All RNAs great and small e-lecture 1
Institute of Genetics and Biotechnology University of Warsaw HISTORY OF RNA
Rinn and Chang, Ann. Rev. Biochem, 2012 RNA – aka My Favorite Molecule
RNA form A helix
- narrow inaccessible major groove (red) - shallow minor groove (green) - versatile and flexible - catalytically active (splicing, translation, modification) - self-sufficient? - labile (regulation of expression) - create complex 3D structures - specific and unspecific interactions with proteins and other RNAs „THE RNA WORLD” hypothesis „primordial soup” „prebiotic soup” pre-RNA world RNA world
RNA+DNA+ proteins RNA+proteins
RNA made via condensation RNA evolution- molecules RNA starts to join aminoacids from ribose and other inorganic learns to replicate and synthesises polypeptides and organic sources and proteins
Proteins aid RNA to replicate and make DNA and proteins take over major roles proteins. dsRNA evolves into stable DNA. as genetic information and enzymes MODERN RNA WORLD RNA vestiges- catalytic RNAs with active centres made of RNA RIBOSOME - protein synthesis SPLICEOSOME - pre-mRNA splicing
5 snRNAs U1, U2, U4, U5, U6
active snRNP center U6 catalytic activity
Ribosome, crystal structure Cryo EM Ditlev Brodersen, Venki Ramakrishnan C complex, Cryo EM Galej et al, Nature, 2016 RNA
• coding: mRNAs • non-coding: ncRNAs • stable • unstable
• structural (rRNA, tRNA) • polyadenylated • regulatory (si/miRNA) • non-polyadenylated
There are no „free” RNAs in the cell All cellular RNAs exist as ribonucleoprotein particles (RNPs) All RNA types are synthesised as precursors and undergo processing
RNA transcription, processing and decay are tightly coordinated Several RNA processing steps occur co-transcriptionally Regulation of RNA biogenesis involves alternative processes: aTSS, aTIS, AS, APA Lecture on ncRNAs by Monika Zakrzewska-Płaczek RNA FLUX Regulation of gene expression
translation 6 1) chromatin 5
degradation 2) transcription 7
splicing 4 3) RNA processing processing transcription 2 3 4) RNA export
5) translation (mRNA) 3 ncRNAs 1 6) protein stability
7) RNA degradation RNA capacity - CATALYTIC RNAs Nobel 1989 RNA enzymes – RIBOZYMES -1981/82 Tom Cech - self-splicing in Tetrahymena rRNA -1982 Sidney Altman - bacterial RNaseP RNA subunit
Thomas Cech Sidney Altman
Tetrahymena group I self-splicing intron
Escherichia coli RNaseP RNA RIBOZYMES Hammerhead, Hairpin, HDV
viroids, eukaryotes plant satellite RNA, 2007 viruses
mRNA splicing-like Genet., Nat. Rev. , , organelles (fungi, plants), l
bacteria, archea Serganov and Pate and Serganov organelles (fungi, plants), bacteria, mitochondria (animals)
2+ Mechanism: nucleophilic attack of the ribose -OH group (H2O, Me ) on the phosphate RNase P RNA – a true enzyme tRNA processing, multiple turnover
E. coli
2006 RNase P RNA
, TiBS, , TiBS,
Evans et et al Evans
; ;
2007
l, Nat Rev Genet, Genet, Rev l, Nat Serganov and Pate and Serganov mRNA SPLICING Nobel 1993
Phil Sharp Richard Roberts
RNAi Nobel 2006
Andrew Fire Craig Mello SPLICEOSOME: pre-mRNA SPLICING
active center
D3 pre-mRNA::snRNA G B Sm/Lsm Luhrmann and Stark, Curr. Op. Str. Biol., 2009 base-pairing E D1 D2 F
snRNPs
SPLICEOSOME -ribonucleoprotein complex (RNP) organised around snRNAs GENE SILENCING - RNAi
DISCOVERY OF 2002: siRNAs/miRNAs: ncRNAs in RNAi - double stranded small noncoding RNAs - complementary to mRNA targets - participate in gene silencing - mediate: TRANSCRIPTIONAL GENE SILENCING (TGS) • transcription inhibition POST-TRANSCRIPTIONAL GENE SILENCING (PTGS) • mRNA cleavage or • translation inhibition or • translation activation RNAs – STRUCTURE AND FUNCTION Nobel 2009
Elizabeth Blackburn Venkatraman Ramakrishnan Jack Szostak Ada Yonath Carol Greider Thomas Steitz
Telomerase - maintaing chromosome ends Crystal structure of the ribosome THE RIBOSOME
Schmeing and Ramakrishnan, Nature, 2009 RNPs - STRUCTURE/METHODOLOGY Nobel 2017 CRYO-EM
Jacques Dubochet , Mol Cell ,2015 Cell Mol
Joachim Frank Scheres Nogales and Nogales
Richard Henderson Lecture on crystallography and CryoEM by Marcin Nowotny CRISPR-Cas: CRISPR-based genome editing Nobel 2020
Emmanuelle Charpentier Max Planck Institute Jenifer Doudna University of California CRISPR/Cas history
Lander, Cell, 2016 CRISPR/Cas history • • • • RNA CRISPR/ Cas CRISPR target invading DNAs or RNAs recognized byor RNAscrRNAtarget invading DNAs „seed assemble intosurveillanceshort crRNAs complexes long CRISPR: - CRISPR CRISPR associated CRISPR - guided Clustered Clustered Regularly Interspaced Short Palindromic Repeat foreign DNAis integratedintothe CRISPRlocus transcripts are processed byCas orRNase III nuclease Cas RNAi adaptive in Bacteria bacterial and immunity Archaea ” are destroyed
Wiedenheft et al, Nature, 2012
CRISPR/Cas adaptive bacterial immunity
et al, Nature, 2012 Nature, et al, Wiedenheft CRISPR/Cas stages
Hille et al, Cell, 2018 CRISPR/Cas stages
Amitai and Sorek, NatRevMicro, 2016 CRISPR/Cas: adaptation and spacer acquisition
PAM protospacer-adjacent motif in most CRISPR-Cas systems - e.g. in type I immunity usually tri-nucleotide (AWG in E. coli) recognized by the Cascade complex (CasA in E. coli) - probably allows tolerance to self (prevents autoimmunity against spacer DNA sequences complementary to crRNAs they encode) Jiang and Marraffini, AnnuRevMicro, 2016 CRISPR/ targeting crRNA biogenesis Cas RNA crispr : crRNA biogenesis crRNA trans - activating activating , targeting
Jiang and Marraffini, AnnuRevMicro, 2016 CRISPR/Cas types
Lander, Cell, 2016; Tamulaitis, TiMicro 2016 CRISPR/Cas types Gene organization
Wright et al, Cell, 2016 CRISPR/Cas types target DNA
targets RNA and actively targets DNA transcribed DNA
Wright et al, Cell, 2016 Interference of Class 2 CRISPR/Cas One protein effector: Cas9, Cas12a or Cas13
Target RNA
Hille et al, Cell, 2018
Main CRISPR/Cas gene editing tools
, 2019
Biol
Cell Cell
Mol
Nat Rev Rev Nat
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Gersbach
Oliver and and Oliver
- Pickar
https://www.youtube.com/watch?v=k99bMtg4zRk&fbclid=IwAR2HcV xLX2v80gQlJQWoOOS6FkWX-- XeIyYhksegRMuotAVOHySouTcGTIY Anti - CRISPR CRISPR systems : Acr proteins
Marino et al, Nature Methods, 2020