1. What is RNA splicing?

2. Genetic information is transferred from genes to the
proteins they encode via a “messenger” RNA
intermediate
DNA GENE
messenger RNA
(mRNA)
protein
transcription
translation

3. Most genes have their protein-coding information
interrupted by non-coding sequences called “introns”. The
coding sequences are then called “exons”
DNA GE NE
intron
exon 1 exon 2
transcription
precursor-mRNA
(pre-mRNA)
intron

4. The intron is also present in the RNA copy of the gene
and must be removed by a process called “RNA
splicing”
protein
translation
mRNA
RNA splicing
pre-mRNA
intron

5. Splicing a pre-mRNA involves two reactions
pre-mRNA
intron branchpoint
A
spliced mRNA
Step 2
intermediates
Step 1
A

6. Splicing occurs in a “spliceosome”
an RNA-protein complex
(simplified)
pre-mRNA spliced mRNA
spliceosome
(~100 proteins + 5 small RNAs)
Splicing works similarly in different organisms, for
example in yeast, flies, worms, plants and animals.

7. RNA is produced in the nucleus of the cell. The
mRNA has to be transported to the cytoplasm to
produce proteins
Ribosomes are RNA-protein machines that make
proteins, translating the coding information in the
mRNA

8. Pre-messenger RNA Processing
cytoplasm
nucleus
mRNA
RNA splicing
M7
G AAAAAAA200
pre-mRNA
intronexon exon
AAAAAAA200
M7
G
transport
M7
G AAAAAAA200
ribosomes
protein
cap poly(A) tail

9. Alternative splicing
In humans, many genes contain multiple introns
3 4 51 2
1 2 3 54
intron 2 intron 3 intron 4intron 1
Usually all introns must be removed before the
mRNA can be translated to produce protein

10. However, multiple introns may be spliced
differently in different circumstances, for
example in different tissues.
1 2 3 5Heart muscle mRNA
1 43 5Uterine muscle mRNA
Thus one gene can encode more than one protein. The proteins are
similar but not identical and may have distinct properties. This is
important in complex organisms
3 5421pre-mRNA

11. Different signals in the pre-mRNA and different proteins
cause spliceosomes to form in particular positions to give
alternative splicing
We are studying how mRNAs and proteins interact in
order to understand how these machines work in general
and, in particular, how RNA splicing is regulated as it
affects which proteins are produced in each cell and
tissue in the body.

12. 765
75
65 7
Fas pre-mRNA
APOPTOSIS
Alternative splicing can generate mRNAs encoding proteins with
different, even opposite functions
(programmed
cell death)
Fas ligand
Soluble Fas
(membrane)
Fas
Fas ligand
(membrane-
associated)
(+)
(-)

13. Alternative splicing can generate tens of thousands of mRNAs
from a single primary transcript
12 48 33 2
Combinatorial selection of one exon at each of four variable regions generates more than
38,000 different mRNAs and proteins in the Drosophila cell adhesion molecule Dscam
The protein variants are important for wiring of the nervous system and for immune response
protein
mRNA
pre-mRNA

14. Examples of the potential consequences of mutations on splicing
3 541 2
A B
CMutations occur
on the DNA
(in a gene)
1 2
mutation A
truncated mRNA
541 2
mutation B
exon 3 skipped
3 541 2
mutation C
longer exon 4
3 541 2
no mutation
normal mRNA
normal protein
active
truncated protein
inactive
protein of different size (smaller or longer)
inactive or aberrant function

15. Pathologies resulting from aberrant splicing can be
grouped in two major categories
Mutations affecting proteins that are involved in splicing
Examples: Spinal Muscular Atrophy
Retinitis Pigmentosa
Myotonic Dystrophy
Mutations affecting a specific messenger RNA and disturbing its
normal splicing pattern
Examples: ß-Thalassemia
Duchenne Muscular Dystrophy
Cystic Fibrosis
Frasier Syndrome
Frontotemporal Dementia and Parkinsonism

16. Therefore, understanding the mechanism of RNA
splicing in normal cells and how it is regulated in
different tissues and at different stages of
development of an organism is essential in order to
develop strategies to correct aberrant splicing in
human pathologies