Eukarya m 2.2,7/ cm5U acp*D mcm5U chm5U acp3y mcm5Um ncmr mchrrfu mcnrrs2U cm5s2U m3Um m5Um macpv Epitranscriptomics:; the role of posttranscriptional modifications in m2,7G yW }?yw hm5C f*A ncm5s2U OHyW m«Am m^m OHyW* lo*A m'Am galQ g'JA hm*A manO yW-72 Ar{p) yW-58 Gr(p) lm ^^\mV m6A m**A ct*A^ ' ieA >^moEU mcmo5U meteA ^ cmo5U se2U ms2i6A nrrrU nm&se2U ms^^A mnm-U nmVu ms2meA cmnm5s2U cmnm6Um RNA metabolism yW-86 |mG imG-14 inm5s2U nm5ges2U mnm5ges2U m42C \ cmnm6ges2U cmnm&se2U ho'JC &C m4Cm acpJU 5 2i i mnm s U mnmJse^U preQ0 preO, pa m Gm ac°A aamatidine C+ Stepanka Vanacova cnnrp U rn ^,v. mimG m s2Um RNA structure and function 2020 archaeos U Trypanosomes have only one mitochondrion A unique mitochondrial DNA architecture The kinetoplast human / ■ / f ■ • wmé. infirm iľif i " 1" 1 í É llfil I'll íl ill trypanosome RNA editing by trypanosomes: The mystery of missing genes Human mitochondrial gene Yeast Trypanosome DNA ACCAGAGAGGAGAG UGAGGAAAGGCG mRNA AUCCAGUAUUGUUUUUUAUGGUUUUUAUGUAGUGAGUUUGUUUUAUUUAUGGCG T. brucei ATPase 6 m RNA edited MFLFFFCD LFWLRLLLCMYYCVWSRLCF I V Y F N C L M L I F D F L L F C L F D L Y L F V G L C L F L L L W F M L F N L Y S L I L Y Y C I T Y L N L Y L L F C I V F L L Y I A F L F L F C F L C D F F L F N N L L V G D S F M D V F F I R F L L C F L E C : F S L L C R C L S T F L R L F C N L L S S H F L L L M F F D F F Y F I F V F F F W C F L LLIYFIYFCVLFLFI ILCVF IFVGFIC R H I T V I Y F L ter Types of RNA editing Insertion/deletion u Insertion or deletion Conversion A C Types of RNA modifications 1. RNA editing 2. Base modifications insertional & deletional many different once substitutional RNA editing Substitutional RNA editing Cytidine NH2 HoO Ribose Adenosine NH N N Ribose N N HoO NH2 OH HN- Ribose NH HN O Ribose NH HN OH NH *N--^N^ Ribose O NH NH Ribose T/BS Substitutional RNA editing A to I ADARs, ADATs (adenosine deaminases acting on RNA/tRNA C to U CDARs (Apobec) (cytosine deaminases acting on RNA) C to U editing often forms additional stop codons apoB-- gene III II 1 11 1 1 1 1 — i CAA Exon 26 apoB mRNA CAA 5'' 1 Proteins NH. 4536 HcOOH ApoB-100 TAA ::= =3' Intestine 5'- NH- CAA-MJAA UAA i 2152 HcOOH ApoB-48 Apolipoprotein B-100 4563 amino acids Function: transport of cholesterol in the blood Apolipoprotein B-48 2152 amino acids Function: absorption of lipids from the intestine Organization of the glutamate-gated ion channel receptors GluR-B subunit M1 M2 M3 M4 N X2D3=C I Y VC XXJ=C Q R =o=c R G Landscape of A-to-l RNA editing occurrence and impact 100 50 - Heterochromatin Miscellaneous unique sequences Simple repeats/ duplications Repetitive elements Introns Exons Hu genome composition Non translated exons Translated exons RNA Localisation Folding Stability Regulation of splice dynamics Splice site elimination/creation Maturation miRNA Stability Targeting Protein Processing Stability " Interaction Localisation Distribution of editing sites Viral replication Cell migration, invasiveness Interferon response Neurotransmission C-protein coupling Aberrant protein expression Ion channel kinetics of activation and deactivation Ion permeability, trafficking, assembly of neurotransmitter receptors Distribution of molecular functional impact Feeding behaviour Cold adaptation Evolution Physiological and pathophysiological links (a) (b) (c) Maas, Stefan(Sep 2013) A-to-l RNA Editing and Human Genetic Disease. In: el_S. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0024625] Types of RNA modifications 1. RNA editing 2. Base modifications insertional & deletional multiple different types substitutional RNA editing Phylogenetic distribution of modified nucleosides in RNA originating from the three domains of life Eukarya cm5U m3Um acp3D mcm5U chm5U m5Um acp3v mcm5Um ncm5U s2Um m5D m3V m8A m2eA ct^A^630^'3 W mo5U mcmo5U m6t6A hoJU m3C l'acpV m2.2.7G mchm5U mcmVU cm5s2U m4C cmnmHj ms2(0eA m27G yW m5s2U mchm5Um tm5U tm5s2U f5C ^Cm ncm5U ncnrllm °2yw hm5C f*A ncmVU OHyW m«Am m> OHyW lo*A m'Am galQ gcA hm6A manQ yW-72 Ar{p) yW-58 Gr(p) Im teA m6A mG m ?A m8U rrr,G ms m ;s2U m5C Inosine yW-86 |mG s2U ac4C Am m3U cmo5U se2U ms2i6A nmsU nmsse2U ms?hn8A mnm5U nm^U ms2m6A cmnm5s2U cmnm5Um inm5U lnm5Um geszU inm5s2U nm5ges2U mnm5ges2U m42C s4U \ cmnm&ges2U cmnmLse2U ho5C I^C m4Cm acp3^! mnm5s2U imG-14 myt ym Gm rnnm:'se2U Cm (jm m'l m2A hn6A ms2hn*A preQ0 preQ, oGv, ac6A mz7Gm m1Gm imG2 agmatidine C+ archaeoane G* mimG cnm5U m42Cm s2Um m5Cm ac4Cm m Im m jGm m'Gm Archaea Motorin, Yuri(May 2015) RNA Modification. In: el_S. John Wiley & Sons Ltd, Chichester, http://www.els.net [doi: 10.1002/9780470015902.a0000528.pub3] RNA modifications Template DNA 0000000000000000<>OC>00000000<>0 I, Gm, Cm, m5C, XV, mcm5U, mcm5s2U, ncm5U, ncm5Um Cellular localisation of RNA:modification enzymes and coordination between RNA (tRNA) maturation and modification Diversity of nucleotide methylation (a) Methylation sites on the chemical structures of the four major ribonucleotides, inosine, and pseudouridine. Multiple modifications may occur sequentially on a single nucleotide. Motorin and Grosjean. tRNA modification. In: Encyclopedia of Life Sciences. 2005 RNA polymer ■ Gm. m1G. rrčG. m7G, rn^G rr^Gm, rn^Gm, imG, mimG m^G. m"-7G m'Gm. wřJGm yW. c^yW. OHyW. OHyW Am, m1A, rr^A. m6A, ms^A, m62A. mBA m62Am, m'Am 0 \ j N >r | I m1l, m'lm, Im Cm. wfc. m^. mfcm ac4Cm, m*C, m*Cm f^Cm, m*2C (hn; n(nh) 4> Urn, m5U. m5Um m^U. s^Jm mHD Tm m1<ť m1acp!3M'. m1* Known sites of RNA methylation Home Modifications Pathways Reactions RNA sequences Proteins snoRNAs Publications Building blocks Search Links Downloads Help Contact Mo do m ics a database of RNA modification pathways MODOMICS and RNApathwaysDB are two complementary resources whichpresent RNA metabolism at different levels. While MODOMICS presents RNA modification pathways on the level of nucleosides, RNApathwaysDB deals with RNA metabolism with respect to whole RNA molecules. Our ultimate goal is to integrate these databases, however at the moment the users are invited to consult both of these complementary resources, depending on their needs and interests. • o • Overview I The RNA Modifi... X \ + C^^j (i) mods.rna.albany.edu/lntroduction/overview fol Most Visited - ^ Gcttirg Started http://support.pol... 3 Google App cCare Service.. IP C Q. Search A App c - Support -... Apple i$ .Mac 3 Amazor L] News - .............18 000 methylation sites 3-5 per mRNA appear in clusters m6A near the „ stop codon * 5' UTR Coding sequence m6A distribution 3' UTR CO ■t± 1 CD 0 ACU Nature Reviews | Molecular Cell Biology I-1-1-1-1-1-1 -2 -1 m6A 1 2 3 4 N6-methyladenosine is recognized by sensing proteins in two modes Dynamic RNA Modifications in Gene Expression Regulation Translation http://dx.doi.Org/10.1016/j.molcel.2014.09.001 The nuclear roles of m6A Cytoptoumc role m*A-*wikh proteins regulate* splicing SPLICING Or vTMrxi HMINIVUHI miRNA PROCESSING i' END PROCESSING NUCLEAR EXPORT ™DC ^SWral NXFl I— m-A r™.-^L-1 1 ■1 ■ i Covelo-Molares et ai, 2018 N6-methyladenosine modification serves multiple functions RNA stability and sequestering to P-bodies RNA export alternative splicing miRNA processing translation efficiency How is methylation at stop codon achieved ? Why are some DRACH sites methylated and other not ? Which factors are able to distinguish methylated and non-methylated RNA ? What is the function of demethylases ? Wang et al., Ce//,2013; Fustin et al., Cell, 2013, Dominissini et al., Nature,2012; Meyer et al., Ce//2012 m6A is enriched at stop codon at DRACH motif >18 000 methylation sites 1-3 per mRNA appear in clusters Conserved motif DRACH D=A/G/U; R=G/A, H=A/C/U Nature Reviews | Molecular Cell Biology mRNA methylation is a reversible process m6a Writers MMW complex METTL3-METTL14 WTAP METTL16 Adenosine m6a readers N6-methyladenosine m6a erasers Gerken et Al., Science, 2007; Dominissimi et Al., Nature, 2012; Meyer et Al., Cell, 2012; Liu et Al., Nat Chem. Biol. ,2014; Wang et Al., Nature, 2014 AlkBH family of Fe(ll)/a-ketoglutarate-dependent dioxygenases n - - HZ] »- J SM M I mm ] mm ] AlkB -E.coli AlkBHI-human AlkBH2-human AlkBH3-human AlkBH4-human AlkBH5-human AlkBH6-human AlkBH7-human AlkBH8-human FTO-human Acting on RNAs I I- Adapted from Muller and Hausinger. 2015 m6A/m6Am demethylases: FTO and AlkBH5 Substrate mbA and mbA m Oxidative With 2 detectable demethylation intermediates mbA Without stable intermediates Highest expression Brain Testis K.O. mouse model Reduce adipose phenotype tissue Aberrant spermatogenesis Fu Y, et al. Nature Com., 2013; Jia et al., Nat. Chem. Biol., 2011; Mauer, J. et al., Nature, 2017; Zheng, et al., Mol. Cell, 2013; Fsicher, et al., Nature, 2009 FTO (Fat mass and Obesity associated) phenotypes associated with obesity SNPs in human: SNP hotspot in FTO intron correlates with diabetes and obesity 1 in 6 adults are homozygous for the risk allele (Frayling et al., 2007, Science) higher weight (>3 kg compared to average) and 1,67-fold increased odds of developing obesity (Frayling et al., 2007, Science) Mouse models: FTO knockout FTO over-expression (Fischer et al- 2QQ9,Nature) (Church et al.,2010, Nature Genetics) FTO Catalytical mutant (Church et al., 2009, PLoS Genetics) Our FTO results 1. FTO binds pre-mRNAs, it likely functions co-transcriptionally. 2. FTO binding is not enriched around the RRACH motif and correlates with adenosine methylation positions at TSS. 3. FTO appears to play a role in 3' end processing. 4. FTO demethylation activity facilitates exon inclusion in a subset of mRNAs. Stability Translation Alternative splicing 3'end processing Bartosovic et al., 2017 FTO specificity for m6A versus m6Am m6A m6Am HN* N- ,CH< HO- OH OH HN' N- // ,CH< HO- .O. w OH O. VCH- Stability Translation (fro)m (no)m ——?i —— m6Am m6A Alternative splicing m6A m6A m6A 3' end processing © m6A RESEARCH RESEARCH ARTICLE SUMMARY MOLECULAR BIOLOGY Cap-specific terminal A^-methylation of RNA by an RNA polymerase II-associated methyltransferase Shinichiro Akichika', Seiichi Hirano . Yuichi Shichino, Takeo Suzuki, Iliroshi Nishimasu, Ryuichiro Ishitani, Ai Sugita, Yutaka Ilirosc, Shintaro Iwasaki, Osamu Nurekif, Tsutomu Suzuki; Resource Molecular Cell Identification of the m6Am Methyltransferase PCIF1 Reveals the Location and Functions of m6Am in the Transcriptome