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Lecture 14

Lecture 14

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Biology (Sci)
BIOL 300
Siegfried Hekimi

th BIOL 300 October 10 2012 Lecture 14 Dr. Shock Splicing consists of removing introns. It was initially discovered through an experiment which hybridized mRNA to genomic DNA of the matching gene. • They isolated mRNA for a certain gene, as well as DNA from the genome; the DNA was denatured to allow hybridization with the mRNA. If there was no modification to the mRNA, there should be perfect hybridization, but instead, they saw some double stranded region with large single stranded loops of genomic DNA which had no matching regions in the mRNA to bind to. • Therefore, what they were able to conclude from this was that not all genomic components of the gene were contained in the final mRNA. We know now that these regions are introns, which are absent in the mature mRNA. This was the initial evidence for splicing. • The poly A tail is a region of mRNA hanging off the double stranded region because there is no matching area on the genomic DNA; this is because the poly A tail is added post-transcriptionally. • This process is very labor intensive; now, we can identify introns by using sequence analysis comparison of cDNA on genomic DNA to find the splice site junctions. Now, there are full cDNA libraries of various genomes which can be compared to genomic DNA to figure out quickly where these exon-intron junctions are. • Following finding the location of these junctions, you can figure out exactly which sequences and how well these sequences are conserved within different genes of the genome, as well as between genomes. This is a zoomed in copy of two exon- intron junctions, one at either end of the intron (in blue). Again, there are cis acting factors, which are RNA sequences required in the primary RNA transcript for splicing, as well as trans acting factors, which are the splicing machinery proteins. Cis acting factors: • There are 5 conserved regions: • GU are within the 5’ intron border, and an AG on the 3’ intron border. • The other conserved nucleotide is the branch point, which is an A, involved in the splicing reaction itself. Initially, this A is spliced and attached to the 5’G to form a lariat; the now free G in a second reaction will interact with the 3’G to correctly splice out the intron completely. • There are also a few other nucleotides surrounding the splice junctions and around the splice site which are highly conserved 1 th BIOL 300 October 10 2012 Lecture 14 Dr. Shock There are two chemical reactions in the splicing mechanism: • The branch point interacts with the 5’ G to form a lariat • This frees up the 5’ G to interact with the 3’G to completely excise the intron from the mRNA There are also splicing factors which are small RNAs (not proteins), called snRNAs, which are responsible for catalyzing the splicing mechanism. This is carried out through the snRNA’s complementarity to the various intron-exon sites mentioned before: • U1 snRNA complements the 5’ border while the U2 snRNA is complementary to the region around the branch point. • Note that the branch point itself is not hybridized to any of the splicing factors allowing the adenine to be free and chemically reactive. Only the neighboring 2 nucleotides are hybridized to the U2 snRNA which causes the A to stick out. • This was discovered through mutation of these important consensus sequences in axons to prevent base pairing of either U1 or U2 snRNA and the entire splicing mechanism falls apart. • The most definitive proof that snRNA binding is necessary for splicing is through a compensatory mutation, in which: • First, when you mutate only 1 nucleotide to an A on the mRNA, there is no splicing because U2 snRNA can’t bind to the mRNA consensus site • However, when you mutate the U2 snRNA to have a U at this position, it restores the function because there can be proper binding again and the splicing function is restored. Proteins are al
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