Water DNA RNA Protein Lipids, sugars and other small molecules THE CELL Molecular Art by David S. Goodsell Structural biology techniques Protein-RNA interactions As seen by structural biology DNA RNA (rRNA,mRNA,tRNA) Protein Transcription Translation RNA Pol I RNA Pol II RNA Pol III RNA packaging, stability 5ʼ capping, RNA editing splicing, alternative splicing 3ʼ end processing (cleaveage and polyadenylation) export RNA biogenesis Gene expression program Water DNA RNA Protein Lipids, sugars and other small molecules THE CELL Molecular Art by David S. Goodsell DNA RNA (rRNA,mRNA,tRNA) Protein Transcription Translation RNA Pol I RNA Pol II RNA Pol III RNA packaging, stability 5ʼ capping, RNA editing splicing, alternative splicing 3ʼ end processing (cleaveage and polyadenylation) export RNA biogenesis Gene expression program Disease-causing mutations in RNAs & RNPs Disease Gene/Mutation Function Prader Willi syndrome SNORD116 ribosome biogenesis Spinal muscular atrophy (SMA) SMN2 splicing Dyskeratosis congenita (X-linked) DKC1 telomerase/translation Diamond-Blackfan anemia RPS19, RPS24 ribosome biogenesis Prostate cancer SNHG5 ribosome biogenesis Myotonic dystrophy, type 2 (DM2) ZNF9 (RNA gain of function) RNA binding Huntingtonʼs disease-like 2 (HDL2) JPH3 (RNA gain of function) ion channel function Fragile X syndrome FMR1 translation/mRNA localization Retinitis pigmentosa HPRP3 splicing Autism 7q22-q33 locus breakpoint noncoding RNA Amyotrophic lateral sclerosis (ALS) TARDBP splicing, transcription Deafness MTRNR1 ribosome biogenesis (mitochondrial) Cancer SFRS1 splicing, translation, export Cancer miR-17-92 cluster RNA miR-372, miR-373 RNA interference Cooper et al. Cell, 2009 5’ 3’ Pre-mRNA RNA binding proteins RNA binding proteins control the fate of the pre-mRNA RNA binding proteins RBD RNA Binding Domain RRM/RBD/RNP N C KH domain N C dsRBM N C Zinc binding proteins cchc cchc ccch N C N C N C RBD protein-proteinCatalytic domain SAM domain RNA binding proteins of two types: - enzymes polymerase, nuclease, modifying enzymes - binding proteins protection, folding (chaperone), gene regulation Modular architecture of RNA-binding proteins Taken from Lunde et al. Nat. Rev Mol. Cell Biol 2007 RNA-binding modules are often combined to perform multiple functional roles Taken from Lunde et al. Nat. Rev Mol. Cell Biol 2007 Coupled protein binding and folding (phosphorylation dependent) What information is recognized by proteins: G A U C U U A A AA G 5ʼ 3ʼ Recognition of RNA sequence A-form duplex of RNA 5’ 3’ A-U G-C C-G U-A A-U G-C C-G U-A A-U G-C 3’ 5’ Recognition of RNA shape What recognition mode is used by proteins: Rigid-body docking + Induced fit + Capping 7meGpppN=cap Transcription 5’UTR 5’ 3’UTR exon1 exon2 intron 3’ Poly-A tail3’end processing Splicing Induced fit recognition Examples 5’cap binding protein, CBP20-CBP80 Mazza et al, EMBO J (2002) CBP20-CBP80 Induced fit binding Induced fit binding CBP20-m7GpppG contacts Induced fit binding Zinc finger-RNA (Lu et al, Nature , 2003) Induced fit binding Zinc finger-RNAZinc finger-DNA (Lu et al, Nature , 2003)(Nolte et al, PNAS , 1998) Induced fit binding A-form duplex of RNA B-form duplex of DNA A-form double-helix vs. B-form double-helix 5’ 3’ A-U G-C C-G U-A A-U G-C C-G U-A A-U G-C 3’ 5’ 5’ 3’ A-T G-C C-G T-A A-T G-C C-G T-A A-T G-C 3’ 5’ Pumilio domain 5’ 3’ hPUM-UGUAUAU (Wang et al, Cell, 2002) Pumilio domain (Wang et al, Cell, 2002) Induced fit binding PAPB RBD2 RBD1 Poly A binding protein (Deo et al, Cell, v98 1999) 5’ 3’ Protein-protein interactions and protein-RNA interactions define the site of spliceosomal assembly Taken from Lunde et al. Nat. Rev Mol. Cell Biol 2007 Rigid body docking Examples Rigid-body dockingViral B2 protein supresses RNAi by masking dsRNA or siRNA. Taken from Chao et al. Nat. Struct. Mol. Biol 2005 SAM domain Sterile Alpha Motif (SAM) SAM domains SAM-DNA SAM-protein SAM-SAM SAM-RNA ? structure of SAMVts1p (yeast) and its RNA substrate (SRE; fruit fly) structure of SAMVts1p with RNA: a shape specific recognition SAM-DNA SAM-protein SAM-SAM SAM-RNA structure of SAMVts1p with RNA: a shape specific recognition potential Vts1p binding targets found in strongly upregulated transcripts in the vts1∆ strain C N G CNGG(N)-type stem-loops G (N) structure of SAMVts1p (yeast) and its RNA substrate (SRE; fruit fly) structure of SAMVts1p with RNA: a shape specific recognition dsRBD-RNA Ryter and Schultz, Embo J, 1998 dsRBMs double-stranded RNA Binding Motif (dsRBM) Ryter & Schultz EMBO J 1998 xlrbpa dsRBM2 with 20 bp RNA duplex A-form duplex of RNA B-form duplex of DNA A-form double-helix vs. B-form double-helix 5’ 3’ A-U G-C C-G U-A A-U G-C C-G U-A A-U G-C 3’ 5’ 5’ 3’ A-T G-C C-G T-A A-T G-C C-G T-A A-T G-C 3’ 5’ ADAR2 mysterious ADAR (Adenosine deaminase acting on RNA) functions of ADARs  to repair incorrect information in genome  to diversify proteome  regulation 5’ 3’ A to I RNA editing 5’ 3’AAAAAAAAAAA A 5’ 3’AAAAAAAAAAI A Adenosine deaminase acting on RNA (ADAR) I Pre-mRNA splicing splicing GluR-B R/G site GluR-B R/G editing site GluRs subunits of AMPA receptors in neurotransmission • Editing-created codon changes in GluRs affect aa positions of critical impact on biophysical properties of glutamate-activated cation channels. GluRs are subunits of AMPA receptors that mediate postsynaptic currents in CNS hyper-editing by ADARs (nonspecific) A AA A A A A A A AA AA I I I I I I I I regulate gene silencing triggered by intramolecular structures in mRNA LA Tonkin and BL Bass (2003) Science, 302, 1725 UTR’s, introns GluR-B R/G site specific editing by ADARs What are the structural determinants that define the adenosine moieties for specific ADAR2-mediated deamination? ADAR2 ADAR2 what does the catalytic domain of ADAR2 do alone? Macbeth et al. Science, 2005 Non-canonical elements of dsRNA change its shape - recognition signal ADAR2 dsRBM1:gluR-C upper SL ADAR2 dsRBM2:gluR-C lower SL Non-canonical elements of dsRNA change its shape - recognition signal Take Home Messages 5’ RNA Pol RNA Pol RNA Pol 5’ 5’ DNA mRNA, rRNA RNA Pol 5’ 3’ RNA-binding proteins protection, folding (chaperone), gene regulation RNA binding specificity RNA binding proteins: RGG (Gar) SR Dimerisation RBD/RRM/RNP KH, Sam dsRBD Arg-rich Zinc knuckle Zinc finger multidomain protein RBD1 RBD2Inter protein enzymatic domain( ) What information is recognized by proteins: G A U C U U A A AA G 5ʼ 3ʼ Recognition of RNA sequence A-form duplex of RNA 5’ 3’ A-U G-C C-G U-A A-U G-C C-G U-A A-U G-C 3’ 5’ Recognition of RNA shape What recognition mode is used by proteins: Rigid-body docking + Induced fit + What recognition mode is used by proteins: Induced fit + Modular architecture of RNA-binding proteins Taken from Lunde et al. Nat. Rev Mol. Cell Biol 2007