Alan Weiner BIOCHEM 530 Wednesday, EE 037 October 8, 2014 RNA structure and function

1. Alan Weiner BIOCHEM 530 Wednesday, EE 037 October 8, 2014 RNA structure and function

2. catalytic(+) strand template(–) strand If RNA can be bothcatalyst and genome,RNA could replicate itself… the first "living" molecule! precursors Lawrence and Bartel (2005) New ligase-derived RNA polymerase ribozymes. RNA 11, 1173-1180; Shechner and Bartel (2011) The structural basis of RNA-catalyzed RNA polymerization. Nat Struct Mol Biol 18, 1036-1042; Shechner et al. (2009) Crystal structure of the catalytic core of an RNA-polymerase ribozyme. Science 326, 1271-1275; Bagby et al. (2009) A class I ligase ribozyme with reduced Mg2+ dependence: Selection, sequence analysis, and identification of functional tertiary interactions. RNA 15, 2129-2146. catalytic (+) strand copies unfolded template (–) strand two catalytic (+) strandsand original template (–) strand

3. Nucleic acids are plausible prebiotic condensation products

4. Nucleic acids are plausible prebiotic condensation products generated by repeated dehydrations O = HN  uracil   = N O P P P O 5' – H2O, glycosidic bond O ribose OH OH – H2O, phosphoester bond (ribose,  phosphate) – H2O, phosphoanhydride bonds (, and ,)

5. Whoops, right impulse, wrong concept! What modern salvage pathways and "prebiotic" chemists do… What Nature probably did… Szostak (2009) Origins of life: Systems chemistry on early Earth. Nature 459, 171-172 [News and Views]

6. "Systems chemistry on early earth"… proposed by Oparin in 1938, tested by Miller in 1953 as his PhD Thesis!

7. small molecules "Systems chemistry on early earth"… Options for pyrimidine ribonucleotide assembly old-fashioned chemical intuition new-fangled systems pathway cyclic intermediates hydrolysis or phosphorolysis products Powner et al. (2009) Nature 459, 239-242

8. Primordial Soup The RNA World If RNA is catalytic, it could function as both genome and replicase, replicating itself, and perhaps also encoding ribozymes that would carry out intermediary metabolism to make more RNA precursors. We can debate whether an RNA World existed, or how complex it might have been, and whether RNA may have been preceded by a simpler RNA-like polymer that could also function as genome and replicase. However, if life did begin in an RNA-like World, it may still be an RNA World today only slightly disguised by a veil of DNA! Prebiotic World • condensation of sugars, bases, phosphate, and random polynucleotides RNA World • RNA genomes • RNA enzymes RNP World • protein synthesis • protein enzymes DNA World • DNA genomes Progenote mt chl Eukaryotes Eubacteria Archaea

9. CH3 CH3 OH N N N N NH2 NH2 N N O + Nucleotide cofactors could be molecular fossils of metabolism in an RNA World S N N –O O CH3 +NH3 HO OH S-adenosylmethionine (SAM) riboflavin-5'-phosphate flavin mononucleotide (FMN) guanine White HB (1976) Coenzymes as fossils of an earlier metabolic state. J Mol Evol 7, 101-104

10. 5' RNA secondary structure in 5’ untranslated region (5’ UTR) is a thermosensor: stable at 30°C, melts at 37°C [Johansson et al. (2002) Cell 110, 551–561] 3' PfrA coding region Riboswitches can regulate mRNA expression without help from proteins or transcription a virulence-activating transcription factor in Listeria monocytogenes "aptamer" RNA secondary structures can bind small metabolites such as SAM, guanine, and FMN, coordinately controlling genes involved in methyl, purine, and cofactor metabolism [Winkler et al. (2003) Nat Struct Biol 10, 701–707] coding region 3' 5' proteins involved in methyl metabolism (SAM), purine metabolism and transport (guanine), and flavin mononucleotide biosynthesis (FMN) in Bacillus subtilis Breaker (2004) Nature 432, 838; (2007) Nature 447, 497

11. catalytic(+) strand template(–) strand If RNA can be bothcatalyst and genome,RNA could replicate itself… the first "living" molecule! precursors Lawrence and Bartel (2005) New ligase-derived RNA polymerase ribozymes. RNA 11, 1173-1180; Shechner and Bartel (2011) The structural basis of RNA-catalyzed RNA polymerization. Nat Struct Mol Biol 18, 1036-1042; Shechner et al. (2009) Crystal structure of the catalytic core of an RNA-polymerase ribozyme. Science 326, 1271-1275; Bagby et al. (2009) A class I ligase ribozyme with reduced Mg2+ dependence: Selection, sequence analysis, and identification of functional tertiary interactions. RNA 15, 2129-2146. catalytic (+) strand copies unfolded template (–) strand two catalytic (+) strandsand original template (–) strand

12. The smallest natural ribozymes are the hepatitis  ribozyme and the hammerhead ribozyme found in plant viroids

13. Australia battled citrus exocortis virus (oranges, lemons, and etrogs on susceptible rootstocks) in the 1940s and 1950s healthy tree stunted by exocortis http://www.dpi.nsw.gov.au/agriculture/horticulture/citrus/health/diseases/citrus-exocortis

14. Hammerhead ribozyme structure hydrolysis cartoon showing stems, loops, and 17 conserved nucleotides backbone trace of actual structure Martick et al. (2006) A discontinuous hammerhead ribozyme embedded in a mammalian [lectin] messenger RNA. Nature 454, 899-902; Chi et al. (2008) Capturing hammerhead ribozyme structures in action by modulating general base catalysis. PLoS Biol 6, e234.

15. attacking R3-OH O beforetetrahedral phosphatewith two ester bonds(phosphodiester) All natural ribozymes (except the ribosome!) use the same mechanism:the attacking hydroxyl group expels the leaving hydroxyl group,making a new phosphoester bond as it breaks the old one P O– R1-O If R3-OH is RNA-OH, the result isRNA isomerization.If R3-OH is H-OH (water), the result is RNA hydrolysis. O-R2 duringattacking hydroxylgenerates a trigonal,bipyramidal intermediate O H R3-O O-R2 P O– R1-O O afterbipyramidal intermediateturns "inside out"like umbrella in high wind("inversion of configuration") R2-OH expelled P O– R3-O O-R1

16. H2O O = If the attacking hydroxyl belongs to H2O, the RNA is hydrolyzed (cleaved). All natural ribozymes (except the ribosome!) use the same mechanism:the attacking hydroxyl group expels the leaving hydroxyl group,making a new phosphoester bond as it breaks the old one O-P-O – O O = + HO-P-O OH – O OH O = If the attacking hydroxyl belongs to RNA, the RNAs are isomerized. O-P-O – O O = O-P-O – O + OH

17. RNA splicing and replication are chemically (almost) identical OH O = If the attacking hydroxyl belongs to RNA, the RNAs are isomerized as in splicing. O-P-O – O O = O O O-P-O – -P-O O -P-O- O + OH O O OH O O If the RNA 3' hydroxyl attacks a ribonucleotide triphosphate (NTP), the reaction is a polymerization as in replication! N O-P-O -P-O- OH O O O O = = = = = = -P-O + – – – – – N O-P-O- OH – O O

18. mRNA splicing is just an isomerization…a new bond is made for every bond broken 2’ 3’ 3’ 2’, 3’, 5’

19. The smallest natural ribozymes are the hepatitis  ribozyme and the hammerhead ribozyme found in plant viroids

20. The "hairpin" ribozyme cleaves the negative strand of tobacco ringspot virus satellite RNA •To crystallize "the" precursor, the attacking 2'-OH was replaced by the methoxy group 2'-OCH3 which cannot dissociate. •To crystallize "the" transition state, the scissile phosphate was deleted, and the ribozyme was complexed with vanadate (Na3VO4) to mimick the pentacovalent bipyramidal intermediate. •The product crystallizes naturally (and apparently does not undergo the reverse reaction!). 3' 2' 5' Rupert, Massey, Sigurdsson, and Ferré-D'Amaré, 2002

21. The transition state is stabilized by more hydrogen bonds than either precursor or product (A) Precursor complex stabilized by two hydrogen bonds (dashed lines) from G8. (B) Transition state mimic complex stabilized by five hydrogen bonds. (The green triangle connects the equatorial oxygens of the vanadate.) Protonation is inferred from the distance between N1 of A38 and the 5'-oxygen. (C) Product complex stabilized by three hydrogen bonds. The hydrogen bond between N1 of A38 and the 5'-OH of G+1 orients the latter for subsequent ligation. Rupert, Massey, Sigurdsson, and Ferré-D'Amaré, 2002

22. Transition State Stabilization by a Catalytic RNA (Rupert, Massey, Sigurdsson, and Ferré-D'Amaré, 2002) Precursor and product differ "infinitesimally" from the transition state… precursor + vanadate model precursor product + vanadate model product

23. The smallest natural ribozymes are the hepatitis  ribozyme and the hammerhead ribozyme found in plant viroids

24. Doudna and Lorsch (2005) Nat Struct Mol Biol 12, 395-402.

25. Ribozyme-catalyzed reactions: very different, and often far better* * think of ribosomes and spliceosomes which require large motions of large complexes with many RNA molecules coming and going! Doudna and Lorsch (2005) Nat Struct Mol Biol 12, 395-402.

26. cytoplasm mRNA life history is regulated at every stepfrom synthesis to destruction nucleus 5' spliced mRNA unsplicedmRNA 5' cap AAAA 3' DNA RNA polymerase

27. default splicing pattern 1 2 3 4 5’ cap AAAAAAAAA 3’ 1 2 3 4 5’ cap AAAAAAAAA 3’ exon skipping or exclusion (+/– exon 2) alternative exons (include exon 2 or 3) Flavors of alternative mRNA splicing

28. exon 412 alternatives exon 648 alternatives exon 933 alternatives exon 172 alternatives Current world record holder for alternative mRNA splicing isthe Drosophila DSCAM gene, whose protein productsfunction in axon guidance and innate immunity in the fly genomic DNA andpre-mRNA mRNA 5' 3' protein N C outside TM inside The 61 kb DSCAM gene generates an 8 kb mRNA containing 24 exons. Exons 4, 6, 9, and 17 are encoded as tandem arrays of mutually exclusive alternative exons, so this one gene could in principle generate as many as 12  48  33  2 or 38,016 different mRNAs and proteins. McManus and Graveley (2011) Curr Op Genet Dev 21, 373-379

29. New answers pose new questions. We assumed that transcriptional control would be the major determinant of complexity, and we asked What wires transcription? Now it turns out that nerves are wired by alternative mRNA splicing, and so we must now ask What wires alternative splicing? Maybe not an endless hall of mirrors, but certainly no end in sight... outside inside Hattori et al. (2007) Dscam diversity is essential for neuronal wiring and self-recognition. Nature 449, 223-228; Meijers et al. (2007) Structural basis of Dscam isoform specificity. Nature 449, 487-491; Du Pasquier (2005) Insects diversify one molecule to serve two systems. Science 309, 1826-1827..

30. exon 412 alternatives exon 648 alternatives exon 933 alternatives exon 172 alternatives Current world record holder for alternative mRNA splicing isthe Drosophila DSCAM gene, whose protein productsfunction in axon guidance and innate immunity in the fly genomic DNA andpre-mRNA mRNA 5' 3' protein N C outside TM inside Immunoglobulin folds with alternative regions (color) and constant regions (black): homophilic interactions between similar DSCAM variants presumably play a role in axon guidance; alternative regions presumably function in innate immunity against microbial pathogens Schmucker et al. (2000) Drosophila Dscam Is an Axon Guidance Receptor Exhibiting Extraordinary Molecular Diversity. Cell 101, 671-684

31. The exon theory of genes: complex genes were assembled from small coding exons, and the assembled exons were then joined together by mRNA splicing. 18 18 18 18 36 Gilbert, 1986 Accidental DNA recombinations occuring over evolutionary time are random and sloppy…but mRNA splicing is smart, and precisely joins each coding region to the next. 18 18 18 18 17 17 X 17 17 18 18 18 18 17 17 17 17

32. mRNA splicing is just an isomerization…a new phosphoester bond is made for each bond broken 2’ 3’ 3’ 2’, 3’, 5’

33. 5’ exon U1, U2, U4, U5, and U6 small nuclear RNA (snRNAs)collaborate to splice out mRNA introns U4 U1 U5 U6 U snRNPs assemble into a spliceosome on mRNA precursor 5’ exon 3’ exon U2 U1 U4 U5 U6 first catalytic step generates an RNA lariat 3’ exon U2 second catalytic step ligates exons and releases lariat intron with U snRNPs U5 U6 + 5’ exon 3’ exon U2

34. The five spliceosomal snRNAs (major class) catalysis U1 U2 branch site recognition 5’ splice site recognition catalysis U6 U5 repressor and (re)assembly factor U4

35. The five spliceosomal snRNAs (major class) catalysis U1 U2 Sm site branch site recognition 5’ splice site recognition Sm site catalysis U6 Sm site U5 repressor and (re)assembly factor U4 Sm site

36. Sm proteins form a heptameric ring, and are found in Archaea! Kambach, Walke, Young, Avis, de la Fortelle, Raker, Lührmann, Li, and Nagai (1999) Cell 96, 375.

37. The snRNA components of the snRNPsrecognize the 5’ and 3’ splice junctionsand catalyze the mRNA splicing reaction 2’ OH U2 snRNA U1 snRNA

38. mRNA splicing is mechanistically identical to "Group II" autocatalytic self-splicing 2’ 3’ 3’ 2’, 3’, 5’

39. U1 U5 Three kinds of RNA splicing Group I (in cis) Group I Group II (in cis) Group II U2/U6 ? 2 U2/branch site 1 2 mRNA (major class) mRNA (in trans) 1

40. Are the five spliceosomal snRNAs really fragments of a Group II self-splicing intron? catalysis U1 U2 branch site recognition 5’ splice site recognition catalysis U6 U5 repressor and (re)assembly factor U4

41. Group II introns in ancient protein coding genes could have evolved in situ into modern mRNA introns by sequential replacement of Group II domains with snRNAs working in trans Jarrell, Dietrich, Perlman (1988) Mol Cell Biol 8, 2361-2366

42. How the RNP got its protein…an offer it could not refuse? independent RNA protein makes offer RNA cannot refuse RNA relaxes, now protein-dependent