1. A very short introduction (in plants)
Alternative Splicing
A very short introduction (in plants)

2. 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

3. One Gene / One Polypeptide
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: 
1940’s ’s

4. Alternative splicing in metazoans
Human splicing statistics
The Alternative Splicing Gallery (ASG): bridging the gap between genome and transcriptome
Nucleic Acids Research, 2004, Vol. 32, No. 13
Estimating rates of alternative splicing in mammals
and invertebrates. NATURE GENETICS VOLUME 36 | NUMBER 9 | SEPTEMBER 2004
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

5. 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

6. Mechanisms of splicing

7. Genome
Pre-mRNA E1 E2 I1 E3 I2 E4
Spliced mRNA E1 E2 E3 E4

8. The majority of AS events have not been functionally characterized
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

9. 5’ Splice Site Pre-mRNA E1 I1 E2 3’ Splice Site
Reddy, S.N. Annu. Rev. Plant Biol :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)

10. 5’ Splice Site S S Pre-mRNA UTR E1 I1 E2 UTR 3’ Splice Site
ATG
ATG S S
Pre-mRNA
UTR E1 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
Mature mRNA
m7G
UTR E1 E2
UTR
AAA...AA

11. There are 5 main types of splicing
Constitutive (familiar/ “normal”)
Alternative Donor site
Alternative Acceptor site
Alternative position
Exon Skipping
Intron retention
m7G
UTR E1 E2
UTR
AAA...AA

12. Genome
Pre-mRNA E1 E2 I1 E3 I2 E4
Spliced mRNA E1 E2 E3 E4
Constitutive splicing

13. Pre-mRNA E1 I1 E2
Pre-mRNA E1 E1 I1 E2
Spliced mRNA E1 E2
Alternative donor site (AltD)

14. Pre-mRNA E1 I1 E2
Pre-mRNA E1 I1 E2 E2
Spliced mRNA E1 E2
Alternative acceptor site (AltA)

15. Pre-mRNA E1 I1 E2
Pre-mRNA E1 I1 I1 E2
Spliced mRNA E1 E2
Alternative Position (AltP)

16. Pre-mRNA E1 I1 E2 I2 E3
Pre-mRNA E1 I1 E2 I2 E3
Spliced mRNA E1 E3
Exon skipping (ExonS)

17. Pre-mRNA E1 I1 E2
Pre-mRNA E1 I1 E2
Spliced mRNA E1
Intron retention (IntronR)

18. How prevalent are these alternative spliceforms?
AS type
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
4,635 (56.1)
3,094 (14.3)
7,774 (53.5)
4,513 (14.6)
Total
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 
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

19. Alternative splicing is far less common in plants
AS type
Arabidopsis
Rice
Maize
Human
AltD 3%
11% 5%
42%
AltA
18%
22%
24%
ExonS
38%
34%
25%
IntronR
41%
33%
35% 9%
Genome-wide analyses of alternative splicing in plants: Opportunities and challenges
Genome Res. 2008. 18:
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

20. differences in intron size and composition
Reddy, S.N. Annu. Rev. Plant Biol :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

21. How are AS events detected?
Splicing in disease: disruption of the splicing code and the
decoding machinery. doi: /nrg2164
High-througput detection is largely based on microarray data provided by
cDNA and EST data
PCR based assays

22. 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/

23. 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

24. 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.)

25. 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).