Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.
Seminar
On

Introns: Structure and functions
By
Yogesh S. Bhagat
Ph. D Scholar
Institute of Agricultural Biotechnology, Un...
Flow of seminar

o Introduction
o History of introns
o Classification of introns
o Structure and splicing mechanism of int...
C Value paradox
C Value paradox
G Value paradox

o Concept emerged from genomic and transcriptomic projects.
oEstimated number of protein coding genes doe...
The Fractal Complexity of Genome
Introns
o An intron is any nucleotide sequence within a gene that is removed by RNA
splicing to generate the final mature ...
Ist evidence for introns
Introns history
Scientist

Gene

Organism

Philip A
Sharp &
Richard J
Roberts

m RNA of beta-globin,
immunoglobulin,
ovalb...
How prevalent are the introns?
1. Early Intron hypothesis
 Introns were an essential feature of the earliest organisms
A...
How prevalent are the introns?
2. Late Intron hypothesis
 Earliest organisms did not contain introns.
Introns are a rela...
Distribution of introns
o The intron distributions in 5’UTR, CDS and 3’UTR are different
for same organism.
o The intron d...
Classification of Introns

S.No

TYPE OF INTRON

LOCATION

SPLICING

1

Group I

rRNA genes, Organell RNAs,
few bacterial ...
Nuclear Pre-mRNA Introns

Introns in nuclear protein-coding genes that are removed by spliceosomes

o Characterized by spe...
Chambons rule
Short consensus sequences at exon – intron junctions (AG-GT) or
AT – AC.
Splice site sequence requirement

Lariat branch site
Splicing reactions
http://mips.helmholtz-muenchen.de/proj/yeast/reviews/intron/spliceo_splicing.html
Group I catalytic introns and its Distribution
oGroup I introns are large self-splicing ribozymes.
oThey catalyze their ow...
Splicing mechanism of Group I introns
Structure of Group I introns

Database: GISSD
Softwares: Rfam
Group II intron
o Abundant in organellar genomes of plants and lower eukaryotes, but
have not yet been found in higher euk...
Proposed history of group II intron
Self-splicing mechanism of Group II intron
Protein assisted splicing mechanism of Group II intron

+ Maturase

After splicing, the RT remains tightly bound to splice...
Structure of Group II intron

RT binds to unspliced intron RNA at a high affinity binding
site in domain 4, and makes seco...
Function and interactions of the six group II intron domain

Scot A. Kelchner, American Journal Of Botany, 89(10): 1651–16...
Factors that can affect the gain and loss of introns

Daniel C. Jeffares and David Penny, Trends in Genetics Vol.22 No.1, ...
Models for intron gain and loss
Intron gain

Daniel et. al.,2006, Trends in Genet., 22: 18-22.
Intron gain
Intron Loss
Why do genes have introns ?

• Alternate splicing
• Regulating Gene expression
• Gene silencing

(miRNA, SiRNA)

Duret L.,...
Alternative splicing

The processing of an RNA transcript into different mRNA
molecules and a single gene might encode man...
Alternative splicing
Interactions: Protein-protein and protein-RNA interactions
Binding of specific regulatory protein to ...
Alternative splicing
Alternative splicing
e. g. SR proteins
-Acts as repressor and activator of splicing in a tissue specific
manner
Stress and...
Gene expression

o First introns :binding sites for transcription factors or
may act as classical transcriptional enhancer...
How introns influence eukaryotic gene expression?
Introns can affect the efficiency of transcription by several
different ...
How introns influence eukaryotic gene expression?

Introns are also required for specific modification of some exon
sequen...
How introns influence eukaryotic gene expression?
Formation and removal of exon junction complexes (EJCs)
• Once processed...
Intron effect on GUS transgene expression in transgenic rice lines
Constructs used in the study

pRESQ4: rubi3 promoter—5’UTR exon1 (67 bp)----5’UTR intron---the GUS coding sequence
pPSRG30...
Southern hybridization and real time PCR
GUS histochemical assay
Conclusion

 Splicing factors bound to the nascent RNA interact with RNA Pol II
C-terminal domain (CTD) and help to regul...
Beneficial effects of introns on recombinant gene expression

Zago, P., 2009, Biotechnology and Applied Biochemistry, (52)...
Intronic MicroRNA

•

Introns releases trans-acting factors such as microRNA
(miRNA) and small nucleolar RNA (snoRNA)

•

...
Intronic MicroRNA

Nearly 97% of the human genome is composed of noncoding DNA,
which varies from one species to another.
...
Biogenesis of intronic MicroRNA
Hinske, L., et al., 2008
Intron-mediated gene silencing

Artificial splicing-competent intron (SpRNAi):
of consensus nucleotide elements representi...
Intron mediated gene silencing in Zebrafish

Why zebrafish?
 Great use the study of aetiology and pathology of human dise...
Intron mediated gene silencing in Zebrafish

Anti GFP

RFP
Reduction in the of green fluorescence protein
Increase in the level of red fluorescence protein
Conclusion
Man-made intronic miRNAs have potential applications in
(a)The analysis of gene function by developing loss-of-...
Introns as a source of polymorphism
• Exons sequences are conserved but introns sequences vary
(length)
• Plant introns ar...
The longest intron identified in plants is Maize pericarp
gene (7 Kb)
Consensus sequence of
5’ splicing site is AG/GTAAGT
...
Development of Intron Polymorphism Markers

Detectable genetic polymorphism or allelic variation at DNA sequence
level.
Tw...
Detection of Intron Polymorphisms
Intron Polymorphism(IP): Polymorphism between allelic introns
Intron Length Polymorphism...
Detection of Intron Polymorphisms

• Amplification of introns with PCR primed on flanking exons
• Detection of ILPs: Separ...
Desirable features of IP markers
• Introns are variable----high polymorphism
SNP frequency in intron is 3~6 times higher t...
Conditions needed for developing IP markers

Known exon sequences: serving as templates for primer design
Known intron pos...
Method for developing IP markers in non-model plants

• Exon sequence: known from EST
• Intron position: predicted from mo...
Advantages of IP markers
IP marker has similar advantages to SSR marker.
In addition, it has some special advantages:
oInt...
Conclusion
Introns: structure and functions
Introns: structure and functions
Introns: structure and functions
Introns: structure and functions
Introns: structure and functions
Introns: structure and functions
Introns: structure and functions
Introns: structure and functions
Introns: structure and functions
Upcoming SlideShare
Loading in …5
×

Introns: structure and functions

7,682 views

Published on

"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)

Published in: Education, Technology
  • Be the first to comment

Introns: structure and functions

  1. 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. 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. 3. C Value paradox
  4. 4. C Value paradox
  5. 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. 6. The Fractal Complexity of Genome
  7. 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. 8. Ist evidence for introns
  9. 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. 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. 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. 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. 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. 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. 15. Chambons rule Short consensus sequences at exon – intron junctions (AG-GT) or AT – AC.
  16. 16. Splice site sequence requirement Lariat branch site
  17. 17. Splicing reactions
  18. 18. http://mips.helmholtz-muenchen.de/proj/yeast/reviews/intron/spliceo_splicing.html
  19. 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. 20. Splicing mechanism of Group I introns
  21. 21. Structure of Group I introns Database: GISSD Softwares: Rfam
  22. 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. 23. Proposed history of group II intron
  24. 24. Self-splicing mechanism of Group II intron
  25. 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. 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. 27. Function and interactions of the six group II intron domain Scot A. Kelchner, American Journal Of Botany, 89(10): 1651–1669. 2005.
  28. 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. 29. Models for intron gain and loss
  30. 30. Intron gain Daniel et. al.,2006, Trends in Genet., 22: 18-22.
  31. 31. Intron gain
  32. 32. Intron Loss
  33. 33. Why do genes have introns ? • Alternate splicing • Regulating Gene expression • Gene silencing (miRNA, SiRNA) Duret L., 2008, Trends in Genetics
  34. 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. 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. 36. Alternative splicing
  37. 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. 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. 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. 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. 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. 42. Intron effect on GUS transgene expression in transgenic rice lines
  43. 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. 44. Southern hybridization and real time PCR
  45. 45. GUS histochemical assay
  46. 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. 47. Beneficial effects of introns on recombinant gene expression Zago, P., 2009, Biotechnology and Applied Biochemistry, (52): 191–198.
  48. 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. 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. 50. Biogenesis of intronic MicroRNA
  51. 51. Hinske, L., et al., 2008
  52. 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. 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. 54. Intron mediated gene silencing in Zebrafish Anti GFP RFP
  55. 55. Reduction in the of green fluorescence protein Increase in the level of red fluorescence protein
  56. 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. 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. 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. 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. 60. Detection of Intron Polymorphisms Intron Polymorphism(IP): Polymorphism between allelic introns Intron Length Polymorphism (ILP) Intron Single Nucleotide Polymorphism (ISNP)
  61. 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. 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. 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. 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. 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. 66. Conclusion

×