Multiple Introns Research Articles

Generation of inheritable A-to-G transitions using adenine base editing and NG-PAM Cas9 in Arabidopsis thaliana.

Towards precise genome editing, base editors have been developed by fusing catalytically compromised Cas9 with deaminase components, mediating C-to-T (cytosine base editors) or A-to-G (adenine base editors) transition. We developed a set of vectors consisting of a 5'-NG-3' PAM-recognising variant of SpCas9 with adenosine deaminases, TadA7.10 or TadA8e. Using a phenotype-based screen in Arabidopsis thaliana targeting multiple PDS3 intron splice sites, we achieved up to 81% somatic A-to-G editing in primary transformants at a splice acceptor site with NGG PAM, while 35% was achieved for the same target adenine with NGA PAM. Among tested vectors, pECNUS4 (Addgene #184887), carrying TadA8e, showed the highest ABE efficiency. With pECNUS4, we recreated a naturally occurring allele of DANGEROUS MIX3 (DM3) in two generations, transgene-free, for NGC PAM. We also simultaneously base-edited four redundant DM1/SSI4 homologs, encoding nucleotide-binding leucine-rich repeat (NLR) proteins, using a single gRNA with NGA PAM targeting the conserved yet functionally crucial P-loop motif of NLR proteins. We found fixation of A-to-G in three NLR genes for all three possible adenine sites within base-editing window 3-9, as the edited genes segregate in T2. Multigene targeting succeeded in rescuing the previously reported autoimmune phenotype in two generations. Mediating desired ABE on seven NLR genes simultaneously was successful as well; above 77% editing was achieved in six of the seven possible targets in a T1 plant, with the remaining having a moderately high (32%) editing. ABE application to specifically inactivate functional motifs is anticipated to expedite the discovery of novel roles for proteins.

An optimised CRISPR Cas9 and Cas12a mutagenesis toolkit for Barley and Wheat

BackgroundCRISPR Cas9 and Cas12a are the two most frequently used programmable nucleases reported in plant systems. There is now a wide range of component parts for both which likely have varying degrees of effectiveness and potentially applicability to different species. Our aim was to develop and optimise Cas9 and Cas12a based systems for highly efficient genome editing in the monocotyledons barley and wheat and produce a user-friendly toolbox facilitating simplex and multiplex editing in the cereal community.ResultsWe identified a Zea mays codon optimised Cas9 with 13 introns in conjunction with arrayed guides driven by U6 and U3 promoters as the best performer in barley where 100% of T0 plants were simultaneously edited in all three target genes. When this system was used in wheat > 90% of T0 plants were edited in all three subgenome targets. For Cas12a, an Arabidopsis codon optimised sequence with 8 introns gave the best editing efficiency in barley when combined with a tRNA based multiguide array, resulting in 90% mutant alleles in three simultaneously targeted genes. When we applied this Cas12a system in wheat 86% & 93% of T0 plants were mutated in two genes simultaneously targeted. We show that not all introns contribute equally to enhanced mutagenesis when inserted into a Cas12a coding sequence and that there is rationale for including multiple introns. We also show that the combined effect of two features which boost Cas12a mutagenesis efficiency (D156R mutation and introns) is more than the sum of the features applied separately.ConclusionBased on the results of our testing, we describe and provide a GoldenGate modular cloning system for Cas9 and Cas12a use in barley and wheat. Proven Cas nuclease and guide expression cassette options found in the toolkit will facilitate highly efficient simplex and multiplex mutagenesis in both species. We incorporate GRF-GIF transformation boosting cassettes in wheat options to maximise workflow efficiency.

Identification and analyses of exonic and copy number variants in spastic paraplegia

Hereditary spastic paraplegias are a diverse group of degenerative disorders that are clinically categorized as isolated; with involvement of lower limb spasticity, or symptomatic, where spastic paraplegia is complicated by further neurological features. We sought to identify the underlying genetic causes of these disorders in the participating patients. Three consanguineous families with multiple affected members were identified by visiting special schools in the Punjab Province. DNA was extracted from blood samples of the participants. Exome sequencing was performed for selected patients from the three families, and the data were filtered to identify rare homozygous variants. ExomeDepth was used for the delineation of the copy number variants. All patients had varying degrees of intellectual disabilities, poor speech development, spasticity, a wide-based gait or an inability to walk and hypertonia. In family RDHR07, a homozygous deletion involving multiple exons and introns of SPG11 (NC000015.9:g.44894055_449028del) was found and correlated with the phenotype of the patients who had spasticity and other complex movement disorders, but not those who exhibited ataxic or indeterminate symptoms as well. In families ANMD03 and RDFA06, a nonsense variant, c.985C > T;(p.Arg329Ter) in DDHD2 and a frameshift insertion‒deletion variant of AP4B1, c.965-967delACTinsC;p.(Tyr322SerfsTer14), were identified which were homozygous in the patients while the obligate carriers in the respective pedigrees were heterozygous. All variants were ultra-rare with none, or very few carriers identified in the public databases. The three loss of function variants are likely to cause nonsense-mediated decay of the respective transcripts. Our research adds to the genetic variability associated with the SPG11 and AP4B1 variants and emphasizes the genetic heterogeneity of hereditary spastic paraplegia.

Intron lariat spliceosomes convert lariats to true circles: implications for intron transposition.

Rare, full-length circular intron RNAs distinct from lariats have been reported in several species, but their biogenesis is not understood. We envisioned and tested a hypothesis for their formation using Saccharomyces cerevisiae, documenting full-length and novel processed circular RNAs from multiple introns. Evidence implicates a previously undescribed catalytic activity of the intron lariat spliceosome (ILS) in which the 3'-OH of the lariat tail (with optional trimming and adenylation by the nuclear 3' processing machinery) attacks the branch, joining the intron 3' end to the 5' splice site in a 3'-5' linked circle. Human U2 and U12 spliceosomes produce analogous full-length and processed circles. Postsplicing catalytic activity of the spliceosome may promote intron transposition during eukaryotic genome evolution.

Characterization of rice aspartic protease genes and induced expression by phytohormones and Xanthomonas oryzae pv. oryzae

Aspartic proteases (APs) are present in both monocotyledonous and dicotyledonous plants. APs consist of nine different families of proteolytic enzymes. APs play important functions in several physiological processes including defense. However, there are limited reports on APs in disease resistance in rice (Oryza sativa). Here, we report that 104 APs were discovered in rice genome. They can be classified into three clades (I, II and III) with APs from Arabidopsis thaliana, and Nymphaea colorata according to phylogenetic relationships. Tandem and segmental duplications were observed to lead to the expansion of OsAP genes in rice. Analysis of gene structure revealed that numerous OsAPs contain multiple exons and introns, however, several OsAPs harbor a single exon and no introns. Synteny analyses demonstrated that four AP genes among O. sativa, A. thaliana, and N. colorata may evolve from a common ancestor of land plants. Ten conserved motifs of amino acids were uncovered from the OsAPs, and two of the motifs are the catalytic sequences, with the conserved Asp-Thr and Ser-Gly amino acids. Within the OsAP promoter sequences, cis-regulatory elements responsive to light, abscisic acid, auxin, gibberellins, methyl jasmonate, and salicylic acid were identified. Stress-responsive elements and tissue-specific expressed elements were also found. Majority of OsAP genes were found to be expressed in diverse tissues and organs. Some of the OsAP genes were observed to be induced by brassinosteroids, jasmonic acid, and salicylic acid. Further, some OsAP genes can be induced by Xanthomonas oryzae pv. oryzae (Xoo), the cause of rice bacterial leaf blight. Among them, OsAP21 was strongly activated by the pathogen, suggesting its potential function in response to the bacterial agent. The results shed light in the potential functions of OsAP genes in rice disease resistance to Xoo.

Identification of the RRM1 gene family in rice (Oryza sativa) and its response to rice blast.

To better understand RNA-binding proteins in rice, a comprehensive investigation was conducted on the RRM1 gene family of rice. It encompassed genome-wide identification and exploration of its role in rice blast resistance. The physicochemical properties of the rice OsRRM1 gene family were analyzed. There genes were also analyzed for their conserved domains, motifs, location information, gene structure, phylogenetic trees, collinearity, and cis-acting elements. Furthermore, alterations in the expression patterns of selected OsRRM1 genes were assessed using quantitative real-time PCR (qRT-PCR). A total of 212 members of the OsRRM1 gene family were identified, which were dispersed across 12 chromosomes. These genes all exhibit multiple exons and introns, all of which encompass the conserved RRM1 domain and share analogous motifs. This observation suggests a high degree of conservation within the encoded sequence domain of these genes. Phylogenetic analysis revealed the existence of five subfamilies within the OsRRM1 gene family. Furthermore, investigation of the promoter region identified cis-regulatory elements that are involved in nucleic acid binding and interaction with multiple transcription factors. By employing GO and KEGG analyses, four RRM1 genes were tentatively identified as crucial contributors to plant immunity, while the RRM1 gene family was also found to have a significant involvement in the complex of alternative splicing. The qRT-PCR results revealed distinct temporal changes in the expression patterns of OsRRM1 genes following rice blast infection. Additionally, gene expression analysis indicates that the majority of OsRRM1 genes exhibited constitutive expressions. These findings enrich our understanding of the OsRRM1 gene family. They also provide a foundation for further research on immune mechanisms rice and the management of rice blast.

Genome-wide identification and analysis of the MAPKK gene family in Chinese mitten crab (Eriocheir sinensis) and its response to bacterial challenge

Protein kinases of the MAPK cascade family (MAPKKK–MAPKK–MAPK) play an important role in the growth and development of organisms and their response to environmental stress. The MAPKK gene families in the Chinese mitten crab Eriocheir sinensis have never been systematically analyzed. We identified four MAPKK family genes, EsMEK, EsMAPKK4, EsMAPKK6, and EsMAPKK7, in E. sinensis and analyzed their molecular features and expression patterns. All four MAPKK genes are composed of multiple exons and introns, all have a conserved domain, and all have 10 conserved motifs (except EsMEK and EsMAPKK7 which are missing motif 10). The four MAPKK genes are on four different chromosomes and have no gene duplications, and the results of phylogenetic tree analysis indicate that the ESMAPKK gene family is highly conserved evolutionarily. The EsMAPKK genes were widely expressed in all the examined tissues with higher expression in hemocytes, hepatopancreas, and gills. Notably, EsMAPKK6 was also highly expressed in the ovary. Vibrio parahaemolyticus infection significantly increased the mRNA levels of the EsMAPKK genes in hemocytes. Further disruption of the EsMAPKK gene family expression affects the expression levels of multiple antimicrobial peptides in hemocytes. Our experimental results provide a starting point for a more in-depth study of the innate immunity functional roles of members of the MAPKK gene families in E. sinensis.

The DEAD-box RNA helicase ZmRH48 is required for the splicing of multiple mitochondrial introns, mitochondrial complex biosynthesis, and seed development in maize.

RNA helicases participate in nearly all aspects of RNA metabolism by rearranging RNAs or RNA-protein complexes in an adenosine triphosphate-dependent manner. Due to the large RNA helicase families in plants, the precise roles of many RNA helicases in plant physiology and development remain to be clarified. Here, we show that mutations in maize (Zea mays) DEAD-box RNA helicase 48 (ZmRH48) impair the splicing of mitochondrial introns, mitochondrial complex biosynthesis, and seed development. Loss of ZmRH48 function severely arrested embryogenesis and endosperm development, leading to defective kernel formation. ZmRH48 is targeted to mitochondria, where its deficiency dramatically reduced the splicing efficiency of five cis-introns (nad5 intron 1; nad7 introns 1, 2, and 3; and ccmFc intron 1) and one trans-intron (nad2 intron 2), leading to lower levels of mitochondrial complexes I and III. ZmRH48 interacts with two unique pentatricopeptide repeat (PPR) proteins, PPR-SMR1 and SPR2, which are required for the splicing of over half of all mitochondrial introns. PPR-SMR1 interacts with SPR2, and both proteins interact with P-type PPR proteins and Zm-mCSF1 to facilitate intron splicing. These results suggest that ZmRH48 is likely a component of a splicing complex and is critical for mitochondrial complex biosynthesis and seed development.

Bioinformatics Analysis of MSH1 Genes of Green Plants: Multiple Parallel Length Expansions, Intron Gains and Losses, Partial Gene Duplications, and Alternative Splicing.

MutS homolog 1 (MSH1) is involved in the recombining and repairing of organelle genomes and is essential for maintaining their stability. Previous studies indicated that the length of the gene varied greatly among species and detected species-specific partial gene duplications in Physcomitrella patens. However, there are critical gaps in the understanding of the gene size expansion, and the extent of the partial gene duplication of MSH1 remains unclear. Here, we screened MSH1 genes in 85 selected species with genome sequences representing the main clades of green plants (Viridiplantae). We identified the MSH1 gene in all lineages of green plants, except for nine incomplete species, for bioinformatics analysis. The gene is a singleton gene in most of the selected species with conserved amino acids and protein domains. Gene length varies greatly among the species, ranging from 3234 bp in Ostreococcus tauri to 805,861 bp in Cycas panzhihuaensis. The expansion of MSH1 repeatedly occurred in multiple clades, especially in Gymnosperms, Orchidaceae, and Chloranthus spicatus. MSH1 has exceptionally long introns in certain species due to the gene length expansion, and the longest intron even reaches 101,025 bp. And the gene length is positively correlated with the proportion of the transposable elements (TEs) in the introns. In addition, gene structure analysis indicated that the MSH1 of green plants had undergone parallel intron gains and losses in all major lineages. However, the intron number of seed plants (gymnosperm and angiosperm) is relatively stable. All the selected gymnosperms contain 22 introns except for Gnetum montanum and Welwitschia mirabilis, while all the selected angiosperm species preserve 21 introns except for the ANA grade. Notably, the coding region of MSH1 in algae presents an exceptionally high GC content (47.7% to 75.5%). Moreover, over one-third of the selected species contain species-specific partial gene duplications of MSH1, except for the conserved mosses-specific partial gene duplication. Additionally, we found conserved alternatively spliced MSH1 transcripts in five species. The study of MSH1 sheds light on the evolution of the long genes of green plants.

Inefficient splicing of long non-coding RNAs is associated with higher transcript complexity in human and mouse

ABSTRACT Recent reports show that long non-coding RNAs (lncRNAs) have inefficient splicing and fewer alternative splice variants than mRNAs. Here, we have explored the efficiency of lncRNAs and mRNAs in producing various splice variants, given the number of exons in humans and mice. Intriguingly, lncRNAs produce more splice variants per exon, referred to as Transcript Complexity, than mRNAs. Most lncRNA splice variants are the product of the alternative last exon and exon skipping. LncRNAs and mRNAs with higher transcript complexity have shorter intron lengths. Longer exon length and GC/AG at 5’/3’ splice sites are associated with higher transcript complexity in lncRNAs. Lastly, our results indicate that inefficient splicing of lncRNAs may facilitate multiple introns splicing and, thus, more spliced products per exon.

Characterization of the genomic and transcriptional structure of chicken NRG4 gene.

Neuregulin 4 (NRG4) is an important adipocytokine, which plays crucial roles in maintaining energy balance, regulating glucose and lipid metabolism, and preventing non-alcoholic fatty liver disease in mammals. At present, the genomic organization, transcript and protein isoforms of human NRG4 gene have been fully explored. Previous studies in our laboratory have shown that the NRG4 gene is expressed in chicken adipose tissue, but the chicken NRG4 (cNRG4) genomic structure, transcript and protein isoforms are still unknown. To this end, in this study, the genomic and transcriptional structure of the cNRG4 gene were systematically investigated using rapid amplification of cDNA ends (RACE) and reverse transcription-polymerase chain reaction (RT-PCR). The results showed that the coding region (CDS) of the cNRG4 gene was small, but it had a very complex transcriptional structure characterized by multiple transcription start sites, alternative splicing, intron retention, cryptic exons, and alternative polyadenylation, thus leading to production of four 5?UTR isoforms (cNRG4 A, cNRG4 B, cNRG4 C, and cNRG4 D) and six 3?UTR isoforms (cNRG4 a, cNRG4 b, cNRG4 c, cNRG4 d, cNRG4 e, and cNRG4 f) of the cNRG4 gene. The cNRG4 gene spanned 21,969 bp of genomic DNA (Chr.10:3,490,314~3,512,282) and consisted of 11 exons and 10 introns. Compared with the cNRG4 gene mRNA sequence (NM_001030544.4), two novel exons and one cryptic exon of the cNRG4 gene were identified in this study. Bioinformatics analysis, RT-PCR, cloning and sequencing analysis showed that the cNRG4 gene could encode three protein isoforms (cNRG4-1, cNRG4-2 and cNRG4-3). This study lays a foundation for further research on the function and regulation of the cNRG4 gene.

High-efficient production of mushroom polyketide compounds in a platform host Aspergillus oryzae

BackgroundOrsellinic acid (2,4-dihydroxy-6-methylbenzoic acid, OA) and its structural analog o-Orsellinaldehyde, have become widely used intermediates in clinical drugs synthesis. Although the research on the biosynthesis of such compounds has made significant progress, due to the lack of suitable hosts, there is still far from the industrial production of such compounds based on synthetic biology.ResultsWith the help of genome mining, we found a polyketide synthase (PKS, HerA) in the genome of the Hericium erinaceus, which shares 60% amino acid sequence homology with ArmB from Armillaria mellea, an identified PKS capable of synthesizing OA. To characterize the function of HerA, we cloned herA and heterologously expressed it in Aspergillus oryzae, and successfully detected the production of OA. Subsequently, the introduction of an incomplete PKS (Pks5) from Ustilago maydis containing only three domains (AMP-ACP-R), which was into herA-containing A. oryzae, the resulted in the production of o-Orsellinaldehyde. Considering the economic value of OA and o-Orsellinaldehyde, we then optimized the yield of these compounds in A. oryzae. The screening showed that when maltose was used as carbon source, the yields of OA and o-Orsellinaldehyde were 57.68 mg/L and 15.71 mg/L respectively, while the yields were 340.41 mg/Kg and 84.79 mg/Kg respectively in rice medium for 10 days.ConclusionsHerein, we successfully expressed the genes of basidiomycetes using A. oryzae heterologous host. As a fungus of ascomycetes, which not only correctly splices genes of basidiomycetes containing multiple introns, but also efficiently produces their metabolites. This study highlights that A. oryzae is an excellent host for the heterologous production of fungal natural products, and has the potential to become an efficient chassis for the production of basidiomycete secondary metabolites in synthetic biology.Graphical

The revealing of a novel lipid transfer protein lineage in green algae

BackgroundNon-specific lipid transfer proteins (nsLTPs) are a group of small and basic proteins that can bind and transfer various lipid molecules to the apoplastic space. A typical nsLTP carries a conserved architecture termed eight-cysteine motif (8CM), a scaffold of loop-linked helices folding into a hydrophobic cavity for lipids binding. Encoded by a multigene family, nsLTPs are widely distributed in terrestrial plants from bryophytes to angiosperms with dozens of gene members in a single species. Although the nsLTPs in the most primitive plants such as Marchantia already reach 14 members and are divergent enough to form separate groups, so far none have been identified in any species of green algae.ResultsBy using a refined searching strategy, we identified putative nsLTP genes in more than ten species of green algae as one or two genes per haploid genome but not in red and brown algae. The analyses show that the algal nsLTPs carry unique characteristics, including the extended 8CM spacing, larger molecular mass, lower pI value and multiple introns in a gene, which suggests that they could be a novel nsLTP lineage. Moreover, the results of further investigation on the two Chlamydomonas nsLTPs using transcript and protein assays demonstrated their late zygotic stage expression patterns and the canonical nsLTP properties were also verified, such as the fatty acids binding and proteinase resistance activities.ConclusionsIn conclusion, a novel nsLTP lineage is identified in green algae, which carries some unique sequences and molecular features that are distinguishable from those in land plants. Combined with the results of further examinations of the Chlamydomonas nsLTPs in vitro, possible roles of the algal nsLTPs are also suggested. This study not only reveals the existence of the nsLTPs in green algae but also contributes to facilitating future studies on this enigmatic protein family.

Gene fusions, micro-exons and splice variants define stress signaling by AP2/ERF and WRKY transcription factors in the sesame pan-genome.

Evolutionary dynamics of AP2/ERF and WRKY genes, the major components of defense response were studied extensively in the sesame pan-genome. Massive variation was observed for gene copy numbers, genome location, domain structure, exon-intron structure and protein parameters. In the pan-genome, 63% of AP2/ERF members were devoid of introns whereas >99% of WRKY genes contained multiple introns. AP2 subfamily was found to be micro-exon rich with the adjoining intronic sequences sharing sequence similarity to many stress-responsive and fatty acid metabolism genes. WRKY family included extensive multi-domain gene fusions where the additional domains significantly enhanced gene and exonic sizes as well as gene copy numbers. The fusion genes were found to have roles in acquired immunity, stress response, cell and membrane integrity as well as ROS signaling. The individual genomes shared extensive synteny and collinearity although ecological adaptation was evident among the Chinese and Indian accessions. Significant positive selection effects were noticed for both micro-exon and multi-domain genes. Splice variants with changes in acceptor, donor and branch sites were common and 6-7 splice variants were detected per gene. The study ascertained vital roles of lipid metabolism and chlorophyll biosynthesis in the defense response and stress signaling pathways. 60% of the studied genes localized in the nucleus while 20% preferred chloroplast. Unique cis-element distribution was noticed in the upstream promoter region with MYB and STRE in WRKY genes while MYC was present in the AP2/ERF genes. Intron-less genes exhibited great diversity in the promoter sequences wherein the predominance of dosage effect indicated variable gene expression levels. Mimicking the NBS-LRR genes, a chloroplast localized WRKY gene, Swetha_24868, with additional domains of chorismate mutase, cAMP and voltage-dependent potassium channel was found to act as a master regulator of defense signaling, triggering immunity and reducing ROS levels.

A manually curated annotation characterises genomic features of P. falciparum lncRNAs

BackgroundImportant regulation occurs at the level of transcription in Plasmodium falciparum and growing evidence suggests that these apicomplexan parasites have complex regulatory networks. Recent studies implicate long noncoding RNAs (lncRNAs) as transcriptional regulators in P. falciparum. However, due to limited research and the lack of necessary experimental tools, our understanding of their role in the malaria-causing parasite remains largely unelucidated. In this work, we address one of these limitations, the lack of an updated and improved lncRNA annotation in P. falciparum.ResultsWe generated long-read RNA sequencing data and integrated information extracted and curated from multiple sources to manually annotate lncRNAs. We identified 1119 novel lncRNAs and validated and refined 1250 existing annotations. Utilising the collated datasets, we generated evidence-based ranking scores for each annotation and characterised the distinct genomic contexts and features of P. falciparum lncRNAs. Certain features indicated subsets with potential biological significance such as 25 lncRNAs containing multiple introns, 335 lncRNAs lacking mutations in piggyBac mutagenic studies and lncRNAs associated with specific biologic processes including two new types of lncRNAs found proximal to var genes.ConclusionsThe insights and the annotation presented in this study will serve as valuable tools for researchers seeking to understand the role of lncRNAs in parasite biology through both bioinformatics and experimental approaches.

Comparative analyses of Flammulina filiformis mitochondrial genomes reveal high length polymorphism in intergenic regions and multiple intron gain/loss in cox1

The golden-needle mushroom Flammulina filiformis is one of the bulk mushroom products in the world. This study obtained complete mitogenomes of 44 wild isolates collected from nine provinces and two artificially bred cultivars of F. filiformis, together with three Flammulina rossica isolates and one Flammulina fennae isolate for comparison. The mitogenome of F. filiformis ranged from 83,540 bp to 90,938 bp, consisting of 14 conserved protein-coding genes (PCGs), two rRNA genes, and 25 tRNA genes. To the best of our knowledge, it contained the highest proportion of intergenic regions compared to the other known Basidiomycota mitogenomes. Introns and intergenic regions were two major contributing factors to the total size of the F. filiformis mitogenome. The conserved PCG cox3 is located in an intron of another conserved PCG, nad5. This is a unique phenomenon in all known fungal mitogenomes. Gain/loss of introns was observed in cox1, nad5, and rnl. Length polymorphism was widely observed in intergenic regions. Accordingly, primers were designed as useful markers for rapid identification of F. filiformis isolates with differentiated mitogenomes. Our findings provide a basis for further studies related to variety identification and population genetics of this economically important mushroom.

Intraspecific Variability and Distribution Difference within the Ribosomal Introns of the Discrete Plasmodiophora brassicae Group in Japan: A Case Study for Complex Dynamics of Intron Evolution

Analysis of the ribosomal introns of Plasmodiophora brassicae populations infecting the cruciferous weed Cardamine occulta revealed the complex dynamics of size, intraspecific variability, and distribution. The results showed that P. brassicae populations from the weed have lost multiple introns in the small and large subunits of the ribosomal RNA genes. Moreover, the retained introns, despite a largely mutual share of conserved parts with the cosmopolitan strains, contained numerous novel structures. These structural differences comprise a high level of polymorphisms, such as transversion point mutations occurring at sites involving the intronic splicing sites or insertions/deletions at the binding sites. Two geographical P. brassicae populations from C. occulta carried a lengthy intron-encoded ORF and putative mobile elements established in the large subunit. A few P. brassicae populations from the Brassica crops also harbored polymorphic introns that shared common mutated motifs with the weed-affecting group. The diversity of ribosomal introns observed from those investigated populations demonstrated the genetic distinction of the P. brassicae populations from C. occulta. The genetic variations might play a key role in the adaptability of the weed-infecting populations and are more likely related to the process of pathogenesis for the cosmopolitan P. brassicae infecting the Brassica crops.

Phylogenetic analysis and expression profiling of Nuclear Factor-Y gene family in Dendrobium catenatum Lindl. (Orchidaceae)

Nuclear factor-Y (NF-Y) are transcription factors that play vital role in various developmental processes such as flowering, embryogenesis, root formation, nodulation and stress tolerance. In the present study, 27 NF-Y genes were identified in Dendrobium catenatum which were grouped into three sub-families: DcNF-YA (5), DcNF-YB (10) and DcNF-YC (12), on the basis of presence of specific conserved domains. The phylogenetic relationship between DcNF-Y protein sequences with their orthologs in Vanilla planifolia and Arabidopsis thaliana confirmed the classification of these proteins into the specified three groups. Physico-chemical characterization reported a wide range in the protein length (111 to 433 amino acids), with DcNF-YAs being longer than the others. Strong interaction between most of the DcNF-Y proteins suggests towards the formation of complexes which might be playing a role in different developmental processes. Gene architectural studies predicted the presence of multiple (3–6) introns in DcNF-YA in contrast with the intron-less status of majority of DcNF-YB and DcNF-YC genes. The abundance of stress related cis-regulatory elements in the promoter regions of DcNF-Y genes indicate the potential of these genes in stress tolerance. Additionally, variable expression of these genes in diverse tissues point towards their widespread role in growth and development. This work can act as a harbinger for functional characterization of NF-Y genes with a view for genetic improvement in Dendrobium catenatum Lindl.

The Evolution of Hemocyanin Genes in Caenogastropoda: Gene Duplications and Intron Accumulation in Highly Diverse Gastropods

Hemocyanin is the oxygen transport protein of most molluscs and represents an important physiological factor that has to be well-adapted to their environments because of the strong influences of abiotic factors on its oxygen affinity. Multiple independent gene duplications and intron gains have been reported for hemocyanin genes of Tectipleura (Heterobranchia) and the caenogastropod species Pomacea canaliculata, which contrast with the uniform gene architectures of hemocyanins in Vetigastropoda. The goal of this study was to analyze hemocyanin gene evolution within the diverse group of Caenogastropoda in more detail. Our findings reveal multiple gene duplications and intron gains and imply that these represent general features of Apogastropoda hemocyanins. Whereas hemocyanin exon–intron structures are identical within different Tectipleura lineages, they differ strongly within Caenogastropoda among phylogenetic groups as well as between paralogous hemocyanin genes of the same species. Thus, intron accumulation took place more gradually within Caenogastropoda but finally led to a similar consequence, namely, a multitude of introns. Since both phenomena occurred independently within Heterobranchia and Caenogastropoda, the results support the hypothesis that introns may contribute to adaptive radiation by offering new opportunities for genetic variability (multiple paralogs that may evolve differently) and regulation (multiple introns). Our study indicates that adaptation of hemocyanin genes may be one of several factors that contributed to the evolution of the large diversity of Apogastropoda. While questions remain, this hypothesis is presented as a starting point for the further study of hemocyanin genes and possible correlations between hemocyanin diversity and adaptive radiation.

Transcription and splicing dynamics during early Drosophila development.

Widespread cotranscriptional splicing has been demonstrated from yeast to human. However, most studies to date addressing the kinetics of splicing relative to transcription used either Saccharomyces cerevisiae or metazoan cultured cell lines. Here, we adapted native elongating transcript sequencing technology (NET-seq) to measure cotranscriptional splicing dynamics during the early developmental stages of Drosophila melanogaster embryos. Our results reveal the position of RNA polymerase II (Pol II) when both canonical and recursive splicing occur. We found heterogeneity in splicing dynamics, with some RNAs spliced immediately after intron transcription, whereas for other transcripts no splicing was observed over the first 100 nt of the downstream exon. Introns that show splicing completion before Pol II has reached the end of the downstream exon are necessarily intron-defined. We studied the splicing dynamics of both nascent pre-mRNAs transcribed in the early embryo, which have few and short introns, as well as pre-mRNAs transcribed later in embryonic development, which contain multiple long introns. As expected, we found a relationship between the proportion of spliced reads and intron size. However, intron definition was observed at all intron sizes. We further observed that genes transcribed in the early embryo tend to be isolated in the genome whereas genes transcribed later are often overlapped by a neighboring convergent gene. In isolated genes, transcription termination occurred soon after the polyadenylation site, while in overlapped genes, Pol II persisted associated with the DNA template after cleavage and polyadenylation of the nascent transcript. Taken together, our data unravel novel dynamic features of Pol II transcription and splicing in the developing Drosophila embryo.