"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)
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
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
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.
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.
20. 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).
24. 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).
27. 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.
28.
29. 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
30. Function and interactions of the six group II intron domain
Scot A. Kelchner, American Journal Of Botany, 89(10): 1651–1669. 2005.
31.
32. Factors that can affect the gain and loss of introns
Daniel C. Jeffares and David Penny, Trends in Genetics Vol.22 No.1, 2006
37. Why do genes have introns ?
• Alternate splicing
• Regulating Gene expression
• Gene silencing
(miRNA, SiRNA)
Duret L., 2008, Trends in Genetics
38. 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).
39. 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
41. 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.
42. 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
43. 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.
44. 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.
45. 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
46.
47. Intron effect on GUS transgene expression in transgenic rice lines
48. 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)
51. 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
52. Beneficial effects of introns on recombinant gene expression
Zago, P., 2009, Biotechnology and Applied Biochemistry, (52): 191–198.
53. 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.
54. 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
57. 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.
58.
59.
60. 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.
62. Reduction in the of green fluorescence protein
Increase in the level of red fluorescence protein
63. 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
64. 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
65. 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.
66. Development of Intron Polymorphism Markers
Detectable genetic polymorphism or allelic variation at DNA sequence
level.
Two types of polymorphism : Length difference
Nucleotide difference (SNP )
67. Detection of Intron Polymorphisms
Intron Polymorphism(IP): Polymorphism between allelic introns
Intron Length Polymorphism (ILP)
Intron Single Nucleotide Polymorphism (ISNP)
68. Detection of Intron Polymorphisms
• Amplification of introns with PCR primed on flanking exons
• Detection of ILPs: Separated by electrophoresis
• Detection of ISNPs:
-Sequencing
-ECOTILLING
69. 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.
70. 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
71.
72. 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
73. 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
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**.