Alternative Splicing

mRNA Splicing

During RNA processing internal segments are removed from the transcript and the remaining segments spliced together.

Internal RNA segments that are removed are named introns; the spliced segments are defined as exons.

Splicing prevalent process in eukaryotes (not 100%, though)

Language erosion: 1-exon genes

Exon ExonIntron

Pre-m

RNA

5’ Splice Site

3’ Splice Site

Reddy, S.N. Annu. Rev. Plant Biol. 2007 58:267-94

Of 1588 examined predicted splice sites in Arabidopsis1470 sites (93%) followed the canonical GT…AGconsensus. (Plant (2004) 39, 877–885)

Canonical splice sites

Multiple splice variants produced from the same gene

An example from A. thaliana

Not a rare event!!!

Alternative Splicing

Removing different segments from mRNAs leads to alternative splice forms of a gene/transcript.

Can occur in any part of the transcript including UTRs and can alter start codons, stop codons, reading frame, CDS, UTRs.

May alter stability-life, translation (time, location, duration), protein sequence, some or all of the above.

Alternative splice forms = Protein isoforms

Contributes to protein diversity

Degree of alternative splicing varies with species

DNALC Clip: http://dnalc.org/resources/3d/

The dogmas – they are a~changing…

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

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

How are AS events detected?

• Based on cDNA and EST data• Alignment against genome sequence• High-throughput RNA-seq• PCR based assays

Alternative splicing in metazoans

• Alternative splicing well characterized in animals.• As many as 96% of human genes may have multiple splice forms.• Functional significance of alternative spicing still poorly understood.

Alternative splicing in animals. Nature Genetics Research 36; 2004

Bridging the gap between genome and transcriptome Nucleic Acids Research 32, 2004.

Splice statistics for human genes

RuBisCo alternative splicing one of first plant examples:

“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….”

Alternative splicing in plants

Biological significance of AS in plants

…includes:- regulation of flowering;- resistance to diseases;- enzyme activity (timing, duration, turn-over time, location).

Most genome databases give alternatively spliced plant gene variants

Example: Disease resistance in tobacco

- Nicotiana tabacum resistance gene N involved in resistance to TMV.- Alternative splicing required to achieve resistance.- Alternative transcripts Ns (short) and NL (long).- NS encodes full-length, NL a truncated protein.- Splicevariants produced by alternative splicing confer resistance (D).- Splicevariants produced by cDNAs do not confer resistance (A, B, C).

ii

Example: Jasmonate signaling in Arabidopsis

- Plant hormone; affects cell division, growth, reproduction and responses to insects, pathogens, and abiotic stress factors.

- Jasmonate Signaling Repressor Protein JAZ 10 splice variants JAZ 10.1, JAZ 10.3 and JAZ 10.4 differ in susceptibility to degradation.

- Phenotypic effects include male sterility, altered root growth.

Example: Jasmonate signaling in Arabidopsis

- Alternative splice sites C’ and D’ lead to different splice variants- JAZ10.3: premature stop codon in D exon, intact JAS domain- JAZ10.4: truncated C exon, protein lacks JAS domain- JAZ 10 encoded by At5G13220

Sequence & course material repository

http://gfx.dnalc.org/files/evidenceDon’t open items, save them to your computer!!

• Annotation (sequences & evidence)• Manuals (DNA, Subway, Apollo, JalView)• Presentations (.ppt files)• Prospecting (sequences)• Readings (Bioinformatics tools, splicing, etc.)• Worksheets (Word docs, handouts, etc.)• BCR-ABL (temporary; not course-related)