6 주차 수업자료

1. 6주차 수업자료

2. Chapter 12. Processing of RNA

3. Types of RNA processing • “RNA world” hypothesis • Coding RNA • mRNA • Non-coding RNA • Ribosomal RNA • Transfer RNA • tmRNA (prok) • Small nuclear or snRNA (U-RNA, euk) • Small nucleolar or snoRNA (U-RNA, euk) • Small cytoplasmic or scRNA • Base modification • Cleavage • Splicing • Capping and tailing (euk)

4. rRNA and tRNA processing in prokaryote Endoribonuclease (RNase III, P, F) • Pre-tRNA processing by RNase E/F, D, P • RNase P: ribozyme, cleaves 5’-end of tRNA No pre-5S rRNA processing is required in eukaryote because it is made separately. Pre-18S, 28S, 5.8S rRNA processing is required as in bacteria.

5. Splicing and maturation of eukaryotic mRNA • Cap • Poly(A) tail • Splicing out intron

6. Capping of eukaryotic mRNA • First step in mRNA maturation • Occurs inside of nucleus • Cap by GTP • +methyl group in G: cap0 • +methyl group in base #1: cap1 • +methyl group in base #2: cap2

7. Adding a poly(A) tail to eukaryotic mRNA • Next (second) step in mRNA maturation • AAUAAA tail recognition sequence is required at 3’-end • RNA pol bypass AAUAAA • Polyadenylation complex assembly • CPSF: binds to AAUAAA • PolyA polymerase • PABP • CST: binds to GU-rich tract • Cleavage of RNA by CPSF/CST at 10-30 bases downstream (right after CA dinucleotide) and polyA (100-200 A) addition by PolyA polymerase • PABP remains bound • Poly(A) tail is required for translation

8. Experimental method: how to isolate eukaryotic mRNA?

9. Removal of intron by splicing #1 • Usual intron: GT…AG • Spliceosome: 5 snurps (small nuclear RNA + protein = small nuclear ribonucleoprotein, snRNP), U1, U2 and U2AF (accessory factor), U4, U5, U6 • snRNAs recognize 5’ splice site (by U1), 3’ splice site (by U2AF), and branch site (by U2) on pre-mRNA by base-pairing.

10. Removal of intron by splicing #2 Lariat structure  degrade • Splicing reaction • Cut at 5’ splice site  Free 5’-end (5’-P) of intron forms loop and joins to A (2’-OH) at branch site • Cut at 3’ splice site  Free 3’-end (3’-OH) of upstream exon joins to 5’-end (5’-P) of downstream exon • Spliceosome assembly

11. Self-splicing of group I and II intron • Self-splicing: by ribozyme activity, no proteins are required • Group I: nucleophilic attack of 5’-splice site (5’-P at 5’-end of intron) by soluble free guanosine or guanosine nucleotide (3’-OH)  Free exon 3’-OH reacts (nucleophilic attack) with 3’-splice site (5’-P at 5’-end of exon) • Group II: nucleophilic attack of 5’-splice site (5’-P at 5’-end of intron) by internal adenosine (2’-OH)  Free exon 3’-OH reacts (nucleophilic attack) with 3’-splice site (5’-P at 5’-end of exon)

13. R-loop analysis to visualize introns

14. Alternative splicing: promoter selection • Two alternative promoters by cell-type specific transcription factors

15. Alternative splicing: tail site selection • Two alternative tail site, used for antibody production

16. Alternative splicing: exon cassette selection • Genuine choice between actual splicing sites

17. Alternative splicing: trans-splicing • Rare • From two different primary transcripts • Observed in Trypanosomes (parasite, single-cell eukaryote)

18. Protein splicing: inteins and exteins • Rare • Intein: like intron • Extein: like exon • Intein splicing requires no accessory enzymes, but requires specific aa (as shown in the figure) at extein/intein boundaries, and a branched intermediate

19. Inteins: mechanism

20. Bizarre role of the intein • dnaE gene: two inteins • Intein mostly works as site-specific DNase (cuts DNA only if intein is deleted) • Intein deletion in bacteria: previously formed intein DNase cuts cell’s DNA  lethal • Intein deletion in eukaryote  intein DNase cuts DNA  can be repaired by “gene conversion”

21. Base modification of rRNA by guide RNA (or snoRNA) • Recognition of modification site in rRNA by base pairing (often nonstandard G-U base pairing observed) with snoRNA • Modification • Methylation of 2’-OH of ribose • Pseudo-uridylation • snoRNA is encoded in the intron

22. RNA editing: alteration of base sequence • CU editing by deaminase + accessory proteins • Liver: no editing  ApoB100 • Intestine: editing  ApoB48 • Other editing possible • RNA editing by insertion or removal of bases in trypanosome (helped by guide RNA)

23. Nuclear export of eukaryotic mRNA • mRNA can exit nucleus through nuclear pore, after capping, poly(A) addition, and splicing • Spliceosome prevents exit of RNA

24. Nuclear pore complex

25. Degradation of mRNA • Prokaryote • Endonuclease (RNase E) • Exonuclease (3’  5’) • Overall 5’  3’ degradation • Eukaryote • Shortening of poly(A) tail to 10-20 bases by exonuclease and PABP release  Cap removal by Dcp1 (decapping protein)  Exonuclease Xrn1 (5’  3’) • Destabilization of mRNA by ARE (AUUUA repeat element) in 3’-UTR & ARE-binding protein  Poly(A) RNase  Endonuclease

26. Nonsense-mediated decay (NMD) of eukaryotic mRNA • Non-sense mutation: stop codon • NMD • protective role to prevent premature termination • Triggered whenever there is a stop codon more than 50-55 nt upstream of the final exon-exon junction • Labeling of exon-exon junction by exon junction complex (EJC) binding to mRNA about 20-24 nt upstream of each exon-exon junction • Upf3 binds to EJC • Nuclear export • Upf2 binds to EJC • During translation, ribosome displaces EJC • If there is a premature stop codon, ribosome stops  RF+Upf1 interact with EJC • Cap removal  Degradation from 5’-end by exonuclease