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【正文】 p) when RNA is only 2530 bp long ? Cap structure is recognized by CBC（ capbinding plex） ? stablize the transcript ? prevent degradation by exonucleases ? stimulate splicing and processing Sometimes methylated Sometimes methylated ? The cap is added after the nascent RNA molecules produced by RNA polymerase II reach a length of 2530 nucleotides. Guanylyltransferase is recruited and activated through binding to the Ser5phosphorylated Pol II CTD. ? The methyl groups are derived from Sadenosylmethionine. ? Capping helps stabilize mRNA and enhances translation, splicing and export into the cytoplasm. Capping of the 5’ end of nascent RNA transcripts with m7G Existing in a single plex Consensus sequence for 3’ process AAUAAA: CstF (cleavage stimulation factor F) GUrich sequence: CPSF (cleavage and polyadenylation specificity factor) Polyadenylation of mRNA at the 3’ end CPSF: cleavage and polyadenylation specificity factor. CStF: cleavage stimulatory factor. CFI amp。 CFII: cleavage factor I amp。 seconds if seldom needed ? t189。 2022 The Nobel Foundation Background: Adenovirus has a DNA genome and makes many mRNAs. Can we determine which part of the genome encodes for each mRNA by making a DNA:RNA hybrid? Experiment: Isolate Adenovirus genomic DNA, isolate one adenovirus mRNA, hybridize and then look by EM at where the RNA hybridizes (binds) to the genomic DNA. Surprise: The RNA is generated from 4 different regions of the DNA! How can we explain this? Splicing!! The discovery of split genes (1977) 1993 Noble Prize in Medicine To Dr. Richard Robert and Dr. Phillip Sharp The matured mRNAs are much shorter than the DNA templates. DNA mRNA Exon and Intron ? Exon is any segment of an interrupted gene that is represented in the mature RNA product. ? Intron is a segment of DNA that is transcribed, but removed from within the transcript by splicing together the sequences (exons) on either side of it. Exons are similar in size Introns are highly variable in size GTAG rule ? GTAG rule describes the presence of these constant dinucleotides at the first two and last two positions of introns of nuclear genes. ? Splice sites are the sequences immediately surrounding the exonintron boundaries ? Splicing junctions are recognized only in the correct pairwise binations The sequence of steps in the production of mature eukaryotic mRNA as shown for the chicken ovalbumin gene. The consensus sequence at the exon–intron junctions of vertebrate premRNAs. 4 major types of introns 4 classes of introns can be distinguished on the basis of their mechanism of splicing and/or characterisitic sequences: – Group I introns in fungal mitochondria, plastids, and in prerRNA in Tetrahymena (selfsplicing) – Group II introns in fungal mitochondria and plastids (selfsplicing) – Introns in premRNA (spliceosome mediated) – Introns in pretRNA Group I and II introns The sequence of transesterification reactions that splice together the exons of eukaryotic premRNAs. Splicing of Group I and II introns ? Introns in fungal mitochondria, plastids, Tetrahymena prerRNA ? Group I – Selfsplicing – Initiate splicing with a G nucleotide – Uses a phosphoester transfer mechanism – Does not require ATP hydrolysis. ? Group II – selfsplicing – Initiate splicing with an internal A – Uses a phosphoester transfer mechanism – Does not require ATP hydrolysis Selfsplicing in prerRNA in Tetrahymena : T. Cech et al. 1981 Exon 1 Exon 2 Intron 1 Exon 1 Exon 2 Intron 1 + prerRNA Spliced exon Intron circle Intron linear prerRNA Nuclear extract GTP + + + + + + + + ?Products of splicing were resolved by gel electrophoresis: Additional proteins are NOT needed for splicing of this prerRNA! Do need a G
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