Investigation on the Interface between Exons and Introns in the Genomic DNA of Arabidopsis thaliana in the Phase Space

In this study,three weight vectors L1,L2 and L3 were set.After calculating the probability of three bases in the exons or introns in the genomic DNA of Arabidopsis thaliana,64-dimensional vector P was obtained.Dot products of P vector and three weight vectors were the feature coordinates for the exons and introns in 3-dimensional phase space.The expression for the interface between the exons and the introns in the genomic DNA of Arabidopsis thaliana in 3-dimensional phase space was established,which could be used to distinguish the exons and the introns in the genomic DNA of Arabidopsis thaliana with an accuracy higher than85%in 3-dimensional phase space.

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

supported by the National Natural Science Fund of China （30970231）;the Genetically Modified Organisms Breeding Major Project of China （2014ZX08003001）

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 triphosphatedependent 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 helicase48(Zm RH48)impair the splicing of mitochondrial introns,mitochondrial complex biosynthesis,and seed development.Loss of Zm RH48 function severely arrested embryogenesis and endosperm development,leading to defective kernel formation.Zm RH48 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 ccm Fc intron 1)and one trans-intron(nad2 intron 2),leading to lower levels of mitochondrial complexes I andⅢ.Zm RH48 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-m CSF1 to facilitate intron splicing.These results suggest that Zm RH48 is likely a component of a splicing complex and is critical for mitochondrial complex biosynthesis and seed development.

A molecular brake that modulates spliceosome pausing at detained introns contributes to neurodegeneration

Emerging evidence suggests that intron-detaining transcripts(IDTs)are a nucleus-detained and polyadenylated mRNA pool for cell to quickly and effectively respond to environmental stimuli and stress.However,the underlying mechanisms of detained intron(DI)splicing are still largely unknown.Here,we suggest that post-transcriptional DI splicing is paused at the Bact state,an active spliceosome but not catalytically primed,which depends on Smad Nuclear Interacting Protein 1(SNIP1)and RNPS1(a serine-rich RNA binding protein)interaction.RNPS1 and Bact components preferentially dock at DIs and the RNPS1 docking is sufficient to trigger spliceosome pausing.Haploinsufficiency of Snip1 attenuates neurodegeneration and globally rescues IDT accumulation caused by a previously reported mutant U2 snRNA,a basal spliceosomal component.Snip1 conditional knockout in the cerebellum decreases DI splicing efficiency and causes neurodegeneration.Therefore,we suggest that SNIP1 and RNPS1 form a molecular brake to promote spliceosome pausing,and that its misregulation contributes to neurodegeneration.

The spliceophilin CYP18-2 is mainly involved in the splicing of retained introns under heat stress in Arabidopsis

Peptidyl-prolyl isomerase-like 1(PPIL1)is associated with the human spliceosome complex.However,its function in pre-mRNA splicing remains unclear.In this study,we show that Arabidopsis thaliana CYCLOPHILIN 18-2(AtCYP18-2),a PPIL1 homolog,plays an essential role in heat tolerance by regulating pre-mRNA splicing.Under heat stress conditions,AtCYP18-2 expression was upregulated in mature plants and GFP-tagged AtCYP18-2 redistributed to nuclear and cytoplasmic puncta.We determined that AtCYP18-2 interacts with several spliceosome complex B^(ACT)components in nuclear puncta and is primarily associated with the small nuclear RNAs U5 and U6 in response to heat stress.The AtCYP18-2 loss-of-function allele cyp18-2 engineered by CRISPR/Cas9-mediated gene editing exhibited a hypersensitive phenotype to heat stress relative to the wild type.Moreover,global transcriptome profiling showed that the cyp18-2 mutation affects alternative splicing of heat stress–responsive genes under heat stress conditions,particularly intron retention(IR).The abundance of most intron-containing transcripts of a subset of genes essential for thermotolerance decreased in cyp18-2 compared to the wild type.Furthermore,the intron-containing transcripts of two heat stress-related genes,HEAT SHOCK PROTEIN 101(HSP101)and HEAT SHOCK FACTOR A2(HSFA2),produced functional proteins.HSP101-IR-GFP localization was responsive to heat stress,and HSFA2-Ⅲ-IR interacted with HSF1 and HSP90.1 in plant cells.Our findings reveal that CYP18-2 functions as a splicing factor within the B~(ACT)spliceosome complex and is crucial for ensuring the production of adequate levels of alternatively spliced transcripts to enhance thermotolerance.

Engineering Introns to Express RNA Guides for Cas9- and Cpfl-Mediated Multiplex Genome Editing

The clustered regularly interspaced short palindromic repeat （CRISPR） system has emerged as the revolu- tionary platform for DNA targeting. This system uses a site-specific RNA guide to direct a CRISPR effector （e.g., Cas9 and Cpfl） to a DNA target. Here, we elaborate a general strategy to simultaneously express multiple guide RNAs （gRNA） and CRISPR RNAs （crRNA） from introns of Cas9 and Cpfl. This method utilizes the endogenous tRNA processing system or crRNA processing activity of Cpfl to cleave the spliced intron that contains tRNA-gRNA polycistron or crRNA-crRNA array. We demonstrated that the tRNA-gRNA intron is able to fuse with Cas9 as one gene. Such a hybrid gene could be expressed using one polymerase II promoter, and exhibited high efficiency and robustness in simultaneously targeting multiple sites. We also implemented this strategy in Cpfl-mediated genome editing using intronic tRNA-crRNA and crRNA-crRNA arrays. Interestingly, hybrid genes containing Cpfl and intronic crRNA array exhibited remarkably increased efficiency compared with the conventional Cpfl vectors. Taken together, this study presents a method to express CRISPR reagents from one hybrid gene to increase genome-editing efficiency and capacity. Owing to its simplicity and versatility, this method could be broadly used to develop sophisticated CRISPR tools in eukaryotes.

Single-Nucleotide Resolution Mapping of the Gossypium raimondii Transcriptome Reveals a New Mechanism for Alternative Splicing of Introns

Alternative splicing （AS） is a vital genetic mechanism that enhances the diversity of eukaryotic transcriptomes. Here, we generated 8.3 Gb high-quality RNA-sequencing data from cotton （Gossypium raimondii） and performed a systematic, comparative analysis of AS events. We mapped 85% of the RNA-sequencing data onto the reference genome and identified 154 368 splice junctions with 16 437 as events in 10197 genes. I ntron retention constituted the majority （40%） of all AS events in G. raimondii. Comparison across 11 eukaryote species showed that intron retention is the most common AS type in higher plants. Although transposable elements （TEs） were found in only 2.9% of all G. raimondii introns, they are present in 43% of the retained introns, suggesting that TE-insertion may be an important mechanism for intron retention during AS. The majority of the TE insertions are concentrated 0-40 nt upstream of the 3＇-splice site, substantially altering the distribution of branch points from preferred positions and reducing the efficiency of intron splicing by decreasing RNA secondary structure flexibility. Our data suggest that TE-insertion-induced changes in branch point-site distribution are important for intron retention-type AS. Our findings may help explain the vast differences in intron-retention frequencies between vertebrates and higher plants.

Comparison between phylogeny of introns and exons in primates

Introns and exons of 7 genes ( epsilon globin, gamma-1 globin, gamma-2 globin, delta globin, beta globin, Immunoglobulin and prepro-insulin) in primates have been separated out and used to infer phylogeny respectively. For each gene, results based on these two parts have been compared and showed that: ( i ) the topology of introns is almost

Introns in higher plant genes

The intron is an important component of eu-karyotic gene. Extensive studies have been conducted to get a better understanding of its structure and function. This paper presents a brief review of the structure and function of introns in higher plant genes. It is shown that higher plant introns possess structural properties shared by all eukaryotic introns, however, they also exhibit a striking degree of diversity. The process of intron splicing in higher plant genes involves interaction between multiple cis -acting elements and trans-acting factors, such as 5’ splicing site, 3’ splicing site and many protein factors. The process of intron splicing is an important level at which gene expression is regulated. Especially alternative splicing of intron can regulate time and space of gene expression. In addition, some introns in higher plant genes also regulate gene expression by affecting the pattern of gene expression, enhancing the level of gene expression and driving the gene expression.

The pentatricopeptide repeat protein EMB1270 interacts with CFM2 to splice specific group Ⅱ introns in Arabidopsis chloroplasts

Chloroplast biogenesis requires the coordinated expression of chloroplast and nuclear genes.Here, we show that EMB1270, a plastid-localized pentatricopeptide repeat(PPR) protein, is required for chloroplast biogenesis in Arabidopsis thaliana.Knockout of EMB1270 led to embryo arrest,whereas a mild knockdown mutant of EMB1270 displayed a virescent phenotype. Almost no photosynthetic proteins accumulated in the albino emb1270 knockout mutant. By contrast, in the emb1270 knockdown mutant, the levels of ClpP1 and photosystem I(PSI) subunits were significantly reduced, whereas the levels of photosystem II(PSII) subunits were normal. Furthermore, the splicing efficiencies of the clpP1.2,ycf3.1, ndhA, and ndhB plastid introns were dramatically reduced in both emb1270 mutants. RNA immunoprecipitation revealed that EMB1270 associated with these introns in vivo. In an RNA electrophoretic mobility shift assay(REMSA), a truncated EMB1270 protein containing the 11 Nterminal PPR motifs bound to the predicted sequences of the clpP1.2, ycf3.1, and ndhA introns.In addition, EMB1270 specifically interacted with CRM Family Member 2(CFM2). Given that CFM2 is known to be required for splicing the same plastid RNAs, our results suggest that EMB1270 associates with CFM2 to facilitate the splicing of specific group II introns in Arabidopsis.

“Too Soon on Earth”: A Biophilosophical Model of Schizophrenia. Some Implications for Humanoid Robots

This paper presents a new explanatory model for schizophrenia based upon philosophical, molecular and neurobiological hypotheses as well as on years of experience in observing and treating these patients. To start with, a novel interpretation of the Hegelian concept of mediation is presented. Mediation is defined as the rejection of non-realizable programs, such as thoughts and ideas, at a certain point in time in the evolution of a living system. Whenever a system treats non-realizable programs as if they were realizable, its ability to “test the reality” is lost, and consequently a loss of ego-boundaries may occur. On the molecular level, I will try to show how “non-splicing” of introns during the mRNA splicing process is equivalent to a loss of the rejection function corresponding to mediation. At the cellular level in the brain, mediation can be explained in terms of glial-neuronal interactions. Glia exert a spatio-temporal boundary setting function determining the grouping of neurons into functional units. Mutations in genes that result in non-splicing of introns can produce truncated (“chimeric”) neurotransmitter receptors. I propose that such dysfunctional receptors are generated in glial cells and that they cannot interact properly with their cognate neurotransmitters. The glia will then lose their inhibitory-rejecting function with respect to the information processing within neuronal networks. This loss of glial boundary setting could be an explanation for the loss of ego or body boundaries in schizophrenia. Pertinent examples of case studies are given attempting to deduce the main symptoms of schizophrenia from the proposed hypothesis. Some implications for the design of delusional robots are also discussed. Finally, the evolutionary potency of non-coding introns is philosophically interpreted that schizophrenics may be “too soon on earth”.

Exploring Splicing Variants and Novel Genes in Sacred Lotus Based on RNA-seq Data

Sacred lotus(Nelumbo nucifera)is a typical aquatic plant,belonging to basal eudicot plant,which is ideal for genome and genetic evolutionary study.Understanding lotus gene diversity is important for the study of molecular genetics and breeding.In this research,public RNA-seq data and the annotated reference genome were used to identify the genes in lotus.A total of 26,819 consensus and 1,081 novel genes were identified.Meanwhile,a comprehensive analysis of gene alternative splicing events was conducted,and a total of 19,983“internal”alternative splicing(AS)events and 14,070“complete”AS events were detected in 5,878 and 5,881 multi-exon expression genes,respectively.Observations made from the AS events show the predominance of intron retention(IR)subtype of AS events representing 33%.IR is followed by alternative acceptor(AltA),alternative donor(AltD)and exon skipping(ES),highlighting the universality of the intron definition model in plants.In addition,functional annotations of the gene with AS indicated its relationship to a number of biological processes such as cellular process and metabolic process,showing the key role for alternative splicing in influencing the growth and development of lotus.The results contribute to a better understanding of the current gene diversity in lotus,and provide an abundant resource for future functional genome analysis in lotus.

Intron Retention Fine-Tunes the Resistance of the Rice Mutant pls4 to Rice Sheath Blight(Rhizotonia solani AG I.1a)

OsPLS4 encodes aβ-ketoacyl carrier protein reductase(KAR).The role of OsPLS4 in rice sheath blight(Rhizoctonia solani)remains unclear.Our preliminary studies showed that premature leaf senescence mutants(pls4)were highly susceptive to sheath blight in the early stage of rice development.To explore the role of this gene in the development of rice sheath blight,the transcriptome profiles of the rice pls4 mutant and wild type were compared by RNA-seq.The results revealed 2,569 differentially expressed genes(DEGs).The down-regulated genes were significantly enriched in the defense response-related biological processes.These down-regulated genes included the chitinase genes and WRKY genes,which were significantly changed in pls4 mutants.Furthermore,467 genes induced significant alternative splicing(AS)events.Among them,intron retention(IR)affected gene expression levels and functions of the vitamin B6(VB6)metabolism pathway related to sheath blight.This result suggests that IR plays an important role in the sheath blight resistance of mutant pls4.Together,these results indicate that pls4 could be involved in the biological process of sheath blight via DEGs and the fine-tuning of IR.The present study provides a molecular basis for further investigation of the resistance of rice to sheath blight.

线粒体nad 1内含子2序列在石斛属植物分子鉴定中的应用(英文)

目的在兰科石斛属药用植物鉴定中应用新的分子标记。方法扩增并测定9种石斛属植物线粒体中NADH脱氢酶亚基1编码基因(nad 1)内含子2(in tron 2)的全长序列。结果比对后的nad 1 in tron 2序列长872bp,其中有17个变异位点,可以鉴别除粉花石斛D end robium lodd ig esii以外的8种植物。结论线粒体nad 1 in tron2序列可以作为一种新的分子标记用于石斛属植物的鉴定。

藏鸡和隐性白羽鸡GH基因的SNP检测及其与生长性状间的关联分析

利用改良等位基因特异性PCR(Modified Allele Specific PCR,M-ASP)结合PCR-RFLP以及直接测序方法对藏鸡和隐性白羽鸡生长激素(Growth Hormone,GH)基因内含子4(Intron4)的一个位点进行了SNP检测,并进行了该基因与藏鸡生长性状的关联分析。检测结果显示,GH基因Intron4的这个位点同时具有M-ASP和SacI-RFLP多态。测序结果表明,该位点发生了C→G的碱基突变,产生了CC、CG和GG三种基因型。两个等位基因和三种基因型在两鸡种中的分布基本一致。其中,基因型CC和等位基因C的频率最高。χ2检验结果表明,基因型频率或者等位基因频率在两鸡种之间没有显著差异(P>0.05),而分别在两鸡种内部却存在显著(P<0.05)或极显著(P<0.01)差异。方差分析结果表明,基因型与藏鸡的2周龄体重等12个生长性状有显著(P<0.05)或极显著(P<0.01)关联;基因型CC与藏鸡的7周龄体重存在显著关联(P<0.05)。这暗示了GH基因是影响藏鸡生长的主效基因或者它与主效基因相关,而该位点的碱基突变可以作为筛选藏鸡高的7周龄体重的分子标记。

内含子的进化及基因表达调节

由于内含子(intron)的进化目前还没有一致的观点,一般认为内含子的进化是基因进化的重要部分,是与物种形成相适应的过程。内含子参与基因的组织特异性表达、蛋白质变异、基因转录等多种生物学过程。

Cloning and Expression Patterns of a Metallothionein-like GenehtMT2 of Helianthus tuberosus

A novel cDNA sequencehtMT2, which encodes a type 2 metallothionein_like protein, was isolated from Helianthus tuberosus L. tuber cDNA library. The whole sequence is 509 bp, including an open reading frame (ORF) of 240 bp, a 5′ UTR of 62 bp and a 3′ UTR of 207 bp. Two genomic sequences covering the coding region ofhtMT2were cloned by PCR reaction. Sequence analysis revealed that the genomic sequences htMTG_1 of 986 bp and htMTG_2 of 982 bp were both composed of three exons and two introns. The deduced protein consisted of 79 amino acid residues with a predicted molecular weight of 7.8 ku (kD). Amino_terminal and carboxy_terminal domains contained 8 and 7 cysteine residues respectively, separated by a central cysteine free spacer. Sequence alignment revealed that the predicted protein ofhtMT2 was homologous to type 2 metallothioneins (MTs) of plants. Southern blotting analysis indicated that htMT2was encoded by a small multi_gene family in H. tuberosus genome. Northern blotting analysis showed that htMT2 transcripts were detected in stems, leaves and leafstalks, but no transcripts were detected in roots. The expression level in stems was the highest among the above tissues. Transcripts in stems were significantly reduced by Cu 2+ treatment. Judging from the homologies between the deduced HtMT2 and other type 2 plant metallothioneins as well as responses to metal ions, we believe thatwere cloned by PCR reaction. Sequence analysis revealed that the genomic sequences htMTG_1 of 986 bp and htMTG_2 of 982 bp were both composed of three exons and two introns. The deduced protein consisted of 79 amino acid residues with a predicted molecular weight of 7.8 ku (kD). Amino_terminal and carboxy_terminal domains contained 8 and 7 cysteine residues respectively, separated by a central cysteine free spacer. Sequence alignment revealed that the predicted protein ofhtMT2 was homologous to type 2 metallothioneins (MTs) of plants. Southern blotting analysis indicated that htMT2was encoded by a small multi_gene family in H. tuberosus genome. Northern blotting analysis showed that htMT2 transcripts were detected in stems, leaves and leafstalks, but no transcripts were detected in roots. The expression level in stems was the highest among the above tissues. Transcripts in stems were significantly reduced by Cu 2+ treatment. Judging from the homologies between the deduced HtMT2 and other type 2 plant metallothioneins as well as responses to metal ions, we believe that[ShtMT2 encodes a new type 2 metallothionein.

Application of Mitochondria DNA Hypervariable Region in Identification of Genetic Relationships among Different Varieties in Musa paradisiaca

[Objective] The aim was to establish an effective method for the identification of genetic relationships among different varieties in Musa paradisaca. [Method] Based on the diversity of mitochondria DNA intron sequence among different varieties of M. paradisaca,an intron of cytochrome oxidase subunit II gene in mitochondria DNA genome was amplified and sequenced. And then the cluster analysis was used to classify 16 varieties of M. paradisaca,which belonged to five genotypes （AAA,AA,AAB,ABB and BB）. [Result] The 16 varieties of M. paradisaca could be divided to three classes：the first class contained one variety,the genotype of which was BB; the second class contained seven varieties,the genotype of which was ABB; the third class contained eight varieties,the genotypes of which included AA,AAA,AAB and BB. The new varieties YiXian 1,2 and 3 showed the nearest relationship with FenZa. [Conclusion] The result of classification was consistent with the genotypes,thus verified the feasibility and effectiveness of the new method in the genetic relationship identification of M. paradisaca germplasm.

GABA transporter 1 transcriptional starting site exhibiting tissue specific difference

GABA transporter 1(GAT1) takes important roles in multiple physiological processes through the uptake and release of GABA, but the regulation of GAT1 gene expression in different tissues is rarely known. To address the question, first, 5’ Rapid amplification of cDNA end (RACE) was used to determine GAT1 transcriptional starting sites in neonatal mouse cerebral cortex and intestine, adult mouse brain and adult rat testis. The products of 5’RACE were confirmed by DNA sequencing. We found that the transcript of GAT1 in neonatal mouse cerebral cortex and adult mouse brain starts at the same site (inside of exon 1), while in mouse intestine, GAT1 starts transcription in intron 1, and in rat testis, the transcript of GAT1 has an additional untranslation exon to the 5’ direction.

Molecular evidence suggests that the enigmatic Sulawesi endemic Geomalia heinrichi belongs in the genus Zoothera (Turdidae, Aves)

The taxonomic status of the Sulawesi endemic Geomalia heinrichi has long been debated, and it has variously been treated as a babbler (Timaliidae) or a turdid (Turdidae). We estimated the phylogeny of 43 taxa in the family Turdidae based on the mitochondrial cytochrome b gene and the nuclear myoglobin intron 2 and ornithine decarboxylase introns 6–7. Geomalia heinrichi was shown to be part of the Zoothera clade with high support. We propose that Geomalia is transferred to Zoothera under the name Zoothera heinrichi.