Gene Structure

Lectures:

1) Alternative Splicing: One Gene, Multiple Polypeptides

2) Introns: Origin and Function

3) Overlapping Genes

4) RNA editing - Things are not always what they seem

“Diversifying the Proteome”

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Dr Tony Southall
t.southall@imperial.ac.uk Q ui
 Lecture 1

Alternative Splicing:
One Gene, Multiple Polypeptides

Dr Tony Southall
t.southall@imperial.ac.uk
 OBJECTIVES

• Understand magnitude of alternative RNA splicing

• Discern differences between categories

• Understand mechanisms behind splicing

• Follow gene hierarchies based on alternative splicing

• Comprehend the levels of control of alternative splicing

What is a very effective way to generate different proteins from
one gene?

"Alternative RNA splicing is like remixing music to

produce a new song or re-editing a movie for a
different effect."
Quick revision of splicing
 Splicing
• Needs no free energy
• Can splice over large distances (10’s of kilobases)

• How are splice sites brought into close proximity?

• How is the correct splice site utilised?
• How are exons skipped?
• How are cryptic splice sites avoided?

The spliceosome and spliceosome co-factors!

 Spliceosome

• Formed of 5 small nuclear RNAs (snRNAs) that associate with proteins

to form small nuclear ribonucleic particles (snRNPs)

• The snRNAs: U1, U2, U4, U5, U6

• snRNPs + accessory proteins (‘spliceosome’)

• Attach to mRNA transcript and form complexes

• Specific complexes are formed in a sequential manner
 Assembly and action of the spliceosome

Step 1 – U1 and U2 assemble onto pre-mRNA in a co-transcriptional manner

Step 2 – The U1 and U2 snRNPs interact with each other to form the pre-spliceosome (complex A)
Assembly and action of the spliceosome (cont.)

Step 3 – the preassembled tri-

snRNP U4–U6•U5 is recruited to
form complex B

Step 4 – complex B undergoes

a series of rearrangements to
form a catalytically active
complex B (complex B*)

Complex B* is
ready to perform
the first catalytic
step!
 Assembly and action of the
spliceosome (cont.)
Step 7 – Release of
spliced mRNA and
lariat

Step 5 – Complex B* then carries out

the first catalytic step of splicing,
generating complex C, which contains
free exon 1 and the intron–exon 2 lariat
intermediate

Step 6 – Complex C undergoes

additional rearrangements and then
carries out the second catalytic step,
resulting in a post-spliceosomal
complex that contains the lariat intron
and spliced exons
 Alternative Splicing
• Until the 1980’s:
• genes thought to have only 1 mRNA
• but: 1° transcripts can have 2 or more splicing pathways
• Related but different mRNAs

~ 0.1% of genes ~ 95% of genes

• So,
The number of genes in an organism’s genome is not a good assessment of protein diversity
 Huge Variation!
• Multiple forms of mRNA transcript variation:
• Exons retained or skipped
• Introns excised or retained
• 5’ & 3’ splice site positions moved: exons longer or shorter

PMID: 18805093

• Example: DSCAM (Down Syndrome Cell Adhesion Molecule) gene

• Membrane-anchored cell surface protein

• A role in neuronal development for axon and dendrite self-avoidance (in Drosophila)
• 24 exons permits 38,016 protein variants
 A Method of Regulation?

• Supplements transcriptional control:

• Insertion/deletion of specific domains
• e.g. it can regulate antibody and neuropeptide production

• Regulates functionality:
• Regulates balance of functional to non-functional RNAs
• Splicing: premature stop codon = RNA degradation
 Effects on mRNAs and Proteins

• Varied!
• Rate of translation of mRNA
• mRNA degradation susceptibility
• Insertion/deletion of amino acids
• Insertion/deletion of functional domains
• Polypeptide truncation due to stop codon

• Protein properties and functions:

• Soluble or membrane bound
• Subcellular location changes
• Affinity changes
 Effects on mRNAs and Proteins

• Consider:
• Selection of alternative splice sites: tissue and developmental-stage specific
• Splice site selection must be tightly regulated
• Many genetic diseases can be caused by point mutations that
activate cryptic splice sites or delete splice sites

SpliceDisease database:
Linking RNA splicing and disease

• Manual curation of 2337 splicing mutations

associated with disease
• 303 genes and 370 diseases
• Supported experimentally in 898 publications

PMID: 22139928
 Basic Categories

• 3 groups of alternatively spliced transcripts:

1) 5’ transcript ends differ from one another

2) 3’ ends differ from one another
3) Middle portions differ

• 5’ transcript ends differ

• Due to different transcriptional start sites
• Then pre-mRNA processed differently
• Recruitment of different splicing complexes?
 1: 5’ End Differences
• Generated through alternative transcriptional
start sites that can promote alternative splicing

Coding exon

• Mouse α-amylase gene

 2: 3’ Ends Differ
• 5’ end the same. 3’ ends differ:
• When different poly(A) sites are utilised for transcriptional termination
• Different use in different tissues
• Alternate splicing can occur when different poly(A) sites used

• Immunoglobulin chains of antibodies

 3: Centre Differs

• 5’ ends the same. 3’ ends the same. Middle different:

• Can’t be explained by differential promoter use or cleavage

• Example: troponin T gene (skeletal muscle)

• 64 different ways (found in different muscle types)

• Tissue-specific splicing factors acting on the pre-mRNA

 Exon shuffling
(different to splicing!)
Many eukaryotic proteins are ‘mosaics of motifs’
• Domains: 40-100 amino acids
• Functions (stabilization, binding, catalysis etc.)
• In different proteins or repeated in the same protein

EGF gene

Exon Exon
shuffling duplication
Fibronectin gene

Exon
Plasminogen gene shuffling
 Exon shuffling
(different to splicing!)

How does exon shuffling happen?

1) Illegitimate non-homologous recombination

2) LINES (Long Interspersed Nuclear Elements)

3) DNA Transposons – similar to LINES – they ‘collect’ exons and can move them
around, inserting them into genes (found in plants)
 Exon shuffling
(different to splicing!)

IMPORTANT! Alternative splicing and exon shuffling are very

different mechanisms for generating proteome diversity

• Alternative splicing acts on RNA to create diversity at a cellular and temporal

resolution during the lifespan of an organism

• Exon shuffling creates diversity on an evolutionary scale over many generations

 Summary (Part 1)
• Splicing occurs through the “spliceosome” complex

• The incorporation of different exons through splicing regulation allows the generation of alternative mRNAs and
proteins (diversifying the proteome..)

• The use of different 5’ exons occurs via alternative transcriptional start sites

• The use of different 3’ exons occurs via alternative termination sites

• Different ‘middle’ exons can be incorporated via alternative splicing

• This can allow the production of proteins with different properties

e.g. - intracellular localisation
- activity
- ability to bind to other proteins
- secretion out of the cell
- solubility

• Exon shuffling is different to alternative splicing and creates genome diversity on an evolutionary scale
Gene Structure Lecture 1 (Part 2)

Regulation of alternative splicing

 Tissue specific splicing factors

• Recognise cis-acting sequences within the RNA transcript

• “Promote” or “inhibit” splice sites in different cases
 Drosophila sex determination
• A gene hierarchy determines sex in fruit flies
• Each gene product controls splicing of the next gene in hierarchy
• Different protein products in males and females

• Sxl (sex lethal) gene

• Sxl is the master sex determination gene in somatic cells
• Inhibits ♂ specific splicing (inhibits splicing in of exon 3, which contains a stop codon)
Drosophila sex determination

• tra (transformer) gene

• SXL binds at proximal splice site (found in intron 1)

• Prevents U2AF binding
• Binds cryptic site in exon 2 (known as the ‘distal splice site’)
• Allows the splicing out of a stop codon
 Drosophila sex determination

• dsx (doublesex) gene

• Encodes a transcriptional repressor that determines development
• Tra is recruited to an Exon Splicing Enhancer (ESE) by Tra2
• This promotes the inclusion of exon 4
• Which leads to transcriptional termination
• ♀ dsx mRNA ends with exon 4 - and a shorter protein isoform with different properties
 Drosophila sex determination

• Tra also regulates the splicing of the transcription factor gene fruitless

PMID: 24052176

• fru (fruitless) gene

• Encodes a transcriptional regulator that determines development
• In ♀ , Tra promotes the splicing from the end of exon 2
• This results in the inclusion of a stop codon
• Which leads to transcriptional termination and no ♂ isoform is produced
 Drosophila sex determination

PMID: 12350340

• Splicing factors:
• Act positively (e.g. Tra) to promote the use of a splice site
• Act negatively (e.g. Sxl) to inhibit the use of a site
 Drosophila sex determination

•FruM controls many aspects of male courtship behaviour

Drosophila sex determination
Alternative splicing of fruitless has a profound effect
on the sexual behaviour of flies

PMID: 15935764
 Response to Signals

PMID: 15340380

• Activation of neurons results in the alternative

splicing of the K+ channel gene SLO
 Regulating Factors

SR proteins
• ‘RNA recognition Motifs’ (RRMs, N-terminal) and SR domain (C-terminal)
• SR proteins: influence splice site selection in 2 ways:
i) Bind 5’ splice site & promote U1 snRNP binding
ii) Bind ESEs within downstream exon and stimulate U2AF binding

Heterogenous nuclear Ribonucleoproteins (hnRNPs) – inhibit splicing (in general)

 Remembering acronyms

ESE - Exonic Splicing Enhancer

ISE - Intronic Splicing Enhancer

ESS - Exonic Splicing Silencer

ISS - Intronic Splicing Silencer

SR proteins – Stimulate splicing

(Serine Arginine repeats)

hnRNPs - hinder splicing

(heterogenous nuclear
RiboNuclearProtein)
 Further Regulation

• So, a summary of regulation so far:

• Some splice factors are constitutively expressed, whilst others are tissue/cell type specific
• Constitutively expressed SR proteins can interact with specifically expressed factors
• eg: Dsx gene splicing needs ♀-specific Tra and the ubiquitously expressed Tra2
• Stabilises U2AF

• Alternative Splicing dictated by:

• RNA sequences,
• Constitutive or tissue-specific trans-acting factors
• Splice site strength:
• Ability to bind general factors (e.g. U1 snRNP)
• Presence or absence of ESE’s
 Link to Transcription

What happens if RNA polymerase is elongating at a slower rate?

 Link to Transcription

• Close link between splicing and Transcription:

• Splicing occurs as transcription is still going on
• Rate of elongation can affect splicing pattern
• Slow: more likely to include exons with weak acceptor sites
• Faster: more likely to skip exons with a weak acceptor site

• Been shown in vivo in Drosophila using a mutant line with a lower

RNA polymerase II elongation rate (PMID: 14536091)
 Identification and Summary

• Genome Wide Analysis:

• microarray (now NGS)

• Massive Variation
- Tissue/cell specific

• Extra level of Control

- responds to cellular signals

• Alternative Splicing: allows related but different protein forms in different tissues

• Depends on a number of factors: RNA sequences e.g. ESE’s and ESS’s

Splicing factor presence and concentration
• Several categories: 5’ different
3’ different
middle different
 Identification: Future methods?

• Alternative Splicing in complex tissues – how do you

know which cells have a specific splice pattern?

• How can you rapidly identify the target genes of

certain splice factors in specific cell types?
Splicing is tightly coupled to transcription
Splicing is tightly coupled to transcription
TaDa – could it be used to profile the interaction of splicing
factors with genome in a cell type-specific manner?

Targeted DamID (TaDa) - PMID: 23792147

Unfortunately, it does not work…

However…
 The splicing factor Sex lethal (Sxl) strongly
associates with a set of transcriptional start sites!

• Can Sxl bring about sexual dimorphism in the nervous system

through transcriptional regulation, as well as alternative splicing?
 Ongoing Research and Development

Recent update…

• No, it cannot!
• Multiple experiments show no evidence for the binding of Sxl to chromatin in
driving sexual dimorphism..

• However, there is the intriguing expression of a splice isoform of Sxl (Sxl-Z)

that is expressed in males, however, only in the adult brain…

• This isoform is truncated and is not able to alternatively splice tra RNA
 Ongoing Research and Development

Expression of Sxl-Z in the

male brain mRNA expression changes when
Sxl-Z is knocked down with RNAi
in the male brain
 Ongoing Research and Development

• Experiments suggest that the Sxl-Z isoform in the male brain

has a neuroprotective role

• This is likely the ancestral role of Sxl (its role in splicing is

relatively recent on an evolutionary timescale)

• How does it do this? We are working on that now!

 References
This lecture is based upon one previously given by Dr Timothy Simpson and Dr Rey
Carabeo (both formerly Imperial College London). All books available in Imperial library.

• Genetics: A Conceptual Approach (2012, 4th Ed) Benjamin A. Pierce. W.H Freeman Palgrave Macmillan
• Genetics: Analysis of Genes and Genomes (2012, 8th Ed) Daniel L. Hartl and Maryellen Ruvolo. Jones & Bartlett
Learning
• Genetics: From Genes to Genomes (2008, 4th Ed) Hartwell, Hood, Goldberg, Reynolds and Silver. McGraw-Hill
International Edition.
• Introduction to Genetic Analysis (2012, 10th Ed) Griffiths, Wessler, Carroll and Doebley. W.H. Freeman and Company,
New York
• Introduction to Genetics: A molecular approach (2012) Terry Brown, Garland Science
• Principles of Biochemistry (2013, 4th Ed) Voet, Voet and Voet, Wiley

Further reading:
Alternative splicing of the trout Pax5 gene and identification of novel B cell populations using Pax5 signatures PMID: 23796789
Cell Signalling and the control of pre-mRNA splicing PMID: 15340380

Bakers Yeast: http://www.microbiologyonline.org.uk/about-microbiology/introducing-microbes/fungi

Immunoglobulin chain splicing diagram: http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-23/23_30.jpg
Dsx splicing: https://upload.wikimedia.org/wikipedia/commons/f/f9/Dsx_splicing.jpg
Drosophila male courtship behaviour: http://www.mdpi.com/insects/insects-05-00439/article_deploy/html/images/insects-05-00439-g001-1024.png
Future: https://cdn.static-economist.com/sites/default/files/images/2014/07/blogs/prospero/20140705_blp510.jpg