GDCB 511-4-3-09 REVIEW0

GDCB 511-4-3-09 REVIEW0 - 14-1GDCB 511: Friday 3 April...

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Unformatted text preview: 14-1GDCB 511: Friday 3 April 2009Review:Chapter 14: Splicing etc.Chapter 15: Capping, PolyAdenylationChapter 16: RNA editing; trans-splicing; post-transcriptional regulation; RNAi; microRNAChapter 17: Iron-regulation of Ferritin expressionExon/intron junctions are defined by the GU-AG rule:A typical intron:5-AG/GU . intron . AG/-3' (donor) (acceptor)Yeast:5-AG/GUAUGU . intron .UACUAAC-YAG/-3(branch site)Mammals:5-AG/GUAAGU. intron .YNCURAC-YnNAG/G-3Y= U or C; R= A or G; Yn= ~9 pyrimidines Minor class introns: (ATAC introns): Have AU..AC instead of GU..AGHow can you find the Splicing Signals?Mechanism of intron excisionHow does splicing happen?Splicing occurs in 2 transesterification reactions in which a OH attacks the RNA phosphodiester backbone:1. 2' OH of branch-site "A" attacks 5' splice donor site, forming a lariat (loop) structure. This contains a 5' - 2' bond connecting the intron 5'-G to the 2' A at the branch site within the intron.2. The OH from upstream exon attacks the phosphodiester bond between intron and exon at 3' splice junction. This ligates the two exons.So how do the snRNPs promote splicing?They help to position three reacting sites in precursor mRNA:5 splice donor siteBranch point sequence3 splice acceptor siteBase-pairing between snRNAs and the pre-mRNA sequences are critical.Overview of snRNA associationsU1 base pairs with the 5 splice siteU2 base pairs with the conserved sequence at the splicing branchpointU4 base pairs with U6 U6 base pairs with the 5 splice site; U6 also base pairs with U2U5 associates with the last nucleotide in one exon and the first nucleotide of the next, presumably lining up the exons for splicingGenetic tests of predicted Yeast U2 Base Pairing with Branchpoint SequenceSource: Adapted from Parker, R., P. G. Sliciano, and C. Guthrie, Recognition of the TACTAAC box during mRNA splicing in yeast involves base pairing to the U2-like snRNA. Cell 49:230, 1987.Are introns removed in a specific order from a particular RNA?(e.g., from one end of the mRNA to the other?)Ovomucoid precursor RNA: 5.5 kb, 7 intronsNorthern blotting of nuclear RNA gives predominant bands indicating that introns are removed in the following order: 5 and 6; then 4 and 7; then 2; then 1 and 3.Therefore: 1. Splicing does not occur randomly, but through a pathway of predominant intron removal.2. Splicing does not occur sequentially from one end of the RNA to the other.(what is the caveat to this study?)Splicing is Essential: most eukaryotic genes contain introns that interrupt the ORF. Splicing is required to restore the ORF and generate functional proteins. Hence, splicing is difficult to study genetically (mutants are lethal)....
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This note was uploaded on 03/08/2010 for the course GDCB 511 taught by Professor Yanhaiyin during the Spring '09 term at Iowa State.

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GDCB 511-4-3-09 REVIEW0 - 14-1GDCB 511: Friday 3 April...

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