Plant Introns Research Articles

PCIS1, Encoded by a Pentatricopeptide Protein Co-expressed Gene, Is Required for Splicing of Three Mitochondrial nad Transcripts in Angiosperms.

Group II introns are large catalytic RNAs, which reside mainly within genes encoding respiratory complex I (CI) subunits in angiosperms' mitochondria. Genetic and biochemical analyses led to the identification of many nuclear-encoded factors that facilitate the splicing of the degenerated organellar introns in plants. Here, we describe the analysis of the pentatricopeptide repeat (PPR) co-expressed intron splicing-1 (PCIS1) factor, which was identified in silico by its co-expression pattern with many PPR proteins. PCIS1 is well conserved in land plants but has no sequence similarity with any known protein motifs. PCIS1 mutant lines are arrested in embryogenesis and can be maintained by the temporal expression of the gene under the embryo-specific ABI3 promoter. The pABI3::PCIS1 mutant plants display low germination and stunted growth phenotypes. RNA-sequencing and quantitative RT-PCR analyses of wild-type and mutant plants indicated that PCIS1 is a novel splicing cofactor that is pivotal for the maturation of several nad transcripts in Arabidopsis mitochondria. These phenotypes are tightly associated with respiratory CI defects and altered plant growth. Our data further emphasize the key roles of nuclear-encoded cofactors that regulate the maturation and expression of mitochondrial transcripts for the biogenesis of the oxidative phosphorylation system, and hence for plant physiology. The discovery of novel splicing factors other than typical RNA-binding proteins suggests further complexity of splicing mechanisms in plant mitochondria.

PlantIntronDB: a database forplant introns that host functional elements.

Although more and more attention has been focused on introns and the important role of plant introns in plant growth and development has been discovered, there is still a lack of an open and comprehensive database on plant introns with functional elements in current research. In order to make full use of large-scale sequencing data and help researchers in related fields to achieve high-throughput functional verification of identified plant introns with functional elements, we designed a database containing five plant species, PlantIntronDB and systematically analyzed 358, 59, 185, 210 and 141 RNA-seq samples from Arabidopsis thaliana (Arabidopsis), Gossypium raimondii (cotton), Zea mays (maize), Brassica napus (oilseed rape) and Oryza sativa Japonica Group (rice). In total, we found 100 126 introns that host functional elements in these five species. Specifically, we found that among all species, the number of introns with functional elements on the positive and negative strands is similar, with a length mostly smaller than 1500 bp, and the Adenine/Thymine (A/T) content is much higher than that of Guanine/Cytosine (G/C). In addition, the distribution of functional elements in introns varies among different species. All the above data can be downloaded for free in this database. This database provides a concise, comprehensive and user-friendly web interface, allowing users to easily retrieve target data based on their needs, using relevant organizational options. The database operation is simple and convenient, aiming to provide strong data support for researchers in related fields to study plant introns that host functional elements, including circular RNAs, lncRNAs, etc. Database URL: http://deepbiology.cn/PlantIntronDB/.

Sequence variations affect the 5' splice site selection of plant introns.

Introns are noncoding sequences spliced out of pre-mRNAs by the spliceosome to produce mature mRNAs. The 5' ends of introns mostly begin with GU and have a conserved sequence motif of AG/GUAAGU that could base-pair with the core sequence of U1 snRNA of the spliceosome. Intriguingly, ∼ 1% of introns in various eukaryotic species begin with GC. This occurrence could cause misannotation of genes; however, the underlying splicing mechanism is unclear. We analyzed the sequences around the intron 5' splice site (ss) in Arabidopsis (Arabidopsis thaliana) and found sequences at the GC intron ss are much more stringent than those of GT introns. Mutational analysis at various positions of the intron 5' ss revealed that although mutations impair base pairing, different mutations at the same site can have different effects, suggesting that steric hindrance also affects splicing. Moreover, mutations of 5' ss often activate a hidden ss nearby. Our data suggest that the 5' ss is selected via a competition between the major ss and the nearby minor ss. This work not only provides insights into the splicing mechanism of intron 5' ss but also improves the accuracy of gene annotation and the study of the evolution of intron 5' ss.

The Importance of Stiff Change of U(T) Content Around Splicing Sites in Efficient Plant Intron Splicing -- A Case Study in Rice

The Importance of Stiff Change of U(T) Content Around Splicing Sites in Efficient Plant Intron Splicing -- A Case Study in Rice

Organellar Introns in Fungi, Algae, and Plants.

Introns are ubiquitous in eukaryotic genomes and have long been considered as ‘junk RNA’ but the huge energy expenditure in their transcription, removal, and degradation indicate that they may have functional significance and can offer evolutionary advantages. In fungi, plants and algae introns make a significant contribution to the size of the organellar genomes. Organellar introns are classified as catalytic self-splicing introns that can be categorized as either Group I or Group II introns. There are some biases, with Group I introns being more frequently encountered in fungal mitochondrial genomes, whereas among plants Group II introns dominate within the mitochondrial and chloroplast genomes. Organellar introns can encode a variety of proteins, such as maturases, homing endonucleases, reverse transcriptases, and, in some cases, ribosomal proteins, along with other novel open reading frames. Although organellar introns are viewed to be ribozymes, they do interact with various intron- or nuclear genome-encoded protein factors that assist in the intron RNA to fold into competent splicing structures, or facilitate the turn-over of intron RNAs to prevent reverse splicing. Organellar introns are also known to be involved in non-canonical splicing, such as backsplicing and trans-splicing which can result in novel splicing products or, in some instances, compensate for the fragmentation of genes by recombination events. In organellar genomes, Group I and II introns may exist in nested intronic arrangements, such as introns within introns, referred to as twintrons, where splicing of the external intron may be dependent on splicing of the internal intron. These nested or complex introns, with two or three-component intron modules, are being explored as platforms for alternative splicing and their possible function as molecular switches for modulating gene expression which could be potentially applied towards heterologous gene expression. This review explores recent findings on organellar Group I and II introns, focusing on splicing and mobility mechanisms aided by associated intron/nuclear encoded proteins and their potential roles in organellar gene expression and cross talk between nuclear and organellar genomes. Potential application for these types of elements in biotechnology are also discussed.

PID: An integrative and comprehensive platform of plant intron

Intron is a non-coding sequence of a broken gene and participates in important biological processes, such as transcription regulation, alternative splicing, and nuclear export. With the development of plant genomes, a comprehensive platform for intron analysis in plants must be established. Plant Intron Database (PID), a publicly available searchable database, was developed to efficiently store, query, analyze, and integrate intron resources in plants. The information of intron, exon, and gene can be searched by key words in PID. Users cannot only view intron length distribution pie chart and 5′ and 3′ splice site sequence feature maps in a statistical interface but can also browse intron information in a graphical visualization interface through JBrowse. ViroBlast for sequence homology searches, Intron detection and sequence interception tools were also provided. PID contains annotated genes from 118 sequenced plants, 24,782,048 introns, 30,843,049 exons, and 414 visual maps. This tool will greatly accelerate research on the distribution, length characteristics, and functions of introns in plants. PID is accessible at http://biodb.sdau.edu.cn/PID/index.php.

CFM1, a member of the CRM-domain protein family, functions in chloroplast group II intron splicing in Setaria viridis.

The chloroplast RNA splicing and ribosome maturation (CRM) domain is a RNA-binding domain found in a plant-specific protein family whose characterized members play essential roles in splicing group I and group II introns in mitochondria and chloroplasts. Together, these proteins are required for splicing of the majority of the approximately 20 chloroplast introns in land plants. Here, we provide evidence from Setaria viridis and maize that an uncharacterized member of this family, CRM Family Member1 (CFM1), promotes the splicing of most of the introns that had not previously been shown to require a CRM domain protein. A Setaria mutant expressing mutated CFM1 was strongly disrupted in the splicing of three chloroplast tRNAs: trnI, trnV and trnA. Analyses by RNA gel blot and polysome association suggest that the tRNA deficiencies lead to compromised chloroplast protein synthesis and the observed whole-plant chlorotic phenotypes. Co-immunoprecipitation data demonstrate that the maize CFM1 ortholog is bound to introns whose splicing is disrupted in the cfm1 mutant. With these results, CRM domain proteins have been shown to promote the splicing of all but two of the introns found in angiosperm chloroplast genomes.

Biogeography and ecology of geographically distant populations of sibling Cryptocephalus leaf beetles

Different populations of two closely related species, Cryptocephalusflavipes and C. bameuli, from western (Alps, Apennines and Pyrenees) and central Europe (Poland, Ukraine and Pannonia) were analysed. On the basis of DNA sequences from two genes, cox1 and ef1-α, distinctiveness of both species was confirmed. Nevertheless, possible hybrids were identified in Carpathian mountains. We found a significant genetic differentiation among populations of C. flavipes and C. bameuli from distant regions but a high genetic similarity between populations of C. bameuli from north and south of the Carpathians. Demographic estimates suggest a past population expansion in the case of C. bameuli and a recent one for C. flavipes, possibly occurred during Pleistocene and Holocene, respectively. Distribution modelling showed that C. flavipes is typically present in the mountain systems, whereas C. bameuli is associated with hilly areas of central and eastern Europe. Based on the present data, Last Glacial Maximum refugia of both species were located in the Alpine region and Black Sea coasts, but on different elevations. The characterization of the insect diet, through a DNA metabarcoding approach targeting the trnL plant intron, demonstrated a significant differentiation of food preferences between the two species, as well as between geographic populations within the species.

OUP accepted manuscript

GrainGenes (https://wheat.pw.usda.gov or https://graingenes.org) is an international centralized repository for curated, peer-reviewed datasets useful to researchers working on wheat, barley, rye and oat. GrainGenes manages genomic, genetic, germplasm and phenotypic datasets through a dynamically generated web interface for facilitated data discovery. Since 1992, GrainGenes has served geneticists and breeders in both the public and private sectors on six continents. Recently, several new datasets were curated into the database along with new tools for analysis. The GrainGenes homepage was enhanced by making it more visually intuitive and by adding links to commonly used pages. Several genome assemblies and genomic tracks are displayed through the genome browsers at GrainGenes, including the Triticum aestivum (bread wheat) cv. 'Chinese Spring' IWGSC RefSeq v1.0 genome assembly, the Aegilops tauschii (D genome progenitor) Aet v4.0 genome assembly, the Triticum turgidum ssp. dicoccoides (wild emmer wheat) cv. 'Zavitan' WEWSeq v.1.0 genome assembly, a T. aestivum (bread wheat) pangenome, the Hordeum vulgare (barley) cv. 'Morex' IBSC genome assembly, the Secale cereale (rye) select 'Lo7' assembly, a partial hexaploid Avena sativa (oat) assembly and the Triticum durum cv. 'Svevo' (durum wheat) RefSeq Release 1.0 assembly. New genetic maps and markers were added and can be displayed through CMAP. Quantitative trait loci, genetic maps and genes from the Wheat Gene Catalogue are indexed and linked through the Wheat Information System (WheatIS) portal. Training videos were created to help users query and reach the data they need. GSP (Genome Specific Primers) and PIECE2 (Plant Intron Exon Comparison and Evolution) tools were implemented and are available to use. As more small grains reference sequences become available, GrainGenes will play an increasingly vital role in helping researchers improve crops.

A plant intron enhances the performance of an infectious clone in planta

Although infectious clones are fundamental tools in virology and plant pathology, their efficacy is often reduced by the instability of viral sequences in Escherichia coli. In this study, we constructed an infectious clone of PepMoV (pPepMoV) in a bacterial binary vector (pSNU1); the clone induces symptoms of PepMoV in agroinfiltrated plants. During its modification and maintenance in E. coli, however, the pPepMoV infectious clone was instable in the bacteria. Manipulation of this unstable clone in the bacterial strain DH10B led to the spontaneous formation of a recombined clone with high stability in the bacteria but with reduced infectivity due to an unwanted insertion of an E. coli sequence in the NIa-protease coding region. Replacement of this sequence with a plant intron restored infectivity and maintained plasmid stability. In addition to restoring plasmid growth in both E. coli and Agrobacterium, the presence of the intron in the PepMoV sequence enhanced the accumulation of PepMoV in agroinfiltrated leaves and resulted in symptom induction in upper systemic leaves that was nearly as strong as with PepMoV sap-inoculation. Plant introns have been previously used to stabilize plasmids in E. coli without any effect or with an unexpected lag in symptom development. In contrast, the current results demonstrated the in vivo enhancement of an infectious clone by a plant intron.

Position-specific intron retention is mediated by the histone methyltransferase SDG725

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

Comprehensive analysis of chloroplast intron-containing genes and conserved splice sites in dicot and monocot plants

Despite the increasing knowledge on the importance of the intron splicing of chloroplast genes during plant growth and stress responses, identification of intron-containing chloroplast genes and determination of splice sites in chloroplast introns are still lacking. Here, we carried out a comprehensive analysis of the chloroplast genome sequences in important plants and crops, including four dicots (Arabidopsis thaliana, Coffea arabica, Nicotiana tabacum, and Panax schinseng) and four monocots (Musa acuminata, Oryza sativa, Triticum aestivum, and Zea mays). The results showed that both dicot and monocot chloroplast genomes harbor 6 intron-containing tRNAs (trnA, trnG, trnI, trnK, trnL, and trnV) and 10-12 intron-containing mRNAs (atpF, rpl2, rpl16, rps16, ndhA, ndhB, petB, petD, rpoC1, rps12, ycf3, and clpP). Notably, rpoC1 and clpP lacked introns in monocot plants, except M. acuminata. Analysis of the nucleotide sequences of chloroplast introns revealed that the 5’-splice sites, 3’-splice sites, and branch-point sites of the chloroplast introns were highly conserved among dicots and monocots. Notably, the 5’-splice sites and 3’-splice sites of the chloroplast introns were similar to those of the nuclear U12 introns, whereas the branch-point sites of the chloroplast introns were homologous to those of the nuclear U2 introns. Taken together, these results indicated that the chloroplast genomes contained strictly limited intron-containing genes with conserved splice sites, suggesting that splicing of chloroplast introns was important for chloroplast biogenesis and function in both dicot and monocot plants.

The Reverse Transcriptase/RNA Maturase Protein MatR Is Required for the Splicing of Various Group II Introns in Brassicaceae Mitochondria.

Group II introns are large catalytic RNAs that are ancestrally related to nuclear spliceosomal introns. Sequences corresponding to group II RNAs are found in many prokaryotes and are particularly prevalent within plants organellar genomes. Proteins encoded within the introns themselves (maturases) facilitate the splicing of their own host pre-RNAs. Mitochondrial introns in plants have diverged considerably in sequence and have lost their maturases. In angiosperms, only a single maturase has been retained in the mitochondrial DNA: the matR gene found within NADH dehydrogenase 1 (nad1) intron 4. Its conservation across land plants and RNA editing events, which restore conserved amino acids, indicates that matR encodes a functional protein. However, the biological role of MatR remains unclear. Here, we performed an in vivo investigation of the roles of MatR in Brassicaceae. Directed knockdown of matR expression via synthetically designed ribozymes altered the processing of various introns, including nad1 i4. Pull-down experiments further indicated that MatR is associated with nad1 i4 and several other intron-containing pre-mRNAs. MatR may thus represent an intermediate link in the gradual evolutionary transition from the intron-specific maturases in bacteria into their versatile spliceosomal descendants in the nucleus. The similarity between maturases and the core spliceosomal Prp8 protein further supports this intriguing theory.

PIECE 2.0: an update for the plant gene structure comparison and evolution database.

PIECE (Plant Intron Exon Comparison and Evolution) is a web-accessible database that houses intron and exon information of plant genes. PIECE serves as a resource for biologists interested in comparing intron–exon organization and provides valuable insights into the evolution of gene structure in plant genomes. Recently, we updated PIECE to a new version, PIECE 2.0 (http://probes.pw.usda.gov/piece or http://aegilops.wheat.ucdavis.edu/piece). PIECE 2.0 contains annotated genes from 49 sequenced plant species as compared to 25 species in the previous version. In the current version, we also added several new features: (i) a new viewer was developed to show phylogenetic trees displayed along with the structure of individual genes; (ii) genes in the phylogenetic tree can now be also grouped according to KOG (The annotation of Eukaryotic Orthologous Groups) and KO (KEGG Orthology) in addition to Pfam domains; (iii) information on intronless genes are now included in the database; (iv) a statistical summary of global gene structure information for each species and its comparison with other species was added; and (v) an improved GSDraw tool was implemented in the web server to enhance the analysis and display of gene structure. The updated PIECE 2.0 database will be a valuable resource for the plant research community for the study of gene structure and evolution.

Utilizing modified ubi1 introns to enhance exogenous gene expression in maize (Zea mays L.) and rice (Oryza sativa L.)

The phenomenon of intron-mediated enhancement (IME) was discovered in 1990 based on the observation that plant introns can stimulate gene expression, particularly in monocots. However, the intrinsic mechanism of IME remains unclear because many studies have yielded various results depending on the promoter, reporter gene, flanking sequences of the intron, and target cell or tissue. In this study, the effect of the first intron of the maize ubiquitin gene (ubi1 intron) was investigated by changing insertion sites, deleting specific regions and mutating individual motifs in maize (Zea mays L.) and rice (Oryza sativa L.) using ubi1 intron-containing GUS (β-glucuronidase) constructs. In maize callus, the integration of the full-length ubi1 intron into the GUS coding sequence at the +13, +115 and +513 positions by particle bombardment increased GUS activity approximately five-, four- and two-fold, respectively. Eight truncated ubi1 introns in the pSG(13i) N construct significantly influenced GUS gene expression to different degrees in transient assays. Notably, the 3′ region deletions significantly reduced the IME effect, whereas a 142-nt deletion, pSG(13i-P5)N, in the 5′ region caused a 1.5-fold enhancement relative to pSG(13i)N. Furthermore, four site-directed mutageneses were performed in pSG(13i-P5)N; these constructs resulted in the up-regulation of GUS gene expression to different levels. The most effective modified ubi1 intron, pSG(13i-M4)N, was further evaluated and proved in rice using transient experiments. In addition, the sequences flanking the GUS insertion significantly influenced the IME effect of the vectors that were constructed. The modified ubi1 intron had the potential application on crop genetic engineering.

Synonymous Codon Usage Bias in Plant Mitochondrial Genes Is Associated with Intron Number and Mirrors Species Evolution

Synonymous codon usage bias (SCUB) is a common event that a non-uniform usage of codons often occurs in nearly all organisms. We previously found that SCUB is correlated with both intron number and exon position in the plant nuclear genome but not in the plastid genome; SCUB in both nuclear and plastid genome can mirror the evolutionary specialization. However, how about the rules in the mitochondrial genome has not been addressed. Here, we present an analysis of SCUB in the mitochondrial genome, based on 24 plant species ranging from algae to land plants. The frequencies of NNA and NNT (A- and T-ending codons) are higher than those of NNG and NNC, with the strongest preference in bryophytes and the weakest in land plants, suggesting an association between SCUB and plant evolution. The preference for NNA and NNT is more evident in genes harboring a greater number of introns in land plants, but the bias to NNA and NNT exhibits even among exons. The pattern of SCUB in the mitochondrial genome differs in some respects to that present in both the nuclear and plastid genomes.

Variability of the trnL plastid gene’s intron in the Faboideae species (Fabaceae)

The intron located between the first and the second nucleotide of the leucine tRNA anticodon is the only representative of the group I introns in higher plants. In this work, the intron sequence of the plastid trnL gene was for the first time described in 16 legumes species and putative secondary structures of the full-length intron and its functional domains were reconstructed. A high level of polymorphism was detected for this intron in the Fabaceae genera and in the species within a single genus. Single nucleotide polymorphisms, have been found in the sequences of the catalytic center which are considered to be highly conservative.

In silico identification and analysis of phytoene synthase genes in plants.

In this study, we examined phytoene synthetase (PSY), the first key limiting enzyme in the synthesis of carotenoids and catalyzing the formation of geranylgeranyl pyrophosphate in terpenoid biosynthesis. We used known amino acid sequences of the PSY gene in tomato plants to conduct a genome-wide search and identify putative candidates in 34 sequenced plants. A total of 101 homologous genes were identified. Phylogenetic analysis revealed that PSY evolved independently in algae as well as monocotyledonous and dicotyledonous plants. Our results showed that the amino acid structures exhibited 5 motifs (motifs 1 to 5) in algae and those in higher plants were highly conserved. The PSY gene structures showed that the number of intron in algae varied widely, while the number of introns in higher plants was 4 to 5. Identification of PSY genes in plants and the analysis of the gene structure may provide a theoretical basis for studying evolutionary relationships in future analyses.

Bioinformatics analysis of plant orthologous introns: identification of an intronic tRNA-like sequence.

Orthologous introns have identical positions relative to the coding sequence in orthologous genes of different species. By analyzing the complete genomes of five plants we generated a database of 40,512 orthologous intron groups of dicotyledonous plants, 28,519 orthologous intron groups of angiosperms, and 15,726 of land plants (moss and angiosperms). Multiple sequence alignments of each orthologous intron group were obtained using the Mafft algorithm. The number of conserved regions in plant introns appeared to be hundreds of times fewer than that in mammals or vertebrates. Approximately three quarters of conserved intronic regions among angiosperms and dicots, in particular, correspond to alternatively-spliced exonic sequences. We registered only a handful of conserved intronic ncRNAs of flowering plants. However, the most evolutionarily conserved intronic region, which is ubiquitous for all plants examined in this study, including moss, possessed multiple structural features of tRNAs, which caused us to classify it as a putative tRNA-like ncRNA. Intronic sequences encoding tRNA-like structures are not unique to plants. Bioinformatics examination of the presence of tRNA inside introns revealed an unusually long-term association of four glycine tRNAs inside the Vac14 gene of fish, amniotes, and mammals.

Arabidopsis PTB1 and PTB2 proteins negatively regulate splicing of a mini-exon splicing reporter and affect alternative splicing of endogenous genes differentially.

This paper examines the function of Arabidopsis thaliana AtPTB1 and AtPTB2 as plant splicing factors. The effect on splicing of overexpression of AtPTB1 and AtPTB2 was analysed in an in vivo protoplast transient expression system with a novel mini-exon splicing reporter. A range of mutations in pyrimidine-rich sequences were compared with and without AtPTB and NpU2AF65 overexpression. Splicing analyses of constructs in protoplasts and RNA from overexpression lines used high-resolution reverse transcription polymerase chain reaction (RT-PCR). AtPTB1 and AtPTB2 reduced inclusion/splicing of the potato invertase mini-exon splicing reporter, indicating that these proteins can repress plant intron splicing. Mutation of the polypyrimidine tract and closely associated Cytosine and Uracil-rich (CU-rich) sequences, upstream of the mini-exon, altered repression by AtPTB1 and AtPTB2. Coexpression of a plant orthologue of U2AF65 alleviated the splicing repression of AtPTB1. Mutation of a second CU-rich upstream of the mini-exon 3' splice site led to a decline in mini-exon splicing, indicating the presence of a splicing enhancer sequence. Finally, RT-PCR of AtPTB overexpression lines with c. 90 known alternative splicing (AS) events showed that AtPTBs significantly altered AS of over half the events. AtPTB1 and AtPTB2 are splicing factors that influence alternative splicing. This occurs in the potato invertase mini-exon via the polypyrimidine tract and associated pyrimidine-rich sequence.