently published barley reference genome indicating that current annotations of protein-coding genes may be refined using GeMoMa predictions.ConclusionsGeMoMa might be of great utility for annotating newly sequenced genomes but also for finding homologs of a specific gene or gene family. GeMoMa has been published under GNU GPL3 and is freely available at http://www.jstacs.de/index.php/GeMoMa.

Evolutionary analysis of the carnitine- and choline acyltransferases suggests distinct evolution of CPT2 versus CPT1 and related variants

Carnitine/choline acyltransferases play diverse roles in energy metabolism and neuronal signalling. Our knowledge of their evolutionary relationships, important for functional understanding, is incomplete. Therefore, we aimed to determine the evolutionary relationships of these eukaryotic transferases. We performed extensive phylogenetic and intron position analyses. We found that mammalian intramitochondrial CPT2 is most closely related to cytosolic yeast carnitine transferases (Sc-YAT1 and 2), whereas the other members of the family are related to intraorganellar yeast Sc-CAT2. Therefore, the cytosolically active CPT1 more closely resembles intramitochondrial ancestors than CPT2. The choline acetyltransferase is closely related to carnitine acetyltransferase and shows lower evolutionary rates than long chain acyltransferases. In the CPT1 family several duplications occurred during animal radiation, leading to the isoforms CPT1A, CPT1B and CPT1C. In addition, we found five CPT1-like genes in Caenorhabditis elegans that strongly group to the CPT1 family. The long branch leading to mammalian brain isoform CPT1C suggests that either strong positive or relaxed evolution has taken place on this node. The presented evolutionary delineation of carnitine/choline acyltransferases adds to current knowledge on their functions and provides tangible leads for further experimental research.

Development of indel markers for molecular authentication of Panax ginseng and P. quinquefolius

Panax ginseng and P. quinquefolius are two kinds of important medicinal herbs. They are morphologically similar but have different pharmacological effects. Therefore, botanical origin authentication of these two ginsengs is of great importance for ensuring pharmaceutical efficacy and food safety. Based on the fact that intron position in orthologous genes is highly conserved across plant species, intron length polymorphisms were exploited from unigenes of ginseng. Specific primers were respectively designed for these two species based on their insertion/deletion sequences of cytochrome P450 and glyceraldehyde 3-phosphate dehydrogenase, and multiplex PCR was conducted for molecular authentication of P.ginseng and P. quinquefolius. The results showed that the developed multiplex PCR assay was effective for molecular authentication of P.ginseng and P. quinquefolius without strict PCR condition and the optimization of reaction system.This study provides a preferred ideal marker system for molecular authentication of ginseng,and the presented method can be employed in origin authentication of other herbal preparations.

Comparative analysis of the mitochondrial genomes of six newly sequenced diatoms reveals group II introns in the barcoding region of cox1

Diatoms are the most diverse lineage of algae and at the base of most aquatic food webs, but only 11 of their mitochondrial genomes have been described. Herein, we present the mitochondrial genomes of six diatom species, including: Melosira undulata, Nitzschia alba, Surirella sp., Entomoneis sp., Halamphora coffeaeformis, and Halamphora calidilacuna. Comparison of these six genomes to the 11 currently published diatom mitochondrial genomes revealed a novel ubiquitous feature block consisting of tatC-orf157-rps11. The presence of intronic retrotransposable elements in the barcoding region of cox1 in the Halamphora genomes may explain historic difficulty (especially PCR) with cox1 as a universal barcode for diatoms. Our analysis suggests that high rates of variability in number and position of introns, in many commonly used coding sequences, prevent these from being universally viable as barcodes for diatoms. Therefore, we suggest researchers examine the chloroplast and/or nuclear genomes for universal barcoding markers.

Hereditary cancer genes are highly susceptible to splicing mutations.

Substitutions that disrupt pre-mRNA splicing are a common cause of genetic disease. On average, 13.4% of all hereditary disease alleles are classified as splicing mutations mapping to the canonical 5′ and 3′ splice sites. However, splicing mutations present in exons and deeper intronic positions are vastly underreported. A recent re-analysis of coding mutations in exon 10 of the Lynch Syndrome gene, MLH1, revealed an extremely high rate (77%) of mutations that lead to defective splicing. This finding is confirmed by extending the sampling to five other exons in the MLH1 gene. Further analysis suggests a more general phenomenon of defective splicing driving Lynch Syndrome. Of the 36 mutations tested, 11 disrupted splicing. Furthermore, analyzing past reports suggest that MLH1 mutations in canonical splice sites also occupy a much higher fraction (36%) of total mutations than expected. When performing a comprehensive analysis of splicing mutations in human disease genes, we found that three main causal genes of Lynch Syndrome, MLH1, MSH2, and PMS2, belonged to a class of 86 disease genes which are enriched for splicing mutations. Other cancer genes were also enriched in the 86 susceptible genes. The enrichment of splicing mutations in hereditary cancers strongly argues for additional priority in interpreting clinical sequencing data in relation to cancer and splicing.

An insight into the evolution of introns in the gyrase A gene of plants

DNA gyrase is a type II topoisomerase essential for replication and transcription in prokaryotes and eukaryotic cell organelles. The functional gyrase enzyme is an A2B2 tetramer encoded by the gyrA and gyrB genes. Most of the eukaryotic gyrase A genes possess introns while they are intron-less in prokaryotes. In the present study, we found out the evolutionary passage of intron development in gyrase A gene with the help of bioinformatics approaches. All the plant gyrase A genes studied by us were found to be a part of the nuclear genome, and their respective proteins were targeted to the organelles. Except the green alga Bathycoccus prasinos, these genes contained introns, and the positions of the homologous introns were found to be highly conserved in diverse plant lineages despite having variation in their nucleotide sequence compositions and lengths. However, in red, brown, and green algae: Chlorella variabilis and Chlamydomonas reinhardtii, homologous intron positions were not conserved, which might be due to the independent acquisition of introns. The study makes it amply evident that the introns appeared in the gene following endosymbiotic gene transfer of the gyrase A to the nuclear genome of an ancestral green plant. The land plants appear to have acquired intron-bearing gyrase A gene from a common ancestral green algae and subsequently lesser re-arrangement of introns at homologous positions resulted in their positional conservation. However, the introns which are known to be under lesser selection pressure evolved differently in various plant species in terms of base composition and lengths.

Novel SNP and Unique Sequences in ATP-binding Cassette Super Family-G Member-2 Transporter (ABCG2) Gene of Vechur cattle (Bos indicus)

In the present study, ABCG2 gene of Vechur cattle of Kerala was sequenced and aligned using Basic Local Alignment Search Tool (BLAST) in National Centre for Biotechnology Information (NCBI) site. The results revealed that ABCG2 gene sequence of Vechur cattle shows 96% similarity with reference NCBI sequences. Addition of eleven base pairs in 13th intron in ABCG2 gene of Vechur cattle which derived a larger amplicon (303 bp) than expected (292 bp). A novel SNP (G4104A) was identified at 4104th position in intron 13 of ABCG2 gene of Vechur cattle. Further research is suggested to depict the association if any of the novel SNP and unique characters of Vechur cattle.

Parallel loss of introns in the ABCB1 gene in angiosperms

BackgroundThe presence of non-coding introns is a characteristic feature of most eukaryotic genes. While the size of the introns, number of introns per gene and the number of intron-containing genes can vary greatly between sequenced eukaryotic genomes, the structure of a gene with reference to intron presence and positions is typically conserved in closely related species. Unexpectedly, the ABCB1 (ATP-Binding Cassette Subfamily B Member 1) gene which encodes a P-glycoprotein and underlies dwarfing traits in maize (br2), sorghum (dw3) and pearl millet (d2) displayed considerable variation in intron composition.ResultsAn analysis of the ABCB1 gene structure in 80 angiosperms revealed that the number of introns ranged from one to nine. All introns in ABCB1 underwent either a one-time loss (single loss in one lineage/species) or multiple independent losses (parallel loss in two or more lineages/species) with the majority of losses occurring within the grass family. In contrast, the structure of the closest homolog to ABCB1, ABCB19, remained constant in the majority of angiosperms analyzed. Using known phylogenetic relationships within the grasses, we determined the ancestral branch-points where the losses occurred. Intron 7, the longest intron, was lost in only a single species, Mimulus guttatus, following duplication of ABCB1. Semiquantitative PCR showed that the M. guttatus ABCB1 gene copy without intron 7 had significantly lower transcript levels than the gene copy with intron 7. We further demonstrated that intron 7 carried two motifs that were highly conserved across the monocot-dicot divide.ConclusionsThe ABCB1 gene structure is highly dynamic, while the structure of ABCB19 remained largely conserved through evolution. Precise removal of introns, preferential removal of smaller introns and presence of at least 2 bp of microhomology flanking most introns indicated that intron loss may have predominantly occurred through non-homologous end-joining (NHEJ) repair of double strand breaks. Lack of microhomology in the exon upstream of lost phase I introns was likely due to release of the selective constraint on the penultimate base (3rd base in codon) of the terminal codon by the splicing machinery. In addition to size, the presence of regulatory motifs will make introns recalcitrant to loss.

Arabidopsis β-Amylase2 Is a K+-Requiring, Catalytic Tetramer with Sigmoidal Kinetics.

The Arabidopsis (Arabidopsis thaliana) genome contains nine β-amylase (BAM) genes, some of which play important roles in starch hydrolysis. However, little is known about BAM2, a plastid-localized enzyme reported to have extremely low catalytic activity. Using conservation of intron positions, we determined that the nine Arabidopsis BAM genes fall into two distinct subfamilies. A similar pattern was found in each major lineage of land plants, suggesting that these subfamilies diverged prior to the origin of land plants. Moreover, phylogenetic analysis indicated that BAM2 is the ancestral member of one of these subfamilies. This finding, along with the conservation of amino acids in the active site of BAM2, suggested that it might be catalytically active. We then identified KCl as necessary for BAM2 activity. Unlike BAM1, BAM3, and BAM5, three Arabidopsis BAMs that all exhibited hyperbolic kinetics, BAM2 exhibited sigmoidal kinetics with a Hill coefficient of over 3. Using multi-angle light scattering, we determined that BAM2 was a tetramer, whereas BAM5 was a monomer. Conserved residues from a diverse set of BAM2 orthologs were mapped onto a homology model of the protein, revealing a large, conserved surface away from the active site that we hypothesize is a secondary carbohydrate-binding site. Introduction of bulky methionine for glycine at two points on this surface reduced catalytic activity significantly without disrupting the tetrameric structure. Expression analysis indicated that BAM2 is more closely coexpressed with other starch degradation enzymes than any other BAM, suggesting that BAM2 may play an important role in starch degradation in plants.

Emergence and loss of spliceosomal twin introns

BackgroundIn the primary transcript of nuclear genes, coding sequences—exons—usually alternate with non-coding sequences—introns. In the evolution of spliceosomal intron–exon structure, extant intron positions can be abandoned and new intron positions can be occupied. Spliceosomal twin introns (“stwintrons”) are unconventional intervening sequences where a standard “internal” intron interrupts a canonical splicing motif of a second, “external” intron. The availability of genome sequences of more than a thousand species of fungi provides a unique opportunity to study spliceosomal intron evolution throughout a whole kingdom by means of molecular phylogenetics.ResultsA new stwintron was encountered in Aspergillus nidulans and Aspergillus niger. It is present across three classes of Leotiomyceta in the transcript of a well-conserved gene encoding a putative lipase (lipS). It occupies the same position as a standard intron in the orthologue gene in species of the early divergent classes of the Pezizomycetes and the Orbiliomycetes, suggesting that an internal intron has appeared within a pre-extant intron. On the other hand, the stwintron has been lost from certain taxa in Leotiomycetes and Eurotiomycetes at several occasions, most likely by a mechanism involving reverse transcription and homologous recombination. Another ancient stwintron present across whole Pezizomycotina orders—in the transcript of the bifunctional biotin biosynthesis gene bioDA—occurs at the same position as a standard intron in many species of non-Dikarya. Nevertheless, also the bioDA stwintron has disappeared from certain lineages within the taxa where it occurs, i.e., Sordariomycetes and Botryosphaeriales. Intriguingly, only the internal intron was lost from the Sordariomycetes bioDA stwintron at all but one occasion, leaving a standard intron in the same position, while where the putative lipase stwintron was lost, no intronic sequences remain.ConclusionsMolecular phylogeny of the peptide product was used to monitor the existence and fate of a stwintron in the transcripts of two neatly defined fungal genes, encoding well conserved proteins. Both defining events—stwintron emergence and loss—can be explained with extant models for intron insertion and loss. We thus demonstrate that stwintrons can serve as model systems to study spliceosomal intron evolution.

Слайдинг и вариабельность длины интронов для генов, обогащенных длинными интронами фазы 1

Due to high mutagenesis of intron sequences, intron evolution is usually considered in terms of evolution of exon-intron structures (EIS). The shifting of intron over short distances (rare evolutionary event called intron sliding) could lead to the change of intron phase, i.e. the intron position relative to the open reading frame. Here we analyze the EIS from four datasets of eukaryotic orthologues in order to find out the preferable choice of intron phase during sliding and to study the correlation between orthologous intron lengths. To identify the orthologous introns we have constructed the alignments of EIS of orthologous genes. Several sliding events with intron phase change were revealed from the analysis; however, our initial hypothesis that in the process of sliding introns prefer to change its phase to 0 more frequently, was not been confirmed. Nevertheless, it is necessary to expand the analysis on a larger dataset for making a proper conclusions. Despite high variability of intron length, some taxonomic groups share the similar length values. Moreover, some length conservation could be observed if instead of intron length L we consider a normalized length N = (L-A)/A, where A is an average length within an orthologous intron group. E.g. for ptprd genes of birds (28 species) the normalized value is in the interval (-0.15, 0.15) for 85.2 % of introns what is significantly higher than the values for random lengths set in accordance with the intron lengths distribution. That length “conservation” leads us to the question what intron length was in the ancient introns.

Activation of a cryptic 5' splice site reverses the impact of pathogenic splice site mutations in the spinal muscular atrophy gene.

Spinal muscular atrophy (SMA) is caused by deletions or mutations of the Survival Motor Neuron 1 (SMN1) gene coupled with predominant skipping of SMN2 exon 7. The only approved SMA treatment is an antisense oligonucleotide that targets the intronic splicing silencer N1 (ISS-N1), located downstream of the 5′ splice site (5′ss) of exon 7. Here, we describe a novel approach to exon 7 splicing modulation through activation of a cryptic 5′ss (Cr1). We discovered the activation of Cr1 in transcripts derived from SMN1 that carries a pathogenic G-to-C mutation at the first position (G1C) of intron 7. We show that Cr1-activating engineered U1 snRNAs (eU1s) have the unique ability to reprogram pre-mRNA splicing and restore exon 7 inclusion in SMN1 carrying a broad spectrum of pathogenic mutations at both the 3′ss and 5′ss of the exon 7. Employing a splicing-coupled translation reporter, we demonstrate that mRNAs generated by an eU1-induced activation of Cr1 produce full-length SMN. Our findings underscore a wider role for U1 snRNP in splicing regulation and reveal a novel approach for the restoration of SMN exon 7 inclusion for a potential therapy of SMA.

An ENU-induced splice site mutation of mouse Col1a1 causing recessive osteogenesis imperfecta and revealing a novel splicing rescue

GU-AG consensus sequences are used for intron recognition in the majority of cases of pre-mRNA splicing in eukaryotes. Mutations at splice junctions often cause exon skipping, short deletions, or insertions in the mature mRNA, underlying one common molecular mechanism of genetic diseases. Using N-ethyl-N-nitrosourea, a novel recessive mutation named seal was produced, associated with fragile bones and susceptibility to fractures (spine and limbs). A single nucleotide transversion (T → A) at the second position of intron 36 of the Col1a1 gene, encoding the type I collagen, α1 chain, was responsible for the phenotype. Col1a1seal mRNA expression occurred at greatly reduced levels compared to the wild-type transcript, resulting in reduced and aberrant collagen fibers in tibiae of seal homozygous mice. Unexpectedly, splicing of Col1a1seal mRNA followed the normal pattern despite the presence of the donor splice site mutation, likely due to the action of a putative intronic splicing enhancer present in intron 25, which appeared to function redundantly with the splice donor site of intron 36. Seal mice represent a model of human osteogenesis imperfecta, and reveal a previously unknown mechanism for splicing “rescue.”

Defective splicing of the RB1 transcript is the dominant cause of retinoblastomas.

Defective splicing is a common cause of genetic diseases. On average, 13.4% of all hereditary disease alleles are classified as splicing mutations with most mapping to the critical GT or AG nucleotides within the 5' and 3' splice sites. However, splicing mutations are underreported and the fraction of splicing mutations that compose all disease alleles varies greatly across disease gene. For example, there is a great excess (46%; ~threefold) of hereditary disease alleles that map to splice sites in RB1 that cause retinoblastoma. Furthermore, mutations in the exons and deeper intronic position may also affect splicing. We recently developed a high-throughput method that assays reported disease mutations for their ability to disrupt pre-mRNA splicing. Surprisingly, 27% of RB1-coding mutations tested also disrupt splicing. High-throughput in vitro spliceosomal assembly assay reveals heterogeneity in which stage of spliceosomal assembly is affected by splicing mutations. 58% of exonic splicing mutations were primarily blocked at the A complex in transition to the B complex and 33% were blocked at the B complex. Several mutants appear to reduce more than one step in the assembly. As RB1 splicing mutants are enriched in retinoblastoma disease alleles, additional priority should be allocated to this class of allele while interpreting clinical sequencing experiments. Analysis of the spectrum of RB1 variants observed in 60,706 exomes identifies 197 variants that have enough potential to disrupt splicing to warrant further consideration.

How introns enhance gene expression.

In many eukaryotes, including mammals, plants, yeast, and insects, introns can increase gene expression without functioning as a binding site for transcription factors. This phenomenon was termed 'intron-mediated enhancement'. Introns can increase transcript levels by affecting the rate of transcription, nuclear export, and transcript stability. Moreover, introns can also increase the efficiency of mRNA translation. This review discusses the current knowledge about these processes. The role of splicing in IME and the significance of intron position relative to the sites of transcription and translation initiation are elaborated. Particular emphasis is placed on the question why different introns, present at the same location of the same genes and spliced at a similar high efficiency, can have very different impacts on expression - from almost no effect to considerable stimulation. This situation can be at least partly accounted for by the identification of splicing-unrelated intronic elements with a special ability to enhance mRNA accumulation or translational efficiency. The many factors that could lead to the large variation observed between the impact of introns in different genes and experimental systems are highlighted. It is suggested that there is no sole, definite answer to the question "how do introns enhance gene expression". Rather, each intron-gene combination might undergo its own unique mixture of processes that lead to the perceptible outcome.

Genome-wide development and utilization of novel intron-length polymorphic (ILP) markers in Medicago sativa

Alfalfa (Medicago sativa L.) is the most widely cultivated forage legume around the world. Though development and application of microsatellite markers in large-scale was reported in this species, a systematic investigation and large-scale exploitation of intron-length polymorphic (ILP) markers has not been conducted. In the present study, the RNA-Seq sequences of alfalfa were aligned with the genomic sequences of Arabidopsis to predict the position of introns and develop ILP markers in alfalfa. A total of 693 putative ILPs were identified, and 502 ILP markers were successfully developed. Furthermore, 100 ILP markers exhibited relatively high levels of transferability to leguminous (40.0%–83.0%) and non-leguminous (21.0%–22.0%) species. Polymorphisms in 40 randomly selected MsILP markers were evaluated in 21 alfalfa accessions and collectively yielded 169 alleles with an average of 4.7 alleles per locus. The polymorphism information content (PIC) ranged from 0.15 to 0.87 with an average of 0.60, which indicated a high level of polymorphism in the MsILP markers. For the first time, we developed large-scale ILP markers in alfalfa and demonstrated their utility in transferability, which will be valuable for genetic relationship assessments, comparative genomic studies and marker-assisted breeding of leguminous and non-leguminous species.

Arginine CGA codons as a source of nonsense mutations: a possible role in multivariant gene expression, control of mRNA quality, and aging.

Methylation of cytosine residues in DNA of higher eukaryotes, including humans, creates "hot spots" of C→T transitions in the genome. The predominantly methylated sequence in mammalian DNAs is CG (CpG). Among CG-containing codons, CGA codons for arginine are unique due to their ability to create stop codons TGA (UGA in mRNA) upon epigenetic-mediated mutation. As such nonsense mutations can have a strong adverse effect on the cell and organism, we have performed a study, on the example of human genes, aimed to characterise the anticipated effects of epigenetic-mediated nonsense mutations CGA→TGA in somatic cells. It is commonly accepted that premature termination codons (PTCs) lead to the biosynthesis of truncated and usually inactive proteins. In addition, transcripts with PTC can be destroyed by a nonsense-mediated mRNA decay (NMD) machinery. We have considered the cell potentialities (gene families, diploidy, and alternative splicing) to overcome the worst consequences of nonsense mutation. As a special case, in the biosynthesis of a particular group of proteins called selenoproteins, the mutation CGA→UGA would not lead to the premature translation termination and NMD but rather to the insertion of selenocysteine or cysteine instead of former arginine. The finding of SECIS (Sel insertion sequence)-like structures in a variety of mRNAs allowed us to postulate the existence of facultative selenoproteins, whose biosynthesis might be coupled with the redefinition of premature UGA stop codons arising upon mutations, as in the case of "classic" selenoproteins. Nevertheless, a detailed structural analysis of 165 transcripts has shown that roughly 80-90% of functional human mRNAs are potential substrates for NMD upon the PTC emergence. A hypothesis was put forward highlighting a role of arginine CGA codons together with glutamine CAA and CAG codons in the control of mRNA quality and life span. According to this hypothesis, the conversion of the ribonucleic codons CGA, CAA, or CAG into stop codons UGA, UAA or UAG owing to spontaneous or enzymatic cytosine deamination might serve as a trigger for the transcript destruction by NMD (C→U control). Thus, the consequences of epigenetic-mediated nonsense mutations are diverse and may largely depend on the structure of the transcript (CGA codon position, the presence and position of introns and SECIS elements, and splicing potential) of the cognate gene. However, this diversity and the presumable role of CGA codons in performing the everyday function by controlling whether genes are expressed correctly do not exclude their long-term role as limiters of the cell and organism life span. Thus, the presumable role of CGA codons in genome functioning and stability opens new perspectives to influence aging and concomitant deceases by codon editing.

Protein 4.1R Exon 16 3′ Splice Site Activation Requires Coordination among TIA1, Pcbp1, and RBM39 during Terminal Erythropoiesis

Exon 16 of protein 4.1R encodes a spectrin/actin-binding peptide critical for erythrocyte membrane stability. Its expression during erythroid differentiation is regulated by alternative pre-mRNA splicing. A UUUUCCCCCC motif situated between the branch point and the 3' splice site is crucial for inclusion. We show that the UUUU region and the last three C residues in this motif are necessary for the binding of splicing factors TIA1 and Pcbp1 and that these proteins appear to act in a collaborative manner to enhance exon 16 inclusion. This element also activates an internal exon when placed in a corresponding intronic position in a heterologous reporter. The impact of these two factors is further enhanced by high levels of RBM39, whose expression rises during erythroid differentiation as exon 16 inclusion increases. TIA1 and Pcbp1 associate in a complex containing RBM39, which interacts with U2AF65 and SF3b155 and promotes U2 snRNP recruitment to the branch point. Our results provide a mechanism for exon 16 3' splice site activation in which a coordinated effort among TIA1, Pcbp1, and RBM39 stabilizes or increases U2 snRNP recruitment, enhances spliceosome A complex formation, and facilitates exon definition through RBM39-mediated splicing regulation.