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