Of Mice and Men....              John Steinbeck (1937) 

        Alternative Splicing of Eukaryotic Genes

  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)
          But, humans still make about 90,000 proteins.     How from only 25,000 genes????
   In 2004 the mouse genome was sequenced and we learned it also has 25K genes (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





- Philip Sharp (MIT) & Richard Roberts (NE Biolabs) discover split genes & the presence of
                introns via DNA-RNA hybridizations and an excision splicing process*.
1980 - Randolph Wall (UCLA) discovers alternate splicing; i.e., some introns left in or exons cut out
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.
      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      
         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
                                       figure*      and         figure*       and     figure C17.11*
         Exons also hold repressor nucleotide sequences [ESS] - Exonic Splicing Suppressor
Next         - when an SR binds @ an ESS it prevents Spliceosome machinery from splicing



Splicing Examples:
   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

   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 

   3. tropomyosin -  via exon skipping different versions of tropomyosin are produced for skeletal
                muscle, smooth muscle, fibroblast cells, live and brain cells.               






Human Genes and Splicing

       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
    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


    back 150 molecular genetics













    These are examples of EXON SKIPPING* - most common form of Alternate Splicing in mammals
    INTRON RETENTION, changing the length of a processed mRNA, is the most common form
        of alternate splicing in plants & lower multicell organisms