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
intron branchpoint
pre-mRNA A
intermediates
Step 1 A
spliced mRNA
Step 2

6. Splicing occurs in a “spliceosome” an RNA-protein complex (simplified)
(~100 proteins + 5 small RNAs)
pre-mRNA
spliced mRNA
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
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
pre-mRNA
intron
exon
AAAAAAA200
M7G
mRNA
RNA splicing
M7G
AAAAAAA200
cap
poly(A) tail
nucleus
transport
M7G
AAAAAAA200
cytoplasm
ribosomes
protein

9. Alternative splicing In humans, many genes contain multiple introns3 4 5 1 2
intron 2
intron 3
intron 4
intron 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 5
Heart muscle mRNA 3 5 4 2 1
pre-mRNA 1 4 3 5
Uterine 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

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. Fas Fas ligand 7 6 5 6 5 7 Fas pre-mRNA APOPTOSIS Fas ligand
Alternative splicing can generate mRNAs encoding proteins with
different, even opposite functions
Fas
Fas ligand
(membrane-
associated) 7 6 5 6 5 7
Fas pre-mRNA
(+)
APOPTOSIS
(programmed
cell death)
(-)
Fas ligand
Soluble Fas
(membrane) 7 5

13. Alternative splicing can generate tens of thousands of mRNAs
from a single primary transcript
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 12 48 33 2
pre-mRNA
mRNA
protein
The protein variants are important for wiring of the nervous system and for immune response

14. Examples of the potential consequences of mutations on splicing3 5 4 1 2 A B C
Mutations occur
on the DNA
(in a gene)
no mutation
normal mRNA
mutation A
truncated mRNA
mutation B
exon 3 skipped
mutation C
longer exon 4 3 5 4 1 2 1 2 5 4 1 2 3 5 4 1 2
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