What is RNA splicing

1.

What is RNA splicing?

2. Genetic information is transferred from genes to the proteins they encode via a “messenger” RNA intermediate

DNA
GENE
transcription
messenger RNA
(mRNA)
translation
protein

3. Most genes have their protein-coding information interrupted by non-coding sequences called “introns”. The coding sequences are

then called “exons”
exon 1
DNA
intron
GE
exon 2
NE
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”

pre-mRNA
intron
RNA splicing
mRNA
translation
protein

5. Splicing a pre-mRNA involves two reactions

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

6. Splicing occurs in a “spliceosome” an RNA-protein complex (simplified)

spliceosome
(~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. 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
M7G
exon
intron
exon
AAAAAAA200
cap
RNA splicing
mRNA
M7G
nucleus
AAAAAAA200
transport
cytoplasm
M7G
AAAAAAA200
ribosomes
protein
poly(A) tail

9. Alternative splicing In humans, many genes contain multiple introns

intron 1
1
intron 3
intron 2
2
3
1
2
3
intron 4
4
4
5
Usually all introns must be removed before the
mRNA can be translated to produce protein
5

10. However, multiple introns may be spliced differently in different circumstances, for example in different tissues.

Heart muscle mRNA
pre-mRNA
1
1
2
3
2
Uterine muscle mRNA
5
3
1
3
4
4
5
5
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.

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

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 splicing
A
Mutations occur
on the DNA
(in a gene)
1
2
no mutation
normal mRNA
1
2 3
4
normal protein
active
B
3
mutation A
truncated mRNA
5
C
1
2
truncated protein
inactive
4
5
mutation B
exon 3 skipped
1
2
4
mutation C
longer exon 4
5
1
2 3
4
protein of different size (smaller or longer)
inactive or aberrant function
5

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