Alternative SplicingA 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

1940’s -------------------------------------------------------------------------------2000’s

Ensembl- What is a gene, post-ENCODE? History and updated definitionGenome Res. 2007. 17: 669-681

Alternative splicing in metazoans

• 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

Estimating rates of alternative splicing in mammalsand 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

Human splicing statistics

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

E1 E2 I2 E4E3I1

E1 E2 E3 E4

Pre-m

RNA

Spliced m

RNA

Genom

e

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

E1 E2I1

Pre-m

RNA

5’ Splice Site

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)

I1

Pre-m

RNA

5’ Splice Site

3’ Splice SiteE1 E2

m7G

UTR UTRAAA...AA

Mature

mRN

A

- Alternative splicing can effect the entire pre-mRNA transcript (UTRs included)

ATG ATG S S

- Alternative splicing can also alter start codons or lead to premature termination codons

E2 UTRE1UTR

There are 5 main types of splicing

Constitutive (familiar/ “normal”) Alternative Donor site Alternative Acceptor site Alternative position Exon Skipping Intron retention

E1 E2

m7G

UTR UTRAAA...AA

E1 E2 I2 E4E3I1

E1 E2 E3 E4

Constitutive splicing

Pre-m

RNA

Spliced m

RNA

Genom

e

E1 E2I1

E1 E2I1E1

E1 E2

Alternative donor site (AltD)

Pre-m

RNA

Pre-m

RNA

Spliced m

RNA

E1 E2I1

E1 E2I1 E2

E1 E2

Alternative acceptor site (AltA)

Pre-m

RNA

Pre-m

RNA

Spliced m

RNA

E1 E2I1

E1 E2

E1 E2

I1I1

Alternative Position (AltP)

Pre-m

RNA

Pre-m

RNA

Spliced m

RNA

E1 E3

E1 E3

I1 I2E2

I1 I2E2

E1 E3

Exon skipping (ExonS)

Pre-m

RNA

Pre-m

RNA

Spliced m

RNA

E1 E2I1

E1 E2I1

E1

Intron retention (IntronR)

Pre-m

RNA

Pre-m

RNA

Spliced m

RNA

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 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 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

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

Alternative splicing is far less common in plants

- 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

How are AS events detected?

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• 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).