RNA Modifications
Claudia Höbartner
Institute of Organic Chemistry University Würzburg
RNA Biology WS 2018 Campus Hubland Süd HIRI Würzburg, November 26, 2018
Functional nucleic acids and RNA synthetic biology @ Höbartner lab
Fluorogenic labeling of RNA modifications In vitro selection of DNA catalysts Mechanism & Structure
Chemical synthesis of RNA Spin labels for EPR and NMR Application of deoxyribozymes Site-specific labeling of RNA
In vitro selection of fluorogenic aptamers Detection of nucleobase modifications
2 The building blocks of RNA – and their modifications
Topics for today
RNA modifications A T - Structures & Functions
- Biosynthesis / Installation
- Methods of Detection GC Nucleotide biosynthesis
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 3
The building blocks of RNA – and their modifications
RNA modifications / modified nucleotides
Many flavors – very broad topic – brainstorming/expectations
- Natural posttranscriptional RNA modifications (m6A, m5C, <,…) - Synthetic / unnatural nucleosides - Modification by reactive electrophiles (structure probing / toxins, lesions) - Bio-orthogonal labeling of RNA (fluorophore attachment) - Modified (oligo)nucleotides as drugs (antivirals; siRNAs, aptamers, mRNA)
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 4 The building blocks of RNA – and their modifications
Learning objectives
1. Recognize and classify modified nucleotides, describe their occurence in natural / synthetic RNA
2. Explain nucleotide biosynthesis and describe mechanisms of RNA methylation / name cofactors and enzymes involved
3. Explain how snoRNAs are involved in installation of RNA modification
4. Describe at least two methods for analysis of modified nucleotides (one global and one site-specific method)
5. Explain the purpose of using modified nucleotides in synthetic/therapeutic RNA
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 5
The building blocks of RNA – Nucleotide triphosphates
Before we start with modifications ….
How is unmodified RNA synthesized in cells?
Formation of phosphodiester bond (Transcription: see lectures day 2) building blocks: NTPs
What about nucleotide metabolism?
NTP de novo biosynthesis + salvage pathways
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 6 Mini-Quiz on Nucleotide metabolism
1. The first nucleotide product in the de novo biosynthetic pathway of purines is A) AMP. C) IMP. B) GMP. D) XMP.
2. Which of the following statements is false concerning purine synthesis? A) N7 is from glycine
B) C2 is form CO2 (bicarbonat) C) N3 is from glutamine D) C8 is from 10-formyl-tetrahydrofolate
3. A ribose sugar is added to the ______rings after their synthesis and to ______rings during their synthesis. A) purine, pyrimidine B) pyrimidine, purine
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 7
Purine biosynthesis
• Precursors • Cost N from Asp, Gln 2 ATP eq for PRPP
C from THF, Gly, CO2 5 more ATP steps
RNA Biology course WS18/19 8 Purine biosynthesis
• Two distinct amination mechanisms
• Salvage is major pathway
• Base is synthesized while attached to ribose
• IMP is common intermediate for AMP and GMP
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 9
Overview of purine metabolism
R R R R R R
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 10 Overview of pyrimidine metabolism
R R R R R
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 11
De novo pyrimidine synthesis
• De novo synthesis is major pathway • UMP is converted to other pyrimidines • Orotidine base is synthesized, then attached to ribose • Aspartate is completely incorporated
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 12 Mini-Quiz on Nucleotide metabolism
1. The first nucleotide product in the de novo biosynthetic pathway of purines is A) AMP C) IMP. B) GMP. D) XMP.
2. Which of the following statements is false concerning purine synthesis? A) N7 is from glycine
B) C2 is form CO2 (bicarbonat) C) N3 is from glutamine D) C8 is from 10-formyl-tetrahydrofolate
3. A ribose sugar is added to the ______rings after their synthesis and to ______rings during their synthesis. A) purine, pyrimidine B) pyrimidine, purine
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 13
Thymidylate Synthase
• dTMP is made from dUMP
• Methylene-tetrahydrofolate as “methyl” donor • Fascinating mechanism Donates methylene and hydride • Inhibited by 5-Fluorouracil antimetabolite
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 14 Postsynthetic modifications of biopolymers
DNA RNA Protein Transcription Translation
DNA methylation RNA modification Protein modification
methylation, acetylation, etc
Epigenetic and epitranscriptomic modifications in mammalian DNA and RNA regulate gene expression
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 15
The chemical diversity of natural RNA modifications
a small selection of >150 known
Site of modification: Nucleobase Watson-Crick edge Hoogsteen edge Ribose
Type: Alkylation, Oxidation, Isomerization Acetylation, Thiolation, Aminoacylation, Ribosylation, ...
Function: Stabilize 2D or 3D structure, alter conformation, change polarity, change base-pairing ability, ...
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 16 The chemical diversity of synthetic RNA modifications – on the nucleobase
Selected examples of functional nucleotides for biophysical studies
Fluorescent nucleoside analogs paramagnetic nucleosides for EPR/NMR spectroscopy
Pyrrolo-C distance measurement via PELDOR
8-vinyl-G 2AP
C-spin
The chemical diversity of synthetic RNA modifications – on the backbone
Selected examples of nucleoside modifications for antisense and siRNA
Goals: Stability against nucleases Enhanced affinity to RNA Improved mismatch discrimination Facilitated delivery
Special case: LNA: locked C3‘-endo (Northern conformation) A-form duplex
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 18 The chemical diversity of synthetic RNA modifications – on the backbone
Selected examples of nucleoside modifications for antisense and siRNA
First approved RNAi drug How are these RNAs prepared ? August 2018, hATTR-Amyloidosis treatment Solid-phase synthesis 2‘-OMe-and PS-modified siRNA, 21bp dsRNA Nat. Biotechnol. 2018 doi: 10.1038/nbt0918-777
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 19
Methylated nucleosides in natural RNA
Site of methylation: N vs O vs C
1 5 2 2 1 m A, m C, m G, m 2G, Cm, Um, m G, etc. in tRNA necessary for folding and decoding
Batey et al. Angew. Chem 1999, 38, 2326.(review) Grosjean et al. BMC Genomics 2008 9:470
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 20 Methylated nucleosides in RNA
Site of methylation: N vs O vs C
1 5 2 2 1 m A, m C, m G, m 2G, Cm, Um, m G, etc.
in tRNA necessary for folding and decoding other heavily modified RNAs: rRNA, snRNA
RNA modification database: http://mods.rna.albany.edu/
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 21
Methylated nucleosides in RNA
Site of methylation: N vs O vs C
1 5 2 2 1 m A, m C, m G, m 2G, Cm, Um, m G, etc.
in tRNA necessary for folding and decoding other heavily modified RNAs: rRNA, snRNA
RNA modification database: http://mods.rna.albany.edu/ http://modomics.genesilico.pl/
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 22 RNA methylation: enzymes and mechanisms
Methyltransferase enzymes Methyl group donor: SAM human: seven NSUN proteins targeting different RNAs
References from our own work: NSUN3 and ABH1 modify the wobble position of mt-tRNAMet to expand codon recognition in mitochondrial translation. EMBO Journal 2016, 35, 2104-2119 NSUN6 is a human RNA methyltransferase that catalyzes formation of m5C72 in specific tRNAs. RNA 2015, 21, 1532-154
Mechanism via covalent RNA-protein intermediate How can target RNAs of specific MTases be identified?
Come up with strategies to trap the covalent intermediate
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 23
RNA methyltransferase NSUN3 methylates C34 in mt-tRNAMet
Mechanism of cytosin methylation
in vivo crosslinking and analysis of cDNA (CRAC)
5-aza-cytidine CRAC
mt-tRNAMet
mt-tRNAPro
mt-tRNAGlu
Haag, Sloan, Ranjan, Warda, ..., Rehling, Rodnina, Höbartner, Bohnsack, EMBO J. 2016 RNA Biology course WS18/19 RNA Modifications / C.Höbartner 24 RNA methylation: enzymes and mechanisms
Methyltransferase enzymes Methyl group donor: SAM Single enzymes or Protein complexes
Wang et al., Cell 2016
Diversity in mechanism and function of tRNA methyltransferases RNA Biology 2015, 12, 398-411 Mechanism via deprotonation for activation of nucleophilic amine 10.1080/15476286.2015.1008358
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 25
RNA methylation: enzymes and mechanisms
Methyltransferase enzymes Methyl group donor: SAM Single enzymes or Protein complexes
Wang et al., Cell 2016
RNA●Protein complex (RNP)
guide RNA + enzyme + protein cofactors
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 26 snoRNA: RNA-guided RNA modification
2'-OMe RNA C/D snoRNP https://www-snorna.biotoul.fr/info.php
Nm in rRNA and snRNA
W.Filipowicz, PNAS 2000, 97,14035 F. Bleichert, et al. Science. 2009, 325, 1384.
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 27
snoRNA: RNA-guided RNA modification
2'-OMe RNA Pseudouridine synthesis
W.Filipowicz, PNAS 2000, 97,14035 Watkins & Bohnsack, WIRES RNA 2012
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 28 Mechansims of pseudouridine synthesis
X. Gu et al., PNAS 1999, 96,14270
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 29
Modified nucleotides in eukaryotic mRNA
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 30 Modified nucleotides in eukaryotic mRNA
Wendy V. Gilbert et al. Science 2016;352:1408-1412
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 31
N6-methyladenosine: m6A
YTH domain
YTHDC1-2 YTHDF1-3
RNA methylation is a dynamic regulator or gene expression m6A RNA methylation is known to be involved in all stages in the life cycle of RNA including pre-mRNA splicing, pri-miRNA processing, through nuclear export, RNA translation modulation and RNA degradation
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 32 N6-methyladenosine: m6A
Influence of m6A on RNA structure and protein recognition m6A binding by YTH proteins
protein
Methyl-Pi interaction within Trp cage
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 33
Analysis of RNA modifications
Fragmentation / digestion into nucleosides followed by 2D TLC, HPLC, and/or LC-MS analysis
Review: specificity of nucleases RNase T1 RNase T2 RNase P1 RNase V1 RNase S1 …
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 34 Analysis of RNA modifications by reverse transcription
Nature Reviews Genetics 18, 275–291 (2017)
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 35
Analysis of m6A by transcriptome wide sequencing methods
Transcriptome-wide sequencing methods for m6A.
Critical dependence on specific antibodies for immunoprecipitation
Issues of site-specificity and quantification
Nature Methods 14, 23–31 (2017)
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 36 Site-specific analysis of RNA modification
SCARLET: Site-specific cleavage and radioactive labeling followed by ligation- assisted extraction and TLC
Digestion to 5‘ mononucleotides
TLC analysis
RNA Biology course WS18/19 M. Helm, Y. Motorin, Nature Reviews Genetics 18, 275–291 (2017) 37
Site-specific analysis of RNA modification
SCARLET: Site-specific cleavage and radioactive labeling followed by ligation- assisted extraction and TLC
Discuss advantages/ disadvantages of SCARLET
Discuss requirements for using DNA enzymes
Requirement for using DNA enzymes: • cut directly at site of modification and release 5’-OH • cut modified and unmodified RNA with equal efficiency has been shown for m5C and pseudouridine in tRNA: M. Hengesbach et al. RNA 2008 RNA Biology course WS18/19 38 DNA-catalyzed RNA cleavage for detection of modifications
DNA-catalyzed RNA cleavage
Detection of nucleobase modifications Distinguish methylated isomers
Site-specific detection and quantification
Direct gel-based readout (or northern blot)
qRT-PCR analysis or input for sequencing
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 39
Methylation-specific RNA cleaving deoxyribozymes
In vitro selection of DNA enzymes from random DNA library with synthetic methylated RNA
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 40 Methylation-specific RNA cleaving deoxyribozymes
In vitro selection of DNA enzymes from random DNA library with synthetic methylated RNA
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 41
Methylation-specific RNA cleaving deoxyribozymes
In vitro selection of DNA enzymes from random DNA library with synthetic methylated RNA
RNA: m6A consensus motif DRACH
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 42 Methylation-specific RNA cleaving deoxyribozymes
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 43
Methylation-specific RNA cleaving deoxyribozymes
Cleavage kinetics of synthetic m6A RNA GG|ACU sequence context
unmodified VMC10 time
substrate RNA
cleaved RNA
6 m A RNA time
substrate RNA
cleaved RNA
cuts unmodified RNA 50x-150x faster Analysis by northern blot and qPCR for endogenous RNA
Sednev, Mykhailiuk, Choudhury, Halang, Sloan, Bohnsack, Höbartner, Angew. Chem. Int. Ed., published Oct 1, 2018. 44 Methylation-specific RNA cleaving deoxyribozymes
Cleavage kinetics of synthetic m6A RNA GG|ACU sequence context
VMA15 VMC10
cuts m6A-RNA 3x-8x faster cuts unmodified RNA 50x-150x faster
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 45
Methylation-specific RNA cleaving deoxyribozymes
C/D box snoRNAs m6A in k-turn motif (Lilley, EMBO Rep. 2016)
m6A interrupts 15.5k protein binding
m6A found in mouse snoRNA (Jaffrey, Nat. Meth. 2015)
46 Methylation-specific RNA cleaving deoxyribozymes
C/D box snoRNAs m6A in k-turn motif (Lilley, EMBO Rep. 2016)
Northern blot RNA Biology course WS18/19 RNA Modifications / C.Höbartner 47
Methylation-specific RNA cleaving deoxyribozymes
Summary and Outlook
• VMA DNA enzymes preferentially cut m6A RNA • VMC DNA enzymes are strongly inhibited by m6A • synthetic RNAs from 20 - 400 nt • endogenous snoRNA and lncRNA • generally applicable for DGACH motifs • Calibration curves for quantitative assessment of methylation level at a specific target site Outlook: DNAzymes with random hexamer binding arm How do DNA enzymes VMA enzymes: find missing GGACU motifs recognize RNA methylation? VMC enzymes: find remaining GGACU motifs
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 48 RNA Modifications: summary & take home message
• RNA modifications alter the physico-chemical properties of nucleotides, e.g. conformation, polarity, hydrophobicity, chemical reactivity and base-pairing interactions
• siRNAs as drug approved to treat human liver disease
• synthetic RNA modifications are necessary for stability and efficacy of therapeutic RNA
• natural RNA modification is performed by highly specific and regulated enzymatic mechanisms involving pure protein enzymes and catalytic RNA–protein complexes (RNPs)
• RNA modification is important for regulation of gene expression
• Transcription-wide RNA modification is a dynamic and regulated cellular process
• Deregulation of RNA modification may lead to human pathologies
• Technological advancements for detection and analysis of RNA modifications
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 49
The building blocks of RNA – and their modifications
Now you should be able to
1. Recognize and classify modified nucleotides, describe their occurence in natural / synthetic RNA
2. Explain nucleotide biosynthesis and describe mechanisms of RNA methylation / name cofactors and enzymes involved
3. Explain how snoRNAs are involved in installation of RNA modification
4. Describe at least two methods for analysis of modified nucleotides (one global and one site-specific method)
5. Explain the purpose of using modified nucleotides in synthetic/therapeutic RNA
RNA Biology course WS18/19 RNA Modifications / C.Höbartner 50