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Of Mice and Men... John Steinbeck (1937)
Alternative Splicing of Eukaryotic Genes |
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| At the
beginning of the 3rd millennium, the estimates of the
number of
human genes was ≈
153,000 making about 90,000 proteins; by the first draft of the Human Genome Sequence (summer of 2002) the number had shrunk to ≈ 30-35,000 protein coding genes. The current estimates of the NHGRI puts the number of human genes at less than 25,000. (& there is actually a betting pool = 21,787) |
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| But, humans still make about 90,000 proteins. How from only 22,000 genes???? | |
| In 2004 the
mouse genome was sequenced and we
learned it also has
25K genes (about the same as man)
and we both share many of the
same exons and
introns. If
Mice and Men are so genomically similar, what makes so vastly different? ---> ALTERNATIVE SPLICING |
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1997 - Philip Sharp (MIT) & Richard Roberts (NE Biolabs) discover split genes & the presence of introns via DNA-RNA hybridizations and an excision splicing process*. |
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| 1980 - Randolph Wall (UCLA) discovers alternate splicing; i.e., some introns left in or exons cut out | |
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1984 - Tom Maniatis &
Mike Green (Harvard)
describe a Splicing Machine =
Splicesome* a highly conserved complex of: 5 small nuclear uridine rich RNA molecules (snRNA - U1, U2, U4, U5, & U6) 150 proteins - can recognize exon/intron interfaces & excise the introns. |
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Splicesomes cut @ exon/intron
interfaces - short nucleotide
sequences 1. 5' splice site 2. 3' end a) branch site b) polypyrimidine site c) 3' splice site |
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| Cutting involves
Regulatory Proteins called
SR
(Splicing Regulator) proteins: - there are some 10 known different SR proteins identified so far - they bind to an Exon nucleotide sequence called Exonic Splicing Enhancers [ESE] - SR binding recruits the Spliceosome Basal Machinery to 5' Splice Site |
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| figure* and figure* and figure C17.11* | |
| Exons also hold repressor nucleotide sequences [ESS]
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Exonic Splicing Suppressor Next - when an SR binds @ an ESS it prevents Spliceosome machinery from splicing |
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| Splicing Examples: | |
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1.
Bcl-x is a gene making a
regulator protein for programmed cell death -
apoptosis gene makes 2 proteins via alternative splicing - a. Bcl-x (L) [larger] -> suppresses apoptosis b. Bcl-x (S) [smaller] -> promotes apoptosis figure* |
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| 2.
sxl
(sex lethal gene of Drosophila) When male sxl (exon-2) is skipped during splicing the gene makes a female specific sxl-protein (figure*) - this protein binds to all subsequent premRNA of same gene resulting in excision of the male exon form all mRNA's --> female flies if male exon is retained in 1st round of splicing --> male sxl protein --> male flies |
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3. tropomyosin - via exon skipping different versions of tropomyosin are produced for skeletal muscle, smooth muscle, fibroblast cells, live and brain cells. figure* |
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Human Genes and Splicing - |
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Average human protein coding gene is about 28,000 nucleotides long with some 8.8 exons, each about 120 nucleotides long separated by some 7.8 introns ranging is size from 100 to 100,000 nucleotides |
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| µ On average -each human gene generates 3 alternately spliced mRNA's | |
| Thus human exons make up only between 1% and 2% of the entire human genome | |
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