1 RNA half-lives vary greatly Protein-RNA complex structures involved in pre-mRNA processing 2 Gene expression and regulation DNA RNA protein Transcription factors Methylation ? Phosphorylation Modification 5’ RNA Pol RNA Pol RNA Pol 5’ 5’ DNA - RNA packaging, stability - 5’ capping - splicing - 3’ end processing (cleaveage and polyadenylation) - export - translation mRNA, rRNA RNA Pol 5’ 3’ RNA-binding proteins RNA editing Alternative-splicing 3 Capping 7meGpppN=cap Transcription 5’UTR 5’ 3’UTR exon1 exon2 intron 3’ Poly-A tail3’end processing Splicing Biophysical, chemical approach RNA binding proteins of two types: - enzymes polymerase, nuclease, modifying enzymes - binding proteins protection, folding (chaperone), gene regulation 4 Protein-RNA structure of the constitutive mRNA processing machinery Yeast Pol II-mRNA-DNA complex Gnatt et al and Kornberg, Science 2001 5 5’ capping Mechanism of capping (viral capping enzyme) PP-RNA + GMP G-PPP-RNA Hakansson et al et Wigley, Cell 1997 6 Mechanism of capping (viral capping enzyme) PP-RNA + GMP G-PPP-RNA Hakansson et al et Wigley, Cell 1997 5’cap binding protein, CBP20-CBP80 Mazza et al, EMBO J (2002) CBP20-CBP80 7 CBP20-m7GpppG contacts Comparison with other 5’cap binding proteins vaccinia virus 8 Collins & Guthrie, Nat.Struct.Biol 2000 Constitutive splicing Pre-mRNA splicing 9 U1snRNP, single particle cryoEM Stark et al, Nature (2001) Stark et al, Nature (2001) U1snRNP, single particle cryoEM U1 snRNA 10 U1A-U1snRNA SLII Oubridge et al, Nature (1994) SM proteins-RNA complex, Archaea, Toro et al, EMBO J (2001) 11 SM protein-RNA complex U2A-U2B’’-U2snRNA stem-loop IV Price et al, Nature (1998) 12 U2A-U2B’’-U2snRNA stem-loop IV Price et al, Nature (1998) The commitment complex 13 SF1-branch point complex Liu et al, Science (2001) 5’ 3’ SF1-branch point (A8) complex 14 3’ end proceessing Cleavage and polyadenylation Bard et al, Science 2001 Yeast PolyA Polymerase-dATP-dATP complex 15 PAPB RBD2 RBD1 Poly A binding protein (Deo et al, Cell, v98 1999) 5’ 3’ Protein-RNA structures of the regulated mRNA processing machinery \ Post-transcriptional gene regulation Unique to each gene 16 Alternative splicing 3 4X In neuronsIn non-neural cells 3 4 3 4X X exon inclusionX exon skipping From Black D.L. Cell (2000), v103 p367 Alternative-splicing of the DSCAM gene Can produce up to 38016 different protein isoforms 17 X X pre-mRNA alternative splicing In neuronsIn non-neural cells 3 4 3 4X X exon inclusionX exon skipping 3 4 U1 U2 U1U2 3 4X U1 U2 U2U1 Chou et al and Black Mol. Cell. (2000) pre-mRNA alternative splicing repression 3 4 N1exon skipping N 3 4 U1 U2 PTB PTB activation 3 4N N1exon inclusion 3 4N U1 U2 U2U1 - + SR 18 Exonic enhancer sequence RRMU2AF35U2AF65 Exonic enhancer sequence RRM U1snRNP U1 70K 5’ Splice-site selection 3’ Splice-site selection intron intron intronintron SR domain 5’ 3’ 5’ 3’ Exon definition by SR proteins U2snRNP + Spectra free- bound Wagner et al (2001) Mol Cell Biol 21, 3281 - 3288 Pyr tract Pyr tract near Branch point PTB is a general Splicing repressor - 19 Model for splicing repression by PTB RBD4 RBD3 3’ 5’ 5’ 3’ - 5’ 3’ 5’ 3’ hnRNPA1- DNA (Ding et al, Gene & Dev, 1999) Splicing repression - 20 RNA editing by adenine deamination A I read as G Pre-mRNA editing by Adenosine deamination From Reenan Trends in Genetics (2001), v17 p53 Human genes Drosophila genes 21 Editing mechanism by ADAR From Reenan Trends in Genetics (2001), v17 p53 5’AUUA GGUGGGUGG AUA UAUAACAAUAU 3’UAGU CCAUCCACC UAU AUAUUGUUGUA GluR-B pre-mRNA A C C G A G ADAR2 Deaminase domainRNA binding domain 1 701 dsRBD1 67 303 dsRBD2 H E C C 394 396 451 526148 228 GC AA U dsRBD2 dsRBD1 Arg (AGG) to Gly (GGG) 22 Drosophila Paralytic gene 13 alternative exons + 11 RNA editing sites = > 1 000 000 potential isoforms Gene regulation at the 3!end 23 RBD1 RBD1 5’ 3’ 5’ 3’ U1A-3’UTR (Varani et al, Nature Struct Biol, v7 2000) Direct interaction with the PAP and inhibition Gene regulation by a feed back mechanism Pumilio domain 5’ 3’ hPUM-UGUAUAU (Wang et al, Cell, 2002) 24 Pumilio domain (Wang et al, Cell, 2002) 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 25 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( ) dsRBD-RNA Ryter and Schultz, Embo J, 1998 26 5’ 3’ N C HIV-1Nucleocapsid- SL3 (De Guzman et al, Science, 1998) HIV-1 RNA packaging Arg-rich-RNA HIV rev-RRE BIV tat-TAR Battiste et al, and Williamson Science 1996 Puglisi et al, Science 1996 27 Zinc finger-RNA (Lu et al, Nature , 2003) Zinc finger-RNA (Lu et al, Nature , 2003) 28 Zinc finger-RNAZinc finger-DNA (Lu et al, Nature , 2003)(Nolte et al, PNAS , 1998) Zinc finger4-RNAZinc finger4-DNA (Lu et al, Nature , 2003)(Nolte et al, PNAS , 1998) 29 Zinc finger6-RNAZinc finger6-DNA (Lu et al, Nature , 2003)(Nolte et al, PNAS , 1998) RNA Binding Domain or RNP domain The most common RNA binding modules found in 224 proteins ( 324 modules) (0.5-1% of the genes)helix 1 helix 2 !"!!"! U1A,U2B!! Pre-mRNA splicing Sex-lethal, HuD Alternative-splicing PABP mRNA stability RBD2RBD1 RBD3 RBD4 Nucleolin Pre-rRNA processing 30 RNA Binding Domain or RNP domain !"!!"! !1 U(F/Y)U*NL "1 ***L***F !2 G*U**Z*U !3 (R/K)G(F/Y)(A/G)(F/Y)V*F "2 Z**AU** !4 G*U*U** ! !1 !2 !3!4 "1 "2 U hydrophobic Z non charged * any residue 75-85 residue long Interaction with U3snoRNA and ribosomal proteins Interaction with ribosomal proteins N terminus RBD1 RGGRBD2 RBD3 RBD4 Nucleolin, the most abundant nucleolar protein NRE RNA binding 31 Nucleolin RNA Targets B1 10-50nM (5!ETS) Mouse B2 50-100nM (5!ETS) Mouse G-C A-U G-C G-C U-G 5! 3! U C C C A G A C 5! 3! C-G G-C U-A A-U C U C C C A G G U Consensus NRE 5! 3! N-N N-N N-N N-N Nx U/G C C C G/A G Ny sNRE 5-20nM 5! 3! C A• A•U G•A G-C G-C G A U C C A G A RBD2-RNA-RBD1 “sandwich” 5’ 3’1 G16 22 F56 5’ 3’ RBD2 RBD1 linker 32 5’ 3’ Structure of the RNA in complex 1 U9 C10 C11 C12 G13 A14 A8 22 Loop E 5’ 3’ F56 and K94 insert in the loop 1 U9 C10 C12 G13 22 33 5’ 3’ Protein side-chain-RNA base stacking 1 U9 C10 C12 G13 22 Y58 F17 Y140 V129 I138 RBD1-RNA interactions 5’ 3’ 1 C12 G13 22 Y58 F17F56 34 RBD1-RNA stem interactions G16 V27 T52 R54 5’ 3’ 1 C12 22 linker-RNA interactions C12 K89 K94 K95 S93 89 100 G13 A14 A15 K89 K95 S93 100 C12 G13 35 RBD2-RNA interactions 5’3’ 1 U9 C10 22 RBD1-RBD2 interactions RBD2 RBD1 5’ 3’ U9K55 D132 K89 E125 C12 G16 36 V129 U/GCCCGA K136 K105U9 C G A K136 I138L103 U9 K105 Sequence specificity: UFU* NL KGFAFV*F !1 !3 UCCCGA Y140 R127 C10 C11 Y140C10C11 U R127 Sequence specificity: G*U**Z*U KGFAYV*F !2 !3 37 Sequence specificity: F56 E86 C12 F17 K89 K55 E86 C12 K89 K55 U UCCCGA UFU* NL RGFAFV*F K *U*U* !1 !3!4 Y58 R49 UCCCGA G13 89 92 Y58 R49 G13 8992 A Sequence specificity: G*U**Z*U KGFAYV*F !2 !3 38 RBD2 5’ 3’ 5’ 5’ 3’ 3’ Poly A binding proteinSex-LethalNucleolin (Handa et al, Nature, 1999) (Deo et al, Cell, 1999) RBD1 3’ 5’ 3’ 5’ 3’5’ (Allain et al, Embo J 2000)