Biol/Chem 473

1. Biol/Chem 473 Schulze lecture 3: Eukaryotic RNA processing

2. Central Dogma: information flow

3. Central Dogma: information flow reverse transcriptase

4. Prokaryotic vs. eukaryotic gene regulation

5. Compartmentalizaton

6. Gene regulation in eukaryotes is compartmentalized

7. Splice out the introns Finish the job Add a cap consisting of a modified guanine nucleotide Final mRNA product

8. Add a cap consisting of a modified guanine nucleotide Final mRNA product

9. 5’ cap CH3

10. “mRNA factory” concept

11. “mRNA factory” concept Unique to RNA pol II

12. Why a cap? • RNA’s transcribed by the other polymerases don’t have caps, partly because the other RNA pols don’t have the CTD. • Uncapped mRNA’s are unstable, and have trouble getting exported into the cytoplasm. • Cap plays a role in translation (eIF4a recognizes cap – important for translation initiation.

13. Mapping the 5’ start of transcripton Poly(A) tail very useful for isolating mRNA’s! Reverse transcriptase will add a bunch of C’s in a cap-dependent manner 5’ 3’

14. Splice out the introns Final mRNA product

15. Polycistronic units are rare in eukaryotes Eukaryotic genes are interrupted Prokaryotic vs. eukaryotic gene organization

16. Eukaryotic genes have introns and exons

17. Processing of the chicken ovalbumin gene

19. Consensus sequences at the exon-intron junction in vertebrate mRNA’s

20. Splicing in theory is energetically cheap

21. However, mRNA splicing is mediated by a spliceosome • The spliceosome consists of 5 short RNA molecules (snRNA’s). • Each snRNA is complexed with about 6-7 proteins to form a snRNP. • snRNPs form the core of the spliceosome that catalyzes the splicing reaction.

22. Spliceosome assembly and rearrangement costs ATP • Fidelity supported by different snRNP’s interacting with the same consensus sequences.

23. How are the right consensus sequences recognized? • Correct sites recognized based on proximity to exons, which contain exonic splicing enhancers (ESE’s) that are binding sites for SR proteins (“exon definition hypothesis”). • Variation in these sites and/or proteins might be involved in alternative splicing.

24. How are the right consensus sequences recognized? • Correct sites recognized based on proximity to exons, which contain exonic splicing enhancers (ESE’s) that are binding sites for SR proteins (“exon definition hypothesis”). • Variation in these sites and/or proteins might be involved in alternative splicing.

25. Alternative splicing generates more protein variants from a single gene

26. Alternative splicing generates more protein variants from a single gene

27. DSCAM: ~40,000 possible proteins from one gene!

28. A splicing cascade regulates Drosophila sex determination

29. Lamin assembly Lamin monomers Lamin dimers Head-to tail assembly Nuclear lamina meshwork EM picture taken from J. Struc. Biol. 122: 42-66

30. Globular domain Rod domain Mutations in A-type lamins are linked with tissue-specific disease Head Domain

31. A silent mutation in the Lamin A gene causes Progeria R482Q R482L R482W H222P H222Y R62G M371K R453W K486N A43T R582H W520S W520C R25P R584H R60G R133P N195K R249Q R336Q V442A G465D R644C R571S Head Domain Globular domain Rod domain G608G G608S I469T V452F K97E I63S T150P E203G E203K Q294P E358K R386K I497T T528K R28W Y45C R541H R541C Y481H R50P R50S R527P R377H R377L G232E N456K N456I L85R E317K R190W L530P • Progeria is a disease of premature aging. • The vast majority of cases result from G608G

32. CaaX-box processing B-type lamins Lamin A Adapted from Nature Medicine 11(7):2005

33. Hutchinson Gilford Progeria Syndrome • Premature ageing syndrome. • Fully penetrant, children die ~age 16. • De novo mutation, NOT inherited. • Mutation results in an improved splicing consensus that ultimately causes a protein missing internal amino acid sequences. • This protein is not properly processed, and accumulates, creating a toxic effect that leads somehow to premature aging. consensus consensus

34. We’ve just finished with these Let’s take a look at these…because they are self-splicing How did splicing evolve?

35. Group I and II introns Group I: G is activated to form the attacking group that will cleave the first of the phosphodiester bonds in the reaction; no lariat. Group II: A is activated to form the attacking group that will cleave the first of the phosphodiester bonds in the reaction; a lariat is formed.

36. Tetrahymena Group I intron splicing reaction • Nucleotide sequence of the intron is critical. • The intron RNA folds into a specific three-dimensional structure which positions the relevant reactive groups together to perform the chemistry of splicing. • Thus group I (and group II) introns are ribosymes.

37. + Exon 1 Intron 1 Exon 2 Exon 1 Exon 2 Intron 1 • Products of splicing were resolved by gel electrophoresis: + + + + pre-rRNA - + - + Nuclear extract - + + - GTP pre-rRNA Spliced exon Intron circle Intron linear Self-splicing in pre-rRNA in Tetrahymena : T. Cech et al. 1981 Additional proteins are NOT needed for splicing of this pre-rRNA! Do need a G nucleotide (GMP, GDP, GTP or Guanosine).

38. Finish the job Final mRNA product

39. Transcription termination

40. Cleavage stimulating factor F Cleavage and polyadenylation specificity factor Doesn’t require a template! Don’t know what determines the length of the poly A tail, but, like the 5’ cap, it is also required later on for proper translation. Transcription termination

41. mRNA export

43. Central Dogma: information flow

44. Not covered, but look it up! • Processing of non-coding RNA’s (e.g.tRNA’s, chemical modifications etc). • snoRNA’s (small nucleolar RNA’s) • RNA editing • Riboswitches Coming attractions in the RNA world • RNA interference • Micro RNA and development