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Introns: structure and functions

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"Introns: Structure and Functions" during November, 2011 (Friday Seminar activity, Department of Biotechnology, University of Agricultural Sciences, Dharwad, Karnataka) by Yogesh S Bhagat (Ph D …

"Introns: Structure and Functions" during November, 2011 (Friday Seminar activity, Department of Biotechnology, University of Agricultural Sciences, Dharwad, Karnataka) by Yogesh S Bhagat (Ph D Scholar)

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  • asfdf
  • Visi bulization of DNA-mRNA hybrids by electron microscopy. DNA-RNA duplexes are more stable than DNA double helices. Thus, if partially denatured double helices are incubated with homologous RNA molecules under appropriate hybridization condition, RNA strands will hybridise with complemenary DNA strands, displacing equivalent DNA strands. The resulting DNA RNA hybrids structure will contain single stranded regions of DNA called R loops. Thus, R loop hybridization and electron microscopy powerful tool for study gene structure.
  • splicing of Group I introns occurs in the absence of proteins, and involves two successive transesterification steps.
    This intron class utilise a guanosine cofactor in catalysis that is sequestered in a globular pre-formed active site, analogous to that of a protein enzymeThe 3'-OH of a guanosine (G, GMP, GDP, GTP all function) acts as a nucleophile, attacks the phosphate at the 5' exon-intron junction, and covalently binds to the excised intron. This step requires metal ions for folding and catalysis.
    Then 3'-OH of the released 5' -exon attacks the 3' junction phosphate, completing the splice.
    Exons are ligated and a free linear intron, with the G nucleoside attached at 5' end, is released. After splicing, intron acts upon itself to circularise.
  • Group I introns are relatively difficult to identify in a genomic context due to very little conservation at the sequence level (Fig.
    1A). The key feature of a group I intron is the P7 stem in the catalytic domain that serves as the binding site for the guanosine
    cofactor. The architecture of P7 is well conserved and can be found using covariation constraints.4 The second key feature is the
    substrate stem (P1) that in almost all introns present a GU pair at the 5'splice site. The apposition of P1 and P7 furthermore reveals
    the presence of two conserved A residues that are part of the active site. Group I introns are 250–500 nt (without insertion elements)
    and have been divided into 13 subgroups based on differences in P7 as well as the peripheral elements.4,5 They have been found
    in genes encoding rRNA, mRNA and tRNA in the genomes of bacteriophages, bacteria, mitochondria, chloroplasts, the nuclei of
    eukaryotic microorganisms, and some eukaryotic viruses. Release 9.1 of the Rfam database (http://rfam.sanger.ac.uk/)6 has 22094
    group I introns annotated. Other major databases with large collections of group I introns are GISSD (http://www.rna.whu.
    edu.ch/gissd)7 and CRW (http://www.rna.ccbb.utexas.edu/).8 The natural history of group I introns with emphasis on distribution
    and phylogeny has been reviewed by Haugen et al.9
    More recently, group I introns have proven to be very useful models for RNA evolution because of their diversity and the rich biology found associated
    with these elements
  • The IEPs of these introns contain four domains denoted reverse transcriptase (RT), a domain required for RNA splicing (maturase) activity (X), DNA
    binding (D), and DNA endonuclease (En)
  • one gene makes one protein of Beadle and Tatum (Schade, 1959) has been modified to one gene makes one polypeptide; many
    polypeptides make a protein(Itano and Pauling, 1961).
    Later, one gene makes multiple polypeptide isoforms as a result of alternative splicing
  • when recombinant human ceruloplasmin was expressed starting from cDNA in baby hamster kidney cells it gave a very low yield due to nuclear
    retention of mRNA. This problem was solved by inserting the second intron of the rabbit b-globin gene upstream of the human ceruloplasmin cDNA. This action was able to alleviate the block of cytoplasmic export and significantly increased recombinant protein expression
    Zago et al. have tested the effect of introduction of two well-characterized introns, an a-tropomyosin derived intron and pCl-neo intron in a naturally
    uninterrupted human gene, interferon beta. Both introns were designed to introduce stop codons in the IFN beta sequence in order to prevent translation from the unspliced mRNA. Both introns were short, 114 bp and 172 bp, with efficient splicing behavior in vivo and in vitro
    In this work, Zago et al. have also tested experimentally different intron positions to obtain the highest impact on the subsequent protein production based
    on the cDNA homology between interferon beta and the loosely related interferon gamma. Intron insertion at the position with the highest homology had the ability to increase mRNA and IFN beta protein levels in both CHO and HeLa cells by 1.5 to 2.5 fold. In this respect, it should also be important to note that in other systems intron positioning can have a profound effect on gene expression and in some cases may even result to be inhibitory
  • The formation of exonic miRNAs involves the precursor (pri-miRNA), e.g., lin-4 and let-7, which is transcribed probably by Pol-III RNA promoters. The intronic miRNAs, however, involves Pol-II promoters. Both intronic miRNA and mRNA are coexpressed in a gene transcript (pre-mRNA) by Pol-II promoters. In the nucleus, the pri-miRNA is excised by Drosha RNase to form a hairpin-like pre-miRNA template and then exported to cytoplasm for further processing by single-stranded RNA-specific Dicer* to form mature miRNAs. Different from the processing of exonic miRNAs, the excision of intronic miRNAs out of premRNA is completed through the process of RNA splicing by spliceosomal snRNPs and the maturation of these miRNAs requires NER proteins as a part of the Dicer**.

Transcript

  • 1. Seminar On Introns: Structure and functions By Yogesh S. Bhagat Ph. D Scholar Institute of Agricultural Biotechnology, University of Agricultural Sciences, Dharwad (Karnataka)
  • 2. Flow of seminar o Introduction o History of introns o Classification of introns o Structure and splicing mechanism of introns o Factors affecting intron gain and loss o Mechanisms of intron gain and loss oRole of introns in regulating the gene expression oBiogenesis and role of intronic miRNAs oConclusion
  • 3. C Value paradox
  • 4. C Value paradox
  • 5. G Value paradox o Concept emerged from genomic and transcriptomic projects. oEstimated number of protein coding genes does not correlate with the organism complexity oe.g Humans and C. elegans have roughly the same number of protein coding genes oOrganism complexity better correlate to the proportion of noncoding DNA
  • 6. The Fractal Complexity of Genome
  • 7. Introns o An intron is any nucleotide sequence within a gene that is removed by RNA splicing to generate the final mature RNA product of a gene. o The term intron refers to both the DNA sequence within a gene, and the corresponding sequence in RNA transcripts. o The word intron is derived from the term intragenic region, i.e. a region inside a gene. Although introns are sometimes called intervening sequences. o The term "intervening sequence" can refer to any of several families of internal nucleic acid sequences that are not present in the final gene product, including inteins, untranslated sequences (UTR), and nucleotides removed by RNA editing, in addition to introns.
  • 8. Ist evidence for introns
  • 9. Introns history Scientist Gene Organism Philip A Sharp & Richard J Roberts m RNA of beta-globin, immunoglobulin, ovalbumin, tRNA and rRNA. Adenovirus P Chambon, P Leader & R A Flavell Beta globin genes, ovulbumin & t RNA genes Chicken Gilbert,W. Introns and exons Tom Cech Self splicing Tetrahymena (Ciliate Protozoan)
  • 10. How prevalent are the introns? 1. Early Intron hypothesis  Introns were an essential feature of the earliest organisms Absence in bacteria: shorter division times of bacterial cells i.e. bacteria have had many more growth cycles in which to evolve. This evolution has brought about the loss of nearly all ancestral introns.
  • 11. How prevalent are the introns? 2. Late Intron hypothesis  Earliest organisms did not contain introns. Introns are a relatively recent arrival in the eukaryotic lineage that to help generate the diversity of regulatory mechanisms that are required to control gene expression in multicellular highly differentiated organisms. In this view, prokaryotes do not have introns because they never had them in the first place.
  • 12. Distribution of introns o The intron distributions in 5’UTR, CDS and 3’UTR are different for same organism. o The intron distribution rules are common for Human, Mouse, Rat, Arabidopsis and Fruit fly. 5’UTR CDS 3’UTR Percentage (sequence have introns) 20% 80% 10% Interval between 2 introns 100nt 140nt uncertain Intron frequency Higher than CDS Higher than 3’UTR Lowest Distribution evenly Shift toward 5’ of CDS Concentrate toward the center of 3’UTR Hong X et. al. Mol Biol Evol. 2008 (12):2392-2404.
  • 13. Classification of Introns S.No TYPE OF INTRON LOCATION SPLICING 1 Group I rRNA genes, Organell RNAs, few bacterial RNAs. Self splicing (Transposase) 2 Group II Chloroplast, mitochondria genes & prokaryotic RNAs. 3 Nuclear- mRNA Nucleus Selfsplicing (Reverse trancriptase & ribozyme) Non Self-splicing (Sn RNAs) 4 t RNA t RNA Enzymatic
  • 14. Nuclear Pre-mRNA Introns Introns in nuclear protein-coding genes that are removed by spliceosomes o Characterized by specific intron sequences located at the boundaries between introns and exons. o These sequences are recognized by spliceosomal RNA molecules o In addition, they contain a branch point o Apart from these three short conserved elements, nuclear pre-mRNA intron sequences are highly variable. Nuclear pre-mRNA introns are often much longer than their surrounding exons.
  • 15. Chambons rule Short consensus sequences at exon – intron junctions (AG-GT) or AT – AC.
  • 16. Splice site sequence requirement Lariat branch site
  • 17. Splicing reactions
  • 18. http://mips.helmholtz-muenchen.de/proj/yeast/reviews/intron/spliceo_splicing.html
  • 19. Group I catalytic introns and its Distribution oGroup I introns are large self-splicing ribozymes. oThey catalyze their own excision from mRNA, tRNA and rRNA precursors in a wide range of organisms. oGroup I introns are widespread……. 1.Mitochondria and plastid genomes of plants and protists (rRNA, tRNA and mRNA genes). 2.Nucleus of certain protists, fungi and lichens (rRNA genes). 3.Eubacteria (tRNA genes) & phages. 4.Metazoans - only in mitochondrial genes of a few anthozoans (e.g., sea anemone).
  • 20. Splicing mechanism of Group I introns
  • 21. Structure of Group I introns Database: GISSD Softwares: Rfam
  • 22. Group II intron o Abundant in organellar genomes of plants and lower eukaryotes, but have not yet been found in higher eukaryotes or in nuclear genomes. o In bacteria, about one quarter of genome contain group II introns. o Also found in archaebacteria o Self-splicing reaction o They encode reverse transcriptase (RT) ORFs and are active mobile elements o Mobile group II introns can insert into defined sites at high efficiencies (called retrohoming), or can invade unrelated sites at low frequencies (retrotransposition).
  • 23. Proposed history of group II intron
  • 24. Self-splicing mechanism of Group II intron
  • 25. Protein assisted splicing mechanism of Group II intron + Maturase After splicing, the RT remains tightly bound to spliced intron, and this RNP particle is the active moiety in subsequent mobility reactions.
  • 26. Structure of Group II intron RT binds to unspliced intron RNA at a high affinity binding site in domain 4, and makes secondary contacts in domains 1, 2 and 6. Together, protein-RNA interactions result in conformational changes in the intron that result in selfsplicing
  • 27. Function and interactions of the six group II intron domain Scot A. Kelchner, American Journal Of Botany, 89(10): 1651–1669. 2005.
  • 28. Factors that can affect the gain and loss of introns Daniel C. Jeffares and David Penny, Trends in Genetics Vol.22 No.1, 2006
  • 29. Models for intron gain and loss
  • 30. Intron gain Daniel et. al.,2006, Trends in Genet., 22: 18-22.
  • 31. Intron gain
  • 32. Intron Loss
  • 33. Why do genes have introns ? • Alternate splicing • Regulating Gene expression • Gene silencing (miRNA, SiRNA) Duret L., 2008, Trends in Genetics
  • 34. Alternative splicing The processing of an RNA transcript into different mRNA molecules and a single gene might encode many proteins. Thus, the acquisition of introns would have been positively selected as a source of functional diversity Introns offer plasticity alternative splicing. to gene expression, through Introns contains functional elements (regulatory elements, alternative promoters).
  • 35. Alternative splicing Interactions: Protein-protein and protein-RNA interactions Binding of specific regulatory protein to pre mRNA Recruitment of specific splicing factors and splicing regulator at the site of transcription
  • 36. Alternative splicing
  • 37. Alternative splicing e. g. SR proteins -Acts as repressor and activator of splicing in a tissue specific manner Stress and temperature SR protein binds to first intron of RNA and recruit TFs Acts against stress in tissue specific manner Reddy, A.S.N. et al., Trends Plant Sci. 2004, 9: 541-547.
  • 38. Gene expression o First introns :binding sites for transcription factors or may act as classical transcriptional enhancers. o Tissue and developmental specific gene expression o First introns : Acts as internal promoter to produce alternate RNA
  • 39. How introns influence eukaryotic gene expression? Introns can affect the efficiency of transcription by several different means. introns can affect transcription is by acting as repositories for transcriptional regulatory elements such as enhancers and repressors Hiret, H. L. et al., 2006, Trends in Biochemical Sci., Parra et. al., 2011, Nucleic Acids Res., 39: 5328-5337.
  • 40. How introns influence eukaryotic gene expression? Introns are also required for specific modification of some exon sequences by RNA editing Interactions between pre-mRNA processing events. The nuclear cap-binding complex promotes the excision of the 5-most intron, whereas interactions between the spliceosome (green) and polyadenylation machinery promote excision of the 3’-most intron and proper 3’-end formation. In many cases, sequences in introns serve as guides for the chemical alteration of exonic nucleotides by RNA editing.
  • 41. How introns influence eukaryotic gene expression? Formation and removal of exon junction complexes (EJCs) • Once processed, EJCs are deposited on mRNAs by splicing at a fixed position 20–24 nucleotides upstream of exon–exon junctions. • Proteins thus far identified as nuclear EJC components. • Interactions between EJCs, TAP/p15 and components of the nuclear pore complex (NPC) facilitate mRNA export. • Upon export, the composition of the EJC changes or is remodeled. • EJCs are removed by ribosomes, translation. during the first round of
  • 42. Intron effect on GUS transgene expression in transgenic rice lines
  • 43. Constructs used in the study pRESQ4: rubi3 promoter—5’UTR exon1 (67 bp)----5’UTR intron---the GUS coding sequence pPSRG30: same as pRESQ4 (except 5’UTR intron)
  • 44. Southern hybridization and real time PCR
  • 45. GUS histochemical assay
  • 46. Conclusion  Splicing factors bound to the nascent RNA interact with RNA Pol II C-terminal domain (CTD) and help to regulate transcriptional initiation and elongation.  proximal intron facilitate the release and rapid recycling of certain transcription initiation factors for new initiation events  Role of EJC in rapid release of transcript from nucleus to cytoplasm
  • 47. Beneficial effects of introns on recombinant gene expression Zago, P., 2009, Biotechnology and Applied Biochemistry, (52): 191–198.
  • 48. Intronic MicroRNA • Introns releases trans-acting factors such as microRNA (miRNA) and small nucleolar RNA (snoRNA) • Term : Mirtrons • miRNA targets include transcription factors and genes involved in stress response, hormone signalling, and cell metabolism. • One fourth of human miRNAs are identified in the introns of pre-mRNAs.
  • 49. Intronic MicroRNA Nearly 97% of the human genome is composed of noncoding DNA, which varies from one species to another. Numerous genes in these non-protein-coding regions encode microRNAs, which are responsible for RNA-mediated gene silencing through RNA interference (RNAi)-like pathways. One fourth of human miRNAs are identified in the introns of premRNAs. Ying., et al., 2010, Methods Mol Biol., 629: 205-237
  • 50. Biogenesis of intronic MicroRNA
  • 51. Hinske, L., et al., 2008
  • 52. Intron-mediated gene silencing Artificial splicing-competent intron (SpRNAi): of consensus nucleotide elements representing:  splice donor and acceptor sites,  branch-point domain,  poly-pyrimidine tract, and  linkers for insertion into gene constructs an insert sequence that is either homologous or complementary to a targeted exon is located within the artificial intron between the splice donor site and the branch-point domain.
  • 53. Intron mediated gene silencing in Zebrafish Why zebrafish?  Great use the study of aetiology and pathology of human diseases  To study diseases underlying molecular mechanism results from the loss of a specific gene function Ying, et. al., 2010, Methods Mol Biol., 629: 205-237.
  • 54. Intron mediated gene silencing in Zebrafish Anti GFP RFP
  • 55. Reduction in the of green fluorescence protein Increase in the level of red fluorescence protein
  • 56. Conclusion Man-made intronic miRNAs have potential applications in (a)The analysis of gene function by developing loss-of-function transgenic animals (b)The evaluation of both the function and effectiveness of miRNA, (c)The design and development of novel gene therapies
  • 57. Introns as a source of polymorphism • Exons sequences are conserved but introns sequences vary (length) • Plant introns are richer in AT bases than their adjacent exons Plant introns are short (80-139nts) Differ from vertebrate and yeast introns(2-3Kb) Resembles to animals like fruit fly and Nematode introns XIE Xianzhi and WU Naihu, Chinese Science Bulletin (47): 17 ,2005
  • 58. The longest intron identified in plants is Maize pericarp gene (7 Kb) Consensus sequence of 5’ splicing site is AG/GTAAGT 3’ splicing site is TGCAG/G It is found that the features of 5 ss, 3 ss and branch site are almost identical between animal and plants. The only obvious difference : Lack of polypyrimidine tract at the 3’ end of plant introns but exists UA-rich sequences throughout the plant intron.
  • 59. Development of Intron Polymorphism Markers Detectable genetic polymorphism or allelic variation at DNA sequence level. Two types of polymorphism : Length difference Nucleotide difference (SNP )
  • 60. Detection of Intron Polymorphisms Intron Polymorphism(IP): Polymorphism between allelic introns Intron Length Polymorphism (ILP) Intron Single Nucleotide Polymorphism (ISNP)
  • 61. Detection of Intron Polymorphisms • Amplification of introns with PCR primed on flanking exons • Detection of ILPs: Separated by electrophoresis • Detection of ISNPs: -Sequencing -ECOTILLING
  • 62. Desirable features of IP markers • Introns are variable----high polymorphism SNP frequency in intron is 3~6 times higher than that in exons in rice Rice: between 93-11 (indica) and Nipponbare (japonica) ILP = 17.98% , ISNP = 51.22% , total = 69.20% Arabidopsis: between Columbia and Landsberg ILP = 18.61% , ISNP = 53.18% , total = 71.79% • Exons are conservative----high specificity Wang et al., 2006, DNA research, 12 (6): 417-427.
  • 63. Conditions needed for developing IP markers Known exon sequences: serving as templates for primer design Known intron position : telling flanking exons for primer design The conditions are available in model plants complete genome sequence and large number of full length cDNAs----known exons and introns
  • 64. Method for developing IP markers in non-model plants • Exon sequence: known from EST • Intron position: predicted from model plant • For any plant, IP marker can be developed as long as it has EST sequence data available
  • 65. Advantages of IP markers IP marker has similar advantages to SSR marker. In addition, it has some special advantages: oIntra-genic marker: IP marker-based genetic map→ linkage relationship among corresponding genes oMainly distributed in gene-rich regions: beneficial for gene mapping and candidate gene approach study oComparable among species based on gene homology: useful for comparative genomics research. Luca et. al., 2010, Diversity, 2: 572-585
  • 66. Conclusion