Introns:

Origin & Function

Gene Structure Lecture 2

Dr Tony Southall
[email protected]
 OBJECTIVES

• Be able to discuss the ideas of ‘Intron Early Theory’ & ‘Intron Late’ and recent
developments

• Be able to discuss possible functions of introns in all “forms” of their existence

Ÿ Harbouring regulatory information

• Timing of gene expression

• Direct control of transcription

• Harbouring non-coding RNAs

• The exon junction complex (EJC)

 Introduction: Origins
Introns: found in “all” eukaryotic genomes
- except, a nucleomorph in a species of free-swimming, biflagellate monads…

• ‘Intron-early theory’
- Walter Gilbert
- Originated in prokaryotes? Then lost introns by ‘genome streamlining’
- Early introns gains- invasive and deleterious?
- ‘Exon theory of gene evolution’
- Shuffling permitted by introns
- Allowed the creation of complex genes (and a large protein collection!)

• ‘Intron- late theory’

- Introns only evolved in eukaryote branch of life
- Archaea and bacteria never had introns or spliceosome
- Random placement into genes
- Not necessarily corresponding to protein structural elements

PMID: 16907971
Truth may be somewhere in between..?

Prokaryote
Archaea like cell

Last Eukaryotic
Common Ancestor
(LECA)
Truth may be somewhere in between..?

PMID: 22507701

• ‘Many Introns Early in Eukaryotic Evolution’

• Last Common Eukaryotic Ancestor (LECA)
- Had intron rich genes
- Thanks to invasion during eukaryotic cell formation or initial stages
- Happened after endosymbiosis (mitochondria formation)?
 Roles during “Life Phases”

(i) Introns can be a host burden

• Spliceosome complex is huge!
• Energy & time ( e.g. RNAP II 60 nt s-1) cost
• Vulnerability e.g. need recognition of
(ii) cis-regulatory sequences

• “Life Phases”
i) Genomic intron
ii) Transcribed intron
(iii) iii) Intron being spliced
iv) Excised intron
v) EJC- harbouring transcript

(iv) (v)
Roles classified can also be classified as :
• ‘Sequence-dependent functions’
• ‘Length-dependent functions’
• ‘Splicing-dependent functions’
PMID: 22518112
“Genomic Introns”
 i) The “Genomic Intron”

• Still in the DNA:

• Location of gene’s cis-regulatory elements

• Transcription initiation (modulate main promoter action)

• Enhancers, silencers, TF binding sites
• Often found in 5’-most introns

Genome wide binding of the

transcription factor HOXC9
PMID: 24274069
 i) The “Genomic Intron”

• Alternative transcription initiation

- Alternative promoter in the intron!
- α-fetoprotein (AFP), plasma protein made in the liver and yolk sack in the foetus
- Regulates osmotic pressure
- Tissue specific expression

PMID: 11000266
 i) The “Genomic Intron”
• Transcription Termination
- Intron sequences can regulate Polyadenylation + cleavage
- eg. in Flt-1 gene

5’ primer

3’ primer

M
S

PMID: 21382012
 i) The “Genomic Intron”

• Nested Genes
- 800 in Drosophila melanogaster
- May have their own promoter & different expression profile
- non-coding RNA & protein-coding genes
- Refer to the ‘Overlapping Gene’ lecture
“Transcribed Introns”
 ii) “Transcribed Introns”

• Timing:
- RNA polymerase II: elongation rate up to 50 kb min -1
- Intron transcription may take hours!
- Time delay between gene activation and translation of the protein
 ii) “Transcribed Introns”

• HES7 gene (Mus muscula)

• Forms a negative feedback loop

• The oscillation of HES7 protein levels is

important for directing mesoderm cells to form
somites during embryonic development
 Transcribed introns

How might introns impact on oscillating gene networks?

 ii) “Transcribed Introns”
• Timing of expression and feedback loops

• HES7 represses its own transcription Oscillations at a

• delays due to time for transcription, splicing and translation
specific frequency
• unstable protein
 ii) “Transcribed Introns”
• Timing of expression and feedback loops

• Introns are very important for ensuring the correct period of oscillation!

PMID: 21300886
 ii) “Transcribed Introns”
• Timing of expression and feedback loops

PMID: 23219549
 “Spliced Introns”

Gene Structure Lecture 2 (Introns Origins &Functions) – Part 2

 iii) “Spliced Introns”
• Splicing is linked to Transcription
• Linked via RNAPII C-terminal domain
• Splicing can affect: Initiation, Elongation and Termination

Initiation:

PMID: 18243121
 iii) “Spliced Introns”
• Splicing factors and spliceosomal components
can interact with transcription elongation factors

Transcription Elongation:

PMID: 11780068
 iii) “Spliced Introns”
Transcription Termination:
• Endonucleolytic cleavage and poly(A) tail addition

PMID: 16857586 and 22770214

“Excised Introns”
 iv) “Excised Introns”

• Once an intron is excised it often undergoes debranching and degradation

• Embedded RNA genes may be expressed upon intron removal

• Non-protein coding RNAs (ncRNAs)

- microRNAs (miRNAs),
- small nucleolar RNAs (snoRNAs)
 iv) “Excised Introns”

“mirtrons”

• Recent studies suggest miRNAs are processed before splicing? (PMID: 19172742)
(Transcribed introns function?!)
Figure: PMID: 22888971
 iv) “Excised Introns”
snoRNAs

• 60-150 nucleotides long

• Fundamental to RNA modifications in archae & eukaryotes
• Modify RNAs (rRNA, snRNA, tRNAs)
• Released after splicing
“EJC-Harbouring transcripts”
 EJC-Harbouring Transcripts
• ‘Exon Junction Complex’
• Binds ~25 nts upstream of exon-exon junction on mRNA transcript
• 4 core proteins (MAGO, YI4, eIF4AIII, MLN51)
• Present from splicing until translation
 EJC-Harbouring Transcripts

• Roles:
1) Nuclear transport
2) Translation activation
3) mRNA localisation
4) nonsense-mediated decay (NMD)

1) Nuclear Transport
• Mature mRNAs bind to mRNA-specific transport factors
• Shuttled through Nuclear pore complexes
• Transport rates x10 for spliced transcripts
• Spliceosome or EJC recruits ALY/REF export factor
 EJC-Harbouring Transcripts

2) Translation activation
• Presence of the EJC on the mature mRNA enhances translation
• Until recently the mechanism was unclear
• Now known that EJC core component MLN51 interacts with eIF3 (PMID: 23530232)

3) Cytoplasmic localisation
• Subcellular regions (targeted within cytoplasm)
• Localisation permitted by shuttling proteins
• oskar mRNA needs EJC for location

PMID: 22426546
 EJC-Harbouring Transcripts

4) Nonsense Mediated Decay

• A surveillance mechanism
• Main role is to degrade mRNAs containing a premature stop codon
• To prevent dominant-negative/gain of function proteins

“mRNA marking” model:

• Splicing dependent
• EJC’s ˃50 nt downstream of a termination codon = premature
EJC-Harbouring Transcripts
Nonsense Mediated Decay
EJC-Harbouring Transcripts
Nonsense Mediated Decay

PMID: 23072888
 EJC-Harbouring Transcripts

PMID: 22518112
 Summary
i) Genomic intron
• Harbouring gene regulatory information
• Alternative transcription initiation sites
• Alternative transcription termination sites
• Nested genes

ii) Transcribed intron

• Timing of gene expression (eg. HES7 negative
feedback loop and oscillations)

iii) Intron being spliced

• Transcription initiation
• Transcription elongation
• Transcription termination

iv) Excised intron

• non-coding RNAs - mirRNAs and snoRNAs

v) EJC- harbouring transcript

• Nuclear transport
• Translation activation
• mRNA localisation
• Nonsense mediated decay (NMD)
 References
This lecture is based upon one previously given by Dr Timothy Simpson
and Dr Rey Carabeo (both formerly Imperial College London).

Chorev, M. and Carmel, L. (2012) The function of introns. Frontiers in Genetics (3): 1-15
Zhou, H and Lin K (2008) Excess of microRNAs in large and very 5’ biased introns. Biochemical and Biophysical Communications 368: 709-
715
Perina, D. et al., (2012) Structural and Functional Characterization of Ribosomal Protein Gene Introns in Sponges. Plos One. 7(8):1-9
Chorev, M and Carmel, L (2013) Computational identification of functional introns: high positional conservation of introns that harbor
RNA genes 41(11) 5604-5613
Kervestin, S. and Jacobson, A (2012) NMD: a multifaceted response to premature translational termination Nature Reviews: Molecular
Cell Biology 13: 700-712
Le Hir, H and Andersen, G.R. (2008) Structural insights into the exon junction complex Protein-nucleic acid interactions 18(1):112-119
Scohy, S., Gabant, P. Szpirer, C. and Szpirer J (2000) Identification of an enhancer and an alternative promoter in the first intron of the α-
fetoprotein gene 28(19): 3743-3751
Chazal, PE et al. (2013) EJC core component MLN51 interacts with eIF3 and activates translation. 110(15):5903-8

Recommended reading:
John S Mattick, “Introns: evolution and function” Current Opinion in Genetics and Development 4: 823-831 (1994)
Walter Gilbert, "Why genes in pieces?" p 501 v 271 Nature. 9 February 1978.

Nucleosomes on DNA: http://www.lifeexplorer.info/wordpress/wp-content/uploads/2014/01/1KX5.png

Stopwatch: http://www.rapitasystems.com/system/files/Timing1.jpg
Mirtrons: http://openi.nlm.nih.gov/imgs/512/263/2631224/2631224_ijbsv05p0097g04.png