Transcript alternative splicing

Alternative Splicing
A very short introduction (in plants)
Alternative Splicing
The exons and introns of a particular gene get shuffled to
create multiple isoforms of a particular protein
•First demonstrated in the late 1970’s in adenovirus
•Fairly well characterized in animals (at least somewhat better than in
plants)
•Contributes to protein diversity
•Affects mRNA stability
One Gene / One Enzyme
One Gene / One Polypeptide
“One Gene / One set of connected transcripts”
Ensembl- What is a gene, post-ENCODE? History and updated definition
Genome Res. 2007. 17: 669-681
1940’s -------------------------------------------------------------------------------2000’s
Alternative splicing in metazoans
Human splicing statistics
Estimating rates of alternative splicing in mammals
and invertebrates. NATURE GENETICS VOLUME 36 | NUMBER 9 | SEPTEMBER 2004
The Alternative Splicing Gallery (ASG): bridging the gap between genome and transcriptome
Nucleic Acids Research, 2004, Vol. 32, No. 13
• Alternative splicing is well characterized in animals
• In humans, the vast majority of genes have multiple spliceforms
• Estimates of up to 80% of human genes are alternatively spliced
Alternative splicing in disease
• By virtue of its widespread involvement in most of the genomic landscape, AS is important
in almost all gene families
• AS (or mis-splicing) is a very important component of genetic diseases
Mechanisms of splicing
Genome
Pre-mRNA
E4
E3
E2
Spliced mRNA
E1
E4
I2
E3
I1
E2
E1
Alternative splicing of RuBisCo was one of the first examples of AS in plants
“The data presented here demonstrate the existence of alternative splicing in plant
systems, but the physiological significance of synthesizing two forms of rubisco
activase remains unclear. However, this process may have important implications in
photosynthesis. if these polypeptides were functionally equivalent enzymes in the
chloroplast, there would be no need for the production of both polypeptides, and
alternative splicing of the rubisco activase mRNA would likely become a dispensable
process.”
The majority of AS events have not been functionally characterized
5’ Splice Site
Pre-mRNA
E1
I1
E2
3’ Splice Site
Reddy, S.N. Annu. Rev. Plant Biol. 2007 58:267-94
- In Arabidopsis out 1470 of 1588 predicted splice sites follow the canonical (GT…AG , CG…AG,
AT…AC )consensus sites. (The Plant Journal (2004) 39, 877–885 Intron retention is a major phenomenon in alternative splicing in Arabidopsis)
5’ Splice Site
Pre-mRNA
ATG
UTR
S
ATG
E1
S
I1
E2
UTR
3’ Splice Site
- Alternative splicing can effect the entire pre-mRNA transcript (UTRs included)
- Alternative splicing can also alter start codons or lead to premature termination codons
m7 G
Mature mRNA
UTR
E1
E2
UTR
AAA...AA
There are 5 main types of splicing
UTR
E1
m7G
Constitutive (familiar/ “normal”)
Alternative Donor site
Alternative Acceptor site
Alternative position
Exon Skipping
Intron retention
E2
UTR
AAA...AA
Genome
Pre-mRNA
E1
Spliced mRNA
Constitutive splicing
E2
E1
I1
E3
E2
I2
E3
E4
E4
Pre-mRNA
E1
I1
E2
Pre-mRNA
E1 E1
I1
E2
Spliced mRNA
E1
Alternative donor site (AltD)
E2
Pre-mRNA
E1
I1
E2
Pre-mRNA
E1
I1
E2 E2
Spliced mRNA
E1
Alternative acceptor site (AltA)
E2
Pre-mRNA
E1
I1
E2
Pre-mRNA
E1
I1
I1
E2
Spliced mRNA
Alternative Position (AltP)
E1
E2
Pre-mRNA
E1
I1
E2
I2
E3
Pre-mRNA
E1
I1
E2
I2
E3
Spliced mRNA
Exon skipping (ExonS)
E1
E3
Pre-mRNA
E1
I1
E2
Pre-mRNA
E1
I1
E2
Spliced mRNA
E1
Intron retention (IntronR)
How prevalent are these alternative spliceforms?
AS type
Events (%)
Genes (%)
Events (%)
Genes (%)
AltD
845 (10.2)
724 (3.3)
1,642 (11.3)
990 (3.2)
AltA
1,810 (21.9)
1,452 (6.7)
2,201 (15.1)
1,698 (5.5)
AltP
308 (3.7)
200 (0.9)
921 (6.3)
562 (1.8)
ExonS
666 (8.1)
379 (1.8)
2,004 (13.8)
999 (3.2)
IntronR
Total
4,635 (56.1) 3,094 (14.3) 7,774 (53.5) 4,513 (14.6)
8,264
4,707 (21.8)
14,542
6,568 (21.2)
Genomewide comparative analysis of alternative splicing in plants
PNAS May 2, 2006 vol. 103 no. 18 7175-7180
21,641 genes and Arabidopsis and 30,917 genes in rice were
interrogated for
Alternative splicing events.
An estimated 1/5th of plant genes undergo alternative splicing
Alternative splicing is far less common in plants
AS type
Arabidopsis
Rice
Maize
Human
AltD
3%
11%
5%
42%
AltA
18%
22%
22%
24%
ExonS
38%
34%
38%
25%
IntronR
41%
33%
35%
9%
Genome-wide analyses of alternative splicing in plants: Opportunities and challenges
Genome Res. 2008. 18:1381-1392
- In humans up to 80% of genes undergo AS (compared to ~20% in plants)
- The types of AS varies across species
- Intron retention is the most common type of AS in plants
Reddy, S.N. Annu. Rev. Plant Biol. 2007 58:267-94
- The plant spliceosome is less well characterized than metazoan mechanisms.
- Plants share similar splice site configurations with animals, but there are significant
differences in intron size and composition
Splicing in disease: disruption of the splicing code and the
decoding machinery. doi:10.1038/nrg2164
How are AS events detected?
• High-througput detection is largely based on microarray data provided by
cDNA and EST data
• PCR based assays
Biological importance of AS
So far, AS has been implicated in a number of biologically important roles including:
- Splicing
- Transcriptions
- Flowering regulation
- Disease resistance
- Enzymatic activity
A database of AS genes is available at plantgdb.org/ASIP/
Some examples: Disease resistance in tobacco
- In tobacco, the N gene confers resistance to Tobacco Mosaic Virus (TMV)
- There are two alternative transcripts Ns and NL (short and long)
- NL lacks 13 of the 14 LRRs that make are a part of the Ns protein
- Infection with TMV causes NL to become more abundant after infection
- Expression of Ns in transgenic plants does not confer TMV resistance
Some examples: Jasmonate signaling in Arabidopsis
• Jasmonate (plant hormone) is involved in cell division and growth, reproduction as
well as defense against insects, pathogens, and abiotic stress.
• AS isoforms (10.4 and 10.3) result in various phenotypic effects (e.g. male sterility,
insensitivity to jasmonate inhibition of root growth, etc.)
Some examples: Jasmonate signaling in Arabidopsis
• The JAZ10.3 isoform results in a premature stop codon in the D exon.
•
The JAZ10.4 (AltD) isoform results in a truncation of the D exon, which leads to the
elimination of an important domain (Jas).