Transcript Splicing - iPlant Pods

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
Canonical splice sites
5’ Splice Site
Intron
Exon
3’ Splice Site
Pre-mRNA
Exon
Reddy, S.N. Annu. Rev. Plant Biol. 2007 58:267-94
Of 1588 examined predicted splice sites in Arabidopsis
1470 sites (93%) followed the canonical GT…AG
consensus. (Plant (2004) 39, 877–885)
An example from A. thaliana
Multiple splice variants produced from the same gene
Alternative Splicing
DNALC Clip: http://dnalc.org/resources/3d/
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
The dogmas – they are a~changing…
One gene, one enzyme
One gene, one polypetide
One gene, one set of transcripts
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
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?
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Based on cDNA and EST data
Alignment against genome sequence
High-throughput RNA-seq
PCR based assays
Alternative splicing in metazoans
Splice statistics for human genes
Alternative splicing in animals. Nature
Genetics Research 36; 2004
Bridging the gap between genome and
transcriptome Nucleic Acids Research 32, 2004.
• 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 plants
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….”
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
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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).
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
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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/evidence
Don’t open items, save them to your computer!!
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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)