1. Study Guide/Outline—RNA Processing RNA Processing: rRNA genes, tRNA genes, self-splicing, spliceosomal Structural genes Pre-RNA Processing What kind of processing must occur to pre-mRNA in eukaryotes? Where does this take place? What happens to the RNA molecule if it does not undergo processing? Purpose of each RNA processing step –What is a methyl guanine “cap”? –How is a polyA tail added? How does this tail contribute to the stability of the mRNA? How are nucleotides numbered in genes with exons and introns? Spliceosomes What is a spliceosome and what class of genes use spliceosomes? What consensus sequences are needed in introns in order for correct splicing to occur? What would happen if there was a mutation in a splice site consensus sequence? What is the significance of the lariat structure in splicing out introns?

2. mRNA and protein synthesis are coupled in bacteria In eukaryotes mRNA must be processed and transported out of nucleus for translation iGenetics, 1 st ed. Russell

3. Prokaryotes vs. Eukaryotes Prokaryotic Polycistronic (one promoter, multiple genes) Introns thought to be non- existent in prokaryotes until very recently Transcription and translation can occur concordantly Exceptions: archaebacteria, bacteriaphage (virus), mitochondria, chloroplasts Eukaryotic Monocistronic (one promoter, one gene) Introns are common High amounts of “junk DNA” in genome. RNA requires significant processing Size of introns is roughly correlated with complexity of the organism.

4. Structure of the methylguanine cap

6. 1 2 3 4 5 O O O 1 2 3 4 5 1 2 3 4 5

7. 14 - 47 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. RNA polymerase II transcribes a gene past the polyA signal sequence. The RNA is cleaved just past the polyA signal sequence. RNA polymerase continues transcribing the DNA. PolyA signal sequence 3′ 5′ 3′ 5′ 3′ Allosteric model: After passing the polyA signal sequence, RNA polymerase II is destabilized due to the release of elongation factors or the binding of termination factors (not shown). Termination occurs. Torpedo model: An exonuclease binds to the 5′ end of the RNA that is still being transcribed and degrades it in a 5′ to 3′ direction. 3′ 5′ 3′ 5′ Exonuclease catches up to RNA polymerase II and causes termination. Exonuclease 3′ 5′ Figure 14.15

8. 5′ 3′ 5′ 3′ Endonuclease cleavage occurs about 20 nucleotides downstream from the AAUAAA sequence. PolyA-polymerase adds adenine nucleotides to the 3′ end. Polyadenylation signal sequence AAUAAA PolyA tail AAAAAAAAAAAA.... Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Brooker Figure 14.22 Consensus sequence in higher eukaryotes

9. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Termination of RNA synthesis in (eukaryotic) RNA Pol II Regular transcript 3’ UTR Site of cleavage and addition of polyA tail

10. Animation of cap addition and poly-A tail addition http://vcell.ndsu.nodak.edu/animations/

11. Length of poly-A Tail and Stability (half-life of mRNA) Prokaryotic Degradation at 5’ end begins immediately (before 3’ transcription is completed) Eukaryotic cFOS mRNA (cell cycle gene) Half-life: 10-30 min Short poly-A tail HemoglobinHalf-life: 24 hoursLong poly-A tail

12. Major types of introns Type of intronGene typeSplicing Mechanism tRNAs and rRNAs tRNA genes Enzymatic Nuclear (pre- mRNA) Protein-encoding genes in nuclear chromosomes Spliceosomal Group I Some rRNA genes Self-splicing Group II Protein-encoding genes in mitochondria Self-splicing

13. Promoter Transcription Cleavage (the light pink regions are degraded) 45S rRNA transcript 18S5.8S28S 5′3′ 18S5.8S28S 18S5.8S28S rRNA Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Functional RNAs that are key in ribosome structure This processing occurs in the nucleolus Brooker Figure 14.16 Processing of ribosomal RNA

14. 3′5′ Endonuclease Exonuclease (RNaseD) P = Pseudouridine T = 4-Thiouridine IP = 2-Isopentenyladenosine Endonuclease (RNaseP) T T A P P IP C C Anticodon m G = Methylguanosine mGmG Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Brooker Figure 14.17 Covalently modified bases Processing of tRNAs

15. RNA-DNA hybrid reveals intron sequences as they “loop out”

16. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Brooker Figure 14.19 3′ 5′ 3′ splice siteBranch site IntronExon UACUUAUCC Py 12 N Py AGG A / C GGU Pu AGUA 5′ splice site Splice site consensus sequences

17. Rough overview of splicing mechanism (formation of lariat structure)

18. Mechanism of Spliceosome

19. U1 3′5′ 5′ splice site3′ splice site Branch site A GU Exon 1Exon 2 U1 binds to 5′ splice site. U2 binds to branch site. AG 3′ 5′ A U4/U6 and U5 trimer binds. Intron loops out and exons are brought closer together. U1 snRNPU2 snRNP 3′ 5′ A U5 snRNP U4/U6 snRNP U2 Intron loops out and exons brought closer together Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Brooker, Fig 14.20 Mechanism of Spliceosome (Brooker)

20. U1 U4 3′ 5′ 3′5′ 5′ splice site is cut. 5′ end of intron is connected to the A in the branch site to form a lariat. U1 and U4 are released. 3′ splice site is cut. Exon 1 is connected to exon 2. The intron (in the form of a lariat) is released along with U2, U5, and U6 (intron will be degraded). A A U5 U6 U5 U6 U2 Intron plus U2, U5, and U6 Two connected exons Exon 1 Exon 2 U2 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Brooker, Fig 14.20 Intron will be degraded and the snRNPs used again Mechanism of Spliceosome (Brooker), cont.

21. Rare mutations in intron sequences can cause a phenotype (e.g. in the large gene, Dystrophin) Duchenne’s Muscular Dystrophy (X-linked) Caused by mutations in Dystrophin gene: 2+ million nt gene (many exons) Relatively high incidence (1/3500 males) due to large size of gene and hemizygosity in males

22. Advanced Duchenne’s Muscular Dystrophy Fig from Medical Genetics, Jorde et al., 3 rd ed.

23. Major types of introns Type of intronGene typeSplicing Mechanism tRNAs and rRNAs tRNA genes Enzymatic Nuclear (pre- mRNA) Protein-encoding genes in nuclear chromosomes Spliceosomal Group I Some rRNA genes Self-splicing Group II Protein-encoding genes in mitochondria Self-splicing

24. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Brooker Fig 14.18a and b H H OH H G G G O CH 2 OH H OH H O CH 2 OH Guanosine HH O OH H CH 2 O O P P A Self-splicing introns (relatively uncommon) Exon 1 Intron Exon 2Exon 1 Intron Exon 2 Guanosine binding site G G P HH O O H CH 2 O O P P P A 3′ OH 3′ 5′3′5′ OH G G HH O O H CH 2 O O P P A RNA 5′ (a) Group I(b) Group II 3′ 2′ 3′ Self splicing Introns

25. Go over lecture outline at end of lecture