introns: structure and functions
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SeminarOn
Introns: Structure and
functions
By
Yogesh S. Bhagat
Ph. D Scholar
Institute of Agricultural Biotechnology, University
of Agricultural Sciences, Dharwad (Karnataka)
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
C Value paradox
C Value paradox
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
The Fractal Complexity of Genome
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.
Introns
Ist evidence for introns
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)
Introns history
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.
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.
How prevalent are the introns?
oThe intron distributions in 5’UTR, CDS and 3’UTR are different for same organism.
oThe 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.
Distribution 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.
Selfsplicing(Reverse
trancriptase & ribozyme)
3 Nuclear- mRNA Nucleus Non Self-splicing(Sn RNAs)
4 t RNA t RNA Enzymatic
Classification of 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.
Nuclear Pre-mRNA Introns
Short consensus sequences at exon – intron junctions (AG-GT) or AT – AC.
Chambons rule
Splice site sequence requirement
Lariat branch site
Splicing reactions
http://mips.helmholtz-muenchen.de/proj/yeast/reviews/intron/spliceo_splicing.html
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).
Group I catalytic introns and its Distribution
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 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).
Proposed history of group II intron
Self-splicing mechanism of Group II intron
After splicing, the RT remains tightly bound to spliced intron, and this RNP particle is the active moiety in
subsequent mobility reactions.
Protein assisted splicing mechanism of Group II intron
+ Maturase
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 self-splicing
Structure of Group II intron
Scot A. Kelchner, American Journal Of Botany, 89(10): 1651–1669. 2005.
Function and interactions of the six group II intron domain
Daniel C. Jeffares and David Penny, Trends in Genetics Vol.22 No.1, 2006
Factors that can affect the gain and loss of introns
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 ?
Duret L., 2008, Trends in Genetics
• Alternate splicing
• Regulating Gene expression
• Gene silencing (miRNA, SiRNA)
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 to gene expression, through alternative splicing.
Introns contains functional elements (regulatory elements, alternative promoters).
Alternative splicing
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
Alternative splicing
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.
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
How introns influence eukaryotic gene expression?
Hiret, H. L. et al., 2006, Trends in Biochemical Sci.,Parra et. al., 2011, Nucleic Acids Res., 39: 5328-5337.
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
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.
Introns are also required for specific modification of some exon sequences by RNA editing
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, during the first round of translation.
How introns influence eukaryotic gene expression?
Intron effect on GUS transgene expression in transgenic rice lines
pRESQ4: rubi3 promoter—5’UTR exon1 (67 bp)----5’UTR intron----the GUS coding sequence
pPSRG30: same as pRESQ4 (except 5’UTR intron)
Constructs used in the study
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 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
Zago, P., 2009, Biotechnology and Applied Biochemistry, (52): 191–198.
Beneficial effects of introns on recombinant gene expression
• 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.
Intronic MicroRNA
Intronic MicroRNA
Ying., et al., 2010, Methods Mol Biol., 629: 205-237
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
pre-mRNAs.
Biogenesis of intronic MicroRNA
Hinske, L., et al., 2008
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.
Intron-mediated gene silencing
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.
Anti GFP
RFP
Intron mediated gene silencing in Zebrafish
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-
function transgenic animals
(b)The evaluation of both the function and effectiveness of
miRNA,
(c)The design and development of novel gene therapies
Introns as a source of polymorphism
Plant introns are short (80-139nts) Differ from vertebrate and yeast introns(2-3Kb) Resembles to animals like fruit fly and Nematode introns
• Exons sequences are conserved but introns sequences vary (length)
• Plant introns are richer in AT bases than their adjacent exons
• Exons sequences are conserved but introns sequences vary (length)
• Plant introns are richer in AT bases than their adjacent exons
XIE Xianzhi and WU Naihu, Chinese Science Bulletin (47): 17 ,2005
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.
Development of Intron Polymorphism Markers
Two types of polymorphism : Length difference Nucleotide difference (SNP )
Detectable genetic polymorphism or allelic variation at DNA sequence level.
Intron Polymorphism(IP): Polymorphism between allelic introns
Intron Length Polymorphism (ILP)
Intron Single Nucleotide Polymorphism (ISNP)
Detection of Intron Polymorphisms
• Amplification of introns with PCR primed on flanking exons
• Detection of ILPs: Separated by electrophoresis
• Detection of ISNPs:-Sequencing-ECOTILLING
Detection of Intron Polymorphisms
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.
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
Conditions needed for developing IP markers
• 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
Method for developing IP markers in non-model plants
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.
Advantages of IP markers
Luca et. al., 2010, Diversity, 2: 572-585
Conclusion
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