Comparative genomics of RNA regulatory elements

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Comparative genomics of RNA regulatory elements

• Mikhail Gelfand
• Research and Training Center Bioinformatics
• Institute for Information Transmission Problems
  Moscow, Russia

September 2006
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Riboflavin biosynthesis pathway
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5 UTR regions of riboflavin genes from various
bacteria
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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
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Attenuation of transcription
Antiterminator
Terminator
The RFN element
Antiterminator
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Attenuation of translation
Antisequestor
SD-sequestor
The RFN element
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RFN the mechanism of regulation

• Transcription attenuation

• Translation attenuation

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Distribution of RFN-elements
Genomes Number of analyzed genomes Number of genomes with RFN Number of the RFN elements
a-proteobacteria 8 4 4
ß-proteobacteria 7 4 4
?-proteobacteria 17 15 15
d- and e-proteobacteria 3 0 0
Bacillus/Clostridium 12 12 19
Actinomycetes 9 4 4
Cyanobacteria 5 0 0
Other eubacteria 7 5 6
Total 68 47 52
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YpaA riboflavin transporter in Gram-positive
bacteria

• 5 predicted transmembrane segments gt a
  transporter
• Upstream RFN element (likely co-regulation with
  riboflavin genes) gt transport of riboflaving or
  a precursor
• S. pyogenes, E. faecalis, Listeria sp. ypaA, no
  riboflavin pathway gt 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)

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Phylogenetic tree of RFN-elements
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thi-box and regulation of thiamine metabolism
genes by thiamine pyrophosphate (Miranda-Rios et
al., 2001)
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Alignment of THI-elements
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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
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THI the mechanism of regulation

• Transcription attenuation

• Bacillus/Clostridium group,
• Thermotoga,
• Fusobacterium,
• Chloroflexus

• Thermus/Deinococcus group,
• CFB group
• Proteobacteria,

• Translation attenuation

• Actinobacteria,
• Cyanobacteria,
• Archaea

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Distribution of THI-elements
Genomes Number of analyzed genomes Number of genomes with THI Number of the THI elements
a-proteobacteria 7 7 15
b-proteobacteria 6 6 12
g-proteobacteria 18 17 38
e- and d-proteobacteria 3 1 1
The Bacillus/Clostridium group 18 18 51
Actinomycetes 9 9 25
Cyanobacteria 5 5 5
Other eubacteria 14 11 11
Archaea (Thermoplasma) 17 3 6
Total 97 77 164
Mandal et al., 2003 THI in 3UTR (plants). THI
in untranslated intron (fungi)
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Metabolic reconstruction of the thiamin
biosynthesis
thiN
Transport of HET
Transport of HMP
(Gram-positive bacteria)
(Gram-negative bacteria)
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Metabolic reconstruction of the thiamin
biosynthesis
thiN
Transport of HET
Transport of HMP
(Gram-positive bacteria)
confirmed (Morett et al., 2003 )
(Gram-negative bacteria)
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The PnuC family of transporters
THI elements
RFN elements
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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

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Conserved RNA secondary structure of the
regulatory B12-element
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The predicted mechanism of the B12-mediated
regulation of cobalamin genes formation of a
pseudoknot
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Distribution of B12-elements in bacterial genomes
B12-element regulates cobalamin biosynthetic
genes and transporters, cobalt transporters and a
number of other cobalamin-related genes.
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Metabolic reconstruction of cobalamin
biosynthesis new enzymes and transporters
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Metabolic reconstruction of cobalamin
biosynthesis new enzymes and transporters
confirmed (Woodson et al., 2004)
recently confirmed (Zayas et al., 2006)
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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 Ribonucleotide reductases
NrdJ (B12-dependent) NrdAB/NrdDG (B12-independent)



Methionine synthase Methionine synthase
MetH (B12-dependent) MetE (B12-independent)



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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)
Ribonucleotide reductases Ribonucleotide reductases
NrdJ (B12-dependent) NrdAB/NrdDG (B12-independent)



Methionine synthase Methionine synthase
MetH (B12-dependent) MetE (B12-independent)



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LYS-element, a.k.a. L-box lysine riboswitch
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Reconstruction of the lysine metabolism
predicted genes are boxed (pathway of acetylated
intermediates in B. subtilis)
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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
• gt acts as activator
• similar architecture of activating adenine
  riboswitch upstream of purine efflux pump ydhL
  (pbuE) in B. subtilis (Mandal and Breaker, 2004)

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S-box (SAM riboswitch)
Grundy and Henkin, 1998
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Reconstruction of the methionine metabolism
predicted genes are boxed and marked by
(transport, salvage cycle)
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A new family of amino acid transporters
S-box (rectangle frame)MetJ (circle
frame)LYS-element (circles)Tyr-T-box
(rectangles)
malate/lactate
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Repression of reverse pathway Met ? Cysin
Clostridium acetobutylicum in the presence of
Cys and absence of Met
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Firmicutes
Loss of S-boxes
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
proteobacteria

• Other genomes with S-boxes the Zoo
• Petrotoga
• actinobacteria (Streptomyces, Thermobifida)
• Chlorobium, Chloroflexus, Cytophaga
• Fusobacterium
• Deinococcus

E.coliTFs
Xanthomonas S-box
alphas SAM-II
Geobacter S-box
Need more genomes
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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
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Riboswitch composition of metagenomes
total per 100 000
contigs 47 27 26
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Riboswitches in metagenomes by taxonomy
62
44
30
26
19
15
11
8
total per 100 000 contigs
3
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Conserved structures of riboswitches (circled
X-ray)
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Mechanisms
gcvT ribozyme, cleaves its mRNA (the Breaker
group)THI-box in plants inhibition of splicing
(the Breaker and Hanamoto groups)
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Characterized riboswitches (more are predicted)
RFN Riboflavin biosynthesis and transport FMN (flavin mononucleotide) Bacillus/Clostridium group, proteobacteria, actinobacteria, other bacteria
THI Biosynthesis and transport of thiamin and related compounds TPP (thiamin pyrophosphate) Bacillus/Clostridium group, proteobacteria, actinobacteria, cyanobacteria, other bacteria, archea (thermoplasmas), plants, fungi
B12 Biosynthesis of cobalamine, transport of cobalt, cobalamin-dependent enzymes Coenzyme B12 (adenosyl-cobalamin) Bacillus/Clostridium group, proteobacteria, actinobacteria, cyanobacteria, spirochaetes, other bacteria
S-boxSAM-IISAM-III Metabolism of methionine and cystein SAM (S-adenosyl- methionine) Bacillus/Clostridium group and some other bacteriaSAM-II (alpha), SAM-III (Streptococci)
LYS Lysine metabolism lysine Bacillus/Clostridium group, enterobacteria, other bacteria
G-box Metabolism of purines purines Bacillus/Clostridium group and some other bacteria
glmS (ribozyme) Synthesis of glucosamine-6-phosphate glucosamine-6-phosphate Bacillus/Clostridium group
gcvT (tandem) Catabolism of glycine glycine Bacillus/Clostridium group
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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?

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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

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Teaser Systematic analysis of T-boxes

• T-boxes the mechanism (Grundy Henkin)

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Partial alignment of predicted T-boxes
TGG T-box
Aminoacyl-tRNA synthetases
Amino acid biosynthetic genes
Amino acid transporters
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continued (in the 5 direction)
anti-anti (specifier) codon
Aminoacyl-tRNA synthetases
Amino acid biosynthetic genes
Amino acid transporters
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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

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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
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Recent duplications and bursts ARG-T-box in
Clostridium difficile
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(No Transcript)
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More duplications THR-T-box in C. difficile
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ASN/ASP/HIS T-boxes Duplications and changes in
specificity
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Blow-up
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Branched-chain amino acids duplications and
changes in specificity
ATC
CTC
ATC
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Blow-up
transporter
ATC
GTC
dual regulation of common enzymes
ATC
CTC
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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)

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• 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