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Lecture 16

Lecture 16

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McGill University
Biology (Sci)
BIOL 300
Siegfried Hekimi

th BIOL 300 October 15 2012 Lecture 16 Dr. Shock As we said before, Tra is an activator protein. It binds to a SR proteins which bind to ESEs in the exon 4 of Dsx, causing splicing to occur. • Dsx is a transcriptional repressor; different version of the protein are produced in each sex; male Dsx repressed female-specific genes and female Dsx repressed male specific genes. The most complicated example is mutually exclusive splicing; an example could be found in muscle cells in the Zasp52 gene which is responsible for proper muscle contraction. The Zasp52 gene has many isoforms, there were at least 13 identified, all containing the result of a mutually exclusive splicing even between exons 7 and 8; these isoforms either have exon 7 or exon 8, but not both. This was discovered through a technique known as RTPCR, in which: • All the mRNA from the organism is isolated and reverse transcribed into cDNA; you can then test using primers to test which sequences are present in the sample mRNA. • What was observed from this when running the results on a gel were 2 bands; the smaller band would be inclusion of exon 7, the larger one would be exon 8 inclusion, but there was no third band which would represent both exons 7 and 8 being present. • These samples were later sequenced to find out what these exons’ functions were. They then did antibody staining with an antibody against the amino acids encoded by exon 8; they saw that the isoforms containing exon 8 were one found in the Z discs of specific muscle types; certain somatic types and heart muscles. • From this, we can hypothesize a function of this exon based on the similarities of these different muscle types containing exon 8. But what are the mechanisms which ensure inclusion of only one of these exons? • The simplest mechanism is an extension of the Sxl pathway; we can see this in the FGFR2 gene which encodes a growth factor receptor which we will see later. • We can see that either the red or green exon is encoded, but not both; this 1 th BIOL 300 October 15 2012 Lecture 16 Dr. Shock selection is tissue or cell-type specific: • In one tissue, you may express a factor which activates splicing of the green and represses splicing of the red, while in another tissue, another factor may be expressed which activates splicing of the red but represses splicing of the green. • In this mechanism, it is important to note that these proteins are not 100% efficient; sometimes, both (or none) or the exons may be included; what usually happens in this case is a premature stop codon which triggers nonsense-mediated decay of these proteins. Another mechanism, known as the steric interference mechanism, can be seen in the alpha-tropomyosin gene which regulates strength of muscle contraction by regulating actin-myosin interactions. • The red exon is found in smooth muscle, which regulates slow, weak contractions, while the green exon if found in other muscle types, which allow faster and stronger contractions. • What they observed is, of course, the red and green exons are never found in the same mature mRNA, and also that the U2 site of the green exon is very close to the U1 site of the red exon. • They hypothesis from this observation is that an mRNA containing both exons will not be able to bind both exon factors because they are too close together and the proteins would not fit. • This is therefore a steric interference which prevents binding of splicing factors. • To test this, they inserted a spacer sequence around the branch point, which is located between the red and green exons of approximately 100 bp, and they observed that splicing of both (or none) or the factors is in fact allowed because U1 and U2 can now fit on the final mRNA containing both the red and green exons. Yet another mechanism is called spliceosome incompatibility, which occurs for example in the human JNK1 gene, which encodes a kinase. • Contained in these exons are binding sites for special SNRNPs known as U11, related to U2, and U12, related to U2. The green exon and the red exon both bind U2 and U11 • This mechanism prevents the green and red exons from being together because U11 is not compatible with U2 (U11 is only compatible with U12 and U1 with U2) • Additional factors will then be responsible for selecting which of the
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