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

Riboflavin biosynthesis pathway

5’ UTR regions of riboflavin genes from various bacteria

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

Attenuation of transcription Terminator The RFN element Antiterminator

Attenuation of translation SD-sequestor The RFN element Antisequestor

RFN: the mechanism of regulation Transcription attenuation Translation attenuation

Distribution of RFN-elements Genomes Number of analyzed genomes Number of genomes with RFN Number of the RFN elements α-proteobacteria844 β-proteobacteria744 γ-proteobacteria1715 δ- and ε-proteobacteria300 Bacillus/Clostridium12 19 Actinomycetes944 Cyanobacteria500 Other eubacteria756 Total684752

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 )

Phylogenetic tree of RFN-elements

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

Alignment of THI-elements

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

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

Distribution of THI-elements Genomes Number of analyzed genomes Number of genomes with THI Number of the THI elements  -proteobacteria 7715  -proteobacteria 6612  -proteobacteria  - and  proteobacteria 311 The Bacillus/Clostridium group18 51 Actinomycetes9925 Cyanobacteria555 Other eubacteria1411 Archaea (Thermoplasma)1736 Total Mandal et al., 2003: THI in 3’UTR (plants). THI in untranslated intron (fungi)

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

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

The PnuC family of transporters RFN elements THI elements

B12-box and 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

Conserved RNA secondary structure of the regulatory B12-element

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

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

Metabolic reconstruction of cobalamin biosynthesis: new enzymes and transporters

recently confirmed (Zayas et al., 2006) confirmed (Woodson et al., 2004)

If a bacterial genome contains B12-dependent and B12- independent isoenzymes, the genes encoding the B12- independent isoenzymes are regulated by B12-elements Ribonucleotide reductases NrdJ (B 12 -dependent (B 12 -dependent) NrdAB/NrdDG (B 12 -independent) +– –+ ++ Methionine synthase MetH (B 12 -dependent) MetE (B 12 -independent) +– –+ ++

If a bacterial genome contains B12-dependent and B12- independent isoenzymes, the genes encoding the B12- independent isoenzymes are regulated by B12-elements nrdAB in Streptomyces coelicolor: experimental confirmation in (Borovok et al., 2005) Ribonucleotide reductases NrdJ (B 12 -dependent (B 12 -dependent) NrdAB/NrdDG (B 12 -independent) +– –+ ++ Methionine synthase MetH (B 12 -dependent) MetE (B 12 -independent) +– –+ ++

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

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

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)

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

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

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

Repression of reverse pathway Met  Cys in Clostridium acetobutylicum in the presence of Cys and absence of Met

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

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

Riboswitch composition of metagenomes total per contigs:

Riboswitches in metagenomes by taxonomy total per contigs

Conserved structures of riboswitches (circled: X-ray)

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

Characterized riboswitches (more are predicted) RFNRiboflavin biosynthesis and transport FMN (flavin mononucleotide) Bacillus/Clostridium group, proteobacteria, actinobacteria, other bacteria THIBiosynthesis and transport of thiamin and related compounds TPP (thiamin pyrophosphate) Bacillus/Clostridium group, proteobacteria, actinobacteria, cyanobacteria, other bacteria, archea (thermoplasmas), plants, fungi B12Biosynthesis of cobalamine, transport of cobalt, cobalamin- dependent enzymes Coenzyme B12 (adenosyl- cobalamin) Bacillus/Clostridium group, proteobacteria, actinobacteria, cyanobacteria, spirochaetes, other bacteria S-box SAM-II SAM-III Metabolism of methionine and cystein SAM (S-adenosyl- methionine) Bacillus/Clostridium group and some other bacteria SAM-II (alpha), SAM-III (Streptococci) LYSLysine metabolismlysineBacillus/Clostridium group, enterobacteria, other bacteria G-boxMetabolism of purines purinesBacillus/Clostridium group and some other bacteria glmS (ribozyme) Synthesis of glucosamine-6- phosphate glucosamine-6- phosphate Bacillus/Clostridium group gcvT (tandem) Catabolism of glycine glycineBacillus/Clostridium group

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?

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 Study scenarios

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

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

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

~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

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

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

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

ASN/ASP/HIS T-boxes: Duplications and changes in specificity

Blow-up

Branched-chain amino acids: duplications and changes in specificity ATC CTC

Blow-up transporter: dual regulation of common enzymes: ATC CTC ATCGTC

Double and one-and-a-half T-boxes TRP: trp operon (Bacillales, C. beijerincki, D. hafniense) TYR: pah (B. cereus) THR: thrZ (Bacillales); hom (C. difficile) ILE: ilv operon (B. cereus) LEU: leuA (C. thermocellum) ILE-LEU: ilvDBNCB-leuACDBA (Desulfotomaculum reducens) TRP: trp operon (T. tengcongensis) PHE: arpLA-pheA (D. reducens, S. wolfei) PHE: trpXY2 (D. reducens) PHE: yngI (D. reducens) TYR: yheL (B. cereus) SER: serCA (D. hafniense) THR: thrZ (S. uberis) THR: brnQ-braB1 (C. thermocellum) HIS: hisXYZ (Lactobacillales) ARG: yqiXYZ (C. difficile)

Andrei Mironov –software genome analysis, conserved RNA patterns Alexei Vitreschak –analysis of RNA structures Dmitry Rodionov –metabolic reconstruction Support: –Howard Hughes Medical Institute –INTAS –Russian Fund of Basic Research –Russian Academy of Sciences