Comparative genomics of RNA regulatory elements

1. Comparative genomics of RNA regulatory elements Mikhail Gelfand Research and Training Center “Bioinformatics” Institute for Information Transmission Problems Moscow, Russia September 2006

2. Riboflavin biosynthesis pathway

3. 5’ UTR regionsof riboflavin genes from various bacteria

4. Conserved secondary structure of the RFN-element Capitals: invariant (absolutely conserved) positions. Lower case letters: strongly conserved positions. Dashes and stars: obligatory and facultative base pairs Degenerate positions: R = A or G; Y = C or U; K = G or U; B= not A; V = not U. N: any nucleotide. X: any nucleotide or deletion

5. Attenuation of transcription Antiterminator Terminator The RFN element Antiterminator

6. Attenuation of translation Antisequestor SD-sequestor The RFN element

7. RFN: the mechanism of regulation • Transcription attenuation • Translation attenuation

8. Distribution of RFN-elements

9. YpaA: riboflavin transporter in Gram-positive bacteria • 5 predicted transmembrane segments => a transporter • Upstream RFN element (likely co-regulation with riboflavin genes) => transport of riboflaving or a precursor • S. pyogenes, E. faecalis, Listeria sp.: ypaA, no riboflavin pathway => transport of riboflavin Prediction: YpaA is riboflavin transporter (Gelfand et al., 1999) Verification: • YpaA transports flavines (riboflavin, FMN, FAD) (by genetic analysis: Kreneva et al., 2000; directly: Burgess et al., 2006) • ypaA is regulated by riboflavin (by microarray expression analysis, Lee et al., 2001) • … via attenuation of transcription (and to some extent inhibition of translaition) (Winkler et al., 2003)

10. Phylogenetic tree of RFN-elements

11. thi-boxand regulation of thiamine metabolism genes by thiamine pyrophosphate (Miranda-Rios et al., 2001)

12. Alignment of THI-elements

13. Conserved secondary structure of the THI-element Capitals: strongly conserved positions. Dashes and points: obligatory and facultative base pairs Degenerate positions: R = A or G; Y = C or U; K = G or U; M= A or C; N = any nucleotide

14. THI: the mechanism of regulation • Transcription attenuation • Bacillus/Clostridium group, • Thermotoga, • Fusobacterium, • Chloroflexus • Thermus/Deinococcus group, • CFB group • Proteobacteria, • Translation attenuation • Actinobacteria, • Cyanobacteria, • Archaea

15. Distribution of THI-elements Mandal et al., 2003: THI in 3’UTR (plants). THI in untranslated intron (fungi)

16. Metabolic reconstruction of the thiamin biosynthesis thiN = Transport of HET Transport of HMP (Gram-positive bacteria) (Gram-negative bacteria)

17. Metabolic reconstruction of the thiamin biosynthesis thiN = Transport of HET Transport of HMP (Gram-positive bacteria) confirmed (Morett et al., 2003) (Gram-negative bacteria)

18. The PnuC family of transporters THI elements RFN elements

19. B12-boxand regulation of cobalamin metabolism genes by cobalamine (Nou & Kadner, 2000; Ravnum & Andersson, 2001; Nahvi et al., 2002) • Long mRNA leader is essential for the regulation of btuB by vitamin B12. • Involvement of a highly conserved B12-box rAGYCMGgAgaCCkGCcd in the regulation of the cobalamin biosynthetic genes (E. coli, S. typhimurium) • Post-transcriptional regulation: RBS-sequestering hairpin is essential for the regulation of the btuB and cbiA • Ado-CBL is an effector molecule involved in the regulation of the cobalamin biosynthesis genes

20. Conserved RNA secondary structure of the regulatory B12-element

21. The predicted mechanism of the B12-mediated regulation of cobalamin genes: formation of a pseudoknot

22. Distribution of B12-elements in bacterial genomes B12-elementregulates cobalamin biosynthetic genes and transporters, cobalt transporters and a number of other cobalamin-related genes.

23. Metabolic reconstruction of cobalamin biosynthesis: new enzymes and transporters

24. Metabolic reconstruction of cobalamin biosynthesis: new enzymes and transporters confirmed (Woodson et al., 2004) recently confirmed (Zayas et al., 2006)

25. If a bacterial genome contains B12-dependent and B12-independent isoenzymes, the genes encoding the B12-independent isoenzymes are regulated by B12-elements

26. If a bacterial genome contains B12-dependent and B12-independent isoenzymes, the genes encoding the B12-independent isoenzymes are regulated by B12-elementsnrdAB in Streptomyces coelicolor: experimental confirmation in (Borovok et al., 2005)

27. LYS-element, a.k.a. L-box: lysine riboswitch

28. Reconstruction of the lysine metabolism predicted genes are boxed (pathway of acetylated intermediates in B. subtilis)

29. Regulation of the lysine catabolism: the first example of an activating riboswitch • LYS-elements upstream of the pspFkamADEatoDA operon in Thermoanaerobacter tengcongensis; kamADElysE operon in Fusobacterium nucleatum • lysine catabolism pathway • LYS element overlaps candidate terminator => acts as activator • similar architecture of activating adenine riboswitch upstream of purine efflux pump ydhL (pbuE) in B. subtilis (Mandal and Breaker, 2004)

30. S-box (SAM riboswitch) Grundy and Henkin, 1998

31. Reconstruction of the methionine metabolism predicted genes are boxed and marked by *(transport, salvage cycle)

32. S-box (rectangle frame)MetJ (circle frame)LYS-element (circles)Tyr-T-box (rectangles) A new family of amino acid transporters malate/lactate

33. Repression of reverse pathway Met  Cysin Clostridium acetobutylicumin the presence of Cys and absence of Met

34. Firmicutes Loss of S-boxes Other genomes with S-boxes: the Zoo • Petrotoga • actinobacteria (Streptomyces, Thermobifida) • Chlorobium, Chloroflexus, Cytophaga • Fusobacterium • Deinococcus Lactobacillales: Met-T-box(Met-tRNA-dependent attenuator) Bacillales: S-box Streptotoccales: MtaR (transcription factor); SAM-III riboswitch (metK) (the Henkin group) Clostridiales: S-box proteobacteria Xanthomonas: S-box E.coli:TFs alphas: SAM-II Geobacter: S-box Need more genomes

35. Riboswitches in metagenomes new functions: S-box: eukaryotic-type translation initiation factor eIF-2B (COG0182) B12-box: fatty-acid desaturase (COG1398) GCVT: malate synthase glcB, phosphoserine aminotransferase serC

36. Riboswitch composition of metagenomes total per 100 000 contigs: 47 27 26

37. Riboswitches in metagenomes by taxonomy 62 44 30 26 19 15 11 8 total per 100 000 contigs 3

38. Conserved structures of riboswitches (circled: X-ray)

39. Mechanisms gcvT: ribozyme, cleaves its mRNA (the Breaker group)THI-box in plants: inhibition of splicing (the Breaker and Hanamoto groups)

40. Characterized riboswitches (more are predicted)

41. Properties of riboswitches • Direct binding of ligands • High conservation • Including “unpaired” regions: tertiary interactions, ligand binding • Same structure – different mechanisms: transcription, translation, splicing, (RNA cleavage) • Distribution in all taxonomic groups • diverse bacteria • archaea: thermoplasmas • eukaryotes:plants and fungi • Correlation of the mechanism and taxonomy: • attenuation of transcription (anti-anti-terminator) – Bacillus/Clostridium group • attenuation of translation (anti-anti-sequestor of translation initiation) – proteobacteria • attenuation of translation (direct sequestor of translation initiation) – actinobacteria • Evolution: horizontal transfer, duplications, lineage-specific loss • Sometimes very narrow distribution: evolution from scratch?

42. Study scenarios • RFN, S-box • early identification of a conserved element • model of regulation from comparative analysis • use for functional annotation • experimental validation • THI, B12, PUR, LYS • scavenging of unexplained published experimental results • models of regulation from comparative analysis • experimental validation • use for functional annotation • GcvT, GlmS • large-scale computational screens • prediction of ligand from functions of regulated genes • experimental validation • SAM-II, SAM-III • gaps in regulatory systems • computational screens • experimental validation • Structures: PUR, THI, S-box

43. Teaser: Systematic analysis of T-boxes • T-boxes: the mechanism (Grundy & Henkin)

44. Partial alignment of predicted T-boxes TGG: T-box Aminoacyl-tRNA synthetases Amino acid biosynthetic genes Amino acid transporters

45. … continued (in the 5’ direction) anti-anti (specifier) codon Aminoacyl-tRNA synthetases Amino acid biosynthetic genes Amino acid transporters

46. ~800 T-boxes in ~90 bacteria • Firmicutes • aa-tRNA synthetases • enzymes • transporters • all amino acids excluding glutamine, glutamate, lysine • Actinobacteria (regulation of translation – predicted) • branched chain (ileS) • aromatic (Atopobium minutum) • Delta-proteobacteria • branched chain (leu – enzymes) • Thermus/Deinococcus group (aa-tRNA synthases) • branched chain (ileS, valS) • glycine • Chloroflexi, Dictyoglomi • aromatic (trp – enzymes) • branched chain (ileS) • threonine

47. Same enzymes – different regulators (common part of the aromatic amino acids biosynthesis pathway) cf. E.coli: AroF,G,H: feedback inhibition by TRP, TYR, PHE; transcriptional regulation by TrpR, TyrR

48. Recent duplications and bursts: ARG-T-box in Clostridium difficile

50. More duplications: THR-T-box in C. difficile