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Lecture 15

Lecture 15

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Biology (Sci)
BIOL 300
Siegfried Hekimi

th BIOL 300 October 12 2012 Lecture 15 Dr. Shock So how to SR proteins bind only to sequences within the exon? Since every exon is different, how do they contain sequences for multiple SRs? • One reason could be that the consensus sequences are very small, so even in very different proteins, there can be one or two identical amino acids corresponding to the ESE sequence A computational study was carried out to try and find these ESE sequences which are conserved in mice and humans and are independent of the coding potential • Remember, one amino acid can be encoded by multiple codons: • E.g. in a serine, the codon is UCx, where the only important nucleotides to have are the U and C, the x can be ay nucleotide • Also, ACx gives threonine and GGx gives glycine. • Therefore, this third position should not affect the amino acid produced, and should therefore be allowed to evolve freely; if there was conservation, this would mean that there is some other significance to this sequence than just being a codon They then tried to find more frequent conservation of codon pairs (snRNAs need about 6 nucleotides to bind to snRNAs, so looking for 2 codons in a row would produce a good snRNA binding motif) than expected by normal amino acid variation; this would hint towards this sequence having a function in splicing • From this, they identified >250 hexamers which fit both criteria above, and then they demonstrated their function in splicing through mutational analysis in which removing them alters splicing function. • There is no single ESE sequence to which SR proteins bind; these is a whole class of SR proteins which can bind to various ESE sequences which must be identified separately. This is a schematic of the human SMN locus responsible for a sever human disease; learning about the splicing of these genes led to development of therapeutic techniques. 15% of human diseases are caused by improper splicing. • This disease is called spinal muscle atrophy, in which motor neuron death in the spinal cord can lead to stopping of muscle function The human SMN (survival of motor neuron) genes, SMN1 and SMN2, are located in close proximity on chromosome 5. They encode the same protein (same codon sequence) and one presumably came from the other from a duplication event during evolution. When they are mutated or incorrectly spliced, these neurons die and these people get the disease. • The SMN genes comprise nine exons and eight introns and encode an identical protein product. During evolution, a silent C–T transition in exon 7 of SMN2 alters a critical exonic splice 1 th BIOL 300 October 12 2012 Lecture 15 Dr. Shock enhancer (ESE) and results in a strong reduction of exon 7 inclusion during splicing, leaving it out of the final protein product. • Consequently, ∼85% of the mature mRNA lacks exon 7 (Δ7), highlighted by the RT–PCR in the bottom panel. In the properly spliced model, you would only have proper SMN, while in the incorrectly spliced model, you have a thin “functional” band and a much thicker lighter band representing the truncated protein. • The truncated protein is defective in SMN self-association and is degraded rapidly. • All of what we talked about happens in NORMAL people; even if SMN2 is not there, there is enough SMNI to carry out a normal function. Spinal muscle atrophy consists of a mutation in the SMN1 gene, for example, in a stop codon (though it could be in any codon). This causes no functional SMN1 product and therefore, you do not have enough functional protein and you have the disease. • Even though there is still some SMN2 in the body, because of the normal mutation in the SMN2 gene, only about 15% of it is functional, and this is not enough to live a healthy adulthood. • Many human diseases are the result of subtle mutations, this is because larger and more significant mutations could not even make it past the embyronic stage of development. This is a schematic of the exon 7 region and the factors involved in the inclusion or exclusion of exon 7 within SMN pre-mRNA. • Components of the machinery are shown in blue. The positively acting sequences and splicing factors are shown in green. The negatively acting sequences and splicing factors are shown in red. • The Tra protein here is an SR protein which binds to an ESE of exon 7. It is responsible for the recruitment of a bunch of other factors, and finally U1 and U2. • The C–T transition at +6 in the SMN2 gene prevents binding of the Tra protein and thus prevents recruitment of the rest of the splicing machinery. Instead, the ESE which contained the C now becomes an ESS which binds a splicing silencer and recruits other silencing proteins, leading to exon 7 not being included in the final protein. Generally, the SMN1 defect is incurable, so instead, as a therapeutic technique, we can try to shift the balance in the amount of properly spliced SMNII • Somehow, we have to raise the 15% of functional product and reduce the 85% of non-functional product as a result of the SMNII gene. This will hopefully either cure the disease, delay its onset, or alleviate the symptoms. We will talk about three strategies of how scientists tried to do this; they all involve oligonucleotides which are complementary to sequences used by the splicing machinery. 2 th BIOL 300 October 12 2012 Lecture 15 Dr. Shock The first strategy is to introduce oligonucleotides to the 3’ splice site of exon 8, reducing binding of U1SNRNA can no longer bind, so that more of exon 7 is spliced, and less of exon 8 (i.e. shift the balance towards exon 7) • A possible problem with this strategy is that through excluding exon 8, you can introduce the same problem as before, where exon 8 begins to be left out of all the final product. However, it is important to note that the stop codon of the SMNII gene is in exon 7, and therefore even if exon 8 is truncated, it won’t make a difference on the final protein product. • Even if we do get complete exon 7 inclusion, you would still miss the polyadenylation site in the pre- mRNA because of exclusion of exon 8, and this may decrease the stability of mRNA and decrease translation. • However, this has been tri
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