1 / 72 Regulatory mechanisms mediated by RNA 8_MB-2017 2 / 72 Functional types of RNA hnRNA rRNA tRNA snRNA, snoRNA, … mRNA siRNA, miRNA, … DNA pre-rRNA pre-tRNA Translation; encodes a sequence of proteins Translation; part of the ribosome Translation; transfer / amino acids activation Splicing / modification of RNA Regulation of gene expression Small non-coding RNAs Long non-coding RNAs TEST 8_MB-2017 3 / 72 encoding genes represent less than 2% of the total genome sequence vs. at least 90% of the human genome is actively transcribed the more complex organism, the more it comprises non-coding RNAs World of noncoding RNAs Recent evidence suggests that the non-coding RNAs (ncRNAs) may play major biological roles in cellular development, physiology and pathologies. NcRNAs could be grouped into two major classes based on the transcript size: small ncRNAs and long ncRNAs. The percentage of protein-coding genes sequences in several eukaryotic and bacterial genomes. 8_MB-2017 4 / 72 Small non-coding RNAs miRNA siRNA piRNA snoRNA PARS tiRNA Long non-coding RNAs lincRNA TERRAs T-UCR microRNA (miRNA) Piwi-interacting RNA (piRNA) small interfering RNA (siRNA) small nucleolar RNA (snoRNAs) tRNA-derived small RNA (tsRNA) small rDNA-derived RNA (srRNA) small nuclear RNA, also commonly referred to as U-RNA 8_MB-2017 5 / 72 RNA interference - RNAi • sequence-specific gene silencing mechanism triggered by double stranded RNA, on the posttranscriptional level or transcriptional level - inhibitory elements are small RNA molecules (miRNAs, siRNAs….) - miRNAs generated by cleavage of larger pre-miRNA molecules • nucleases Drosha and DICER, which are compiled into multiprotein complex RISC (RNA-induced silencing complex) with proteins Argonaut • RNA interference is a process by which noncoding RNA molecules interfere (pair) with target regions of mRNA, resulting in prevention of gene expression of these mRNAs. • For short, this proces is also called RNAi. We rank him among posttransriptional mechanisms of gene expression. • Most eucaryotic organisms is capable of RNA interference, the process was first studied in the C. elegans. Pri- miRNA 8_MB-2017 6 / 72 RNA interference RISC has helicase activity, thanks to which miRNA is loosened; only one chain remains associated with the complex that allows sequence-specific binding of the whole complex to the target complementary mRNA nuclease activity of RISC complex cleaves the mRNA - its degradation occurs Originally protecting cells against viruses common in eukaryotic cells useful for targeted inactivation of genes: research of gene functions 8_MB-2017 7 / 72 Cenorhabditis elegans Discovery of RNA interference (1998) - silencing of gene expression with dsRNA 8_MB-2017 8 / 72 Antisense RNA (= RNA komplementární k mRNA) can silence gene expression (již počátek 80. let 20. století) - direct introduction of antisense RNA (or transcription in reverse orientation) - interaction of mRNA and antisense RNA, formation of dsRNA 8_MB-2017 9 / 72 What is the mechanism behind? Original (!) hypotheses: - antisense RNA mechanically prevents translation - dsRNA is degraded (RNases) 8_MB-2017 10 / 72 Cosuppression in Petunia Result: loss of pigmentation in flower segments Napoli et al. 1990 Plant Cell 2:279–289 Aim: increase expression of pigment-synthetizing enzym 8_MB-2017 11 / 72 Expression of antisense RNA was less efficient!!! Cosuppression in Petunia Napoli et al. 1990 Plant Cell 2:279–289 Mechanism see later! 8_MB-2017 12 / 72 What they get the Nobel prize for? - making proper controls pays off! - Introduction of even very small amount of dsRNA induce specific silencing (antisence RNA is less efficient!) dsRNA has to be a signal! - for sequence specific silencing Andrew J. Hamilton, David C. Baulcombe*(1999): A Species of Small Antisense RNA in Posttranscriptional Gene Silencing in Plants, Science 286 (5441): 950-9528_MB-2017 13 / 72 RNA interference (RNAi) = silencing of gene expression mediated by small RNAs (small RNA, sRNA) in plants predominantly - 21-24 nt The precise role of 25-nt RNA in PTGS remains to be determined. However, because they are long enough to convey sequence specificity yet small enough to move through plasmodesmata, it is possible that they are components of the systemic signal and specificity determinants of PTGS (Hamilton and Baulcombe, 1999). 8_MB-2017 13 14 / 72 RNA interference (RNAi) gene silencing at • transcriptional level (TGS) (transcriptional gene silencing) - induction of DNA methylation (mRNA not formed) • posttranscriptional level (PTGS) (posttranscriptional gene silencing) - transcript cleavage - block of translation 8_MB-2017 14 15 / 72 Mechanism of action of small RNAdepends on the length of sRNA, biogenesis (precursor), ... PTGS (posttranscriptional gene silencing ): - specific transcription degradation or translation blocking TGS (transcriptional gene silencing ): - methylation of cytosines in the promoter (RdDM), heterochromatinization, inhibition of transcription factor binding Pol VPol II TGS PTGS PTGS TEST 8_MB-2017 15 16 / 72 Basic mechanism of RNAi dsRNA in cell is cleaved by RNase DICER into short dsRNA fragments – sRNA Argonaute with a single strand (from sRNA) mediates recognision of complementary sequences, which should be silenced (TGS, PTGS) 8_MB-2017 16 17 / 72 Small RNA in plants/animals - 3’ end of sRNA methylated (HEN1) - protection • miRNA (micro) – from transcipts of RNA Pol II (pre-miRNA) – hunderds MIR genes (in trans) Pol II DROSHA (Rnaze III), PASHA (RNA binding protein), DICER • siRNA (small interfering) – from dsRNA of various origin (both internal and external – thousands types (both in cis and in trans) ….. (+ piRNA in animals) pre-miRNA Wang et al. 2004 8_MB-2017 17 18 / 72 miRNA biogenesis 8_MB-2017 18 19 / 728_MB-2017 19 siRNA Dicer, also known as endoribonuclease Dicer or helicase with RNase motif, is an enzyme that in humans is encoded by the DICER1 gene. Being part of the RNase III family, Dicer cleaves double-stranded RNA (dsRNA) and pre-microRNA (pre-miRNA) into short double-stranded RNA fragments called small interfering RNA and microRNA, respectively. These fragments are approximately 20- 25 base pairs long with a two-base overhang on the 3' end. Dicer facilitates the activation of the RNA-induced silencing complex (RISC), which is essential for RNA interference. RISC has a catalytic component argonaute, which is an endonuclease capable of degrading messenger RNA (mRNA). • siRNA (small interfering) from dsRNA of various origin (both internal and external – thousands types (both in cis and in trans) ….. (+ piRNA in animals) DICER 20 / 72 Argonaute RNA binding protein (20-26 nt RNA) - strand selection (5’ nt, participation of HSP90) - 10 genes in Arabidopsis - main component of RISC (RNA induced silencing complex) - block of translation or slicer (RNAse H-like endonuclease - PIWI doména) - role in TGS (RdDM) (RNA directed DNA methylation) 8_MB-2017 20 21 / 72 Mechanism of small RNA action - overview PTGS (21-22 nt): - specific cleavage of transcript - - block of translation TGS (24 nt): - methylation of promoter, heterochromatin formation - preventing interaction of transcription factors Pol V 8_MB-2017 22 / 72 sRNA mode of action also depends on complementarity - incomplementarity in cleavage site prevents RNase activity 8_MB-2017 23 / 72 dsRNA formation (MIR genes) ? • RdRP = RNA-dependent RNA Polymerase – synthesis of compl. RNA strand templates: - transcripts cleaved by RISC - impaired mRNAs (without polyA or cap) - transcripts of RNA polymerase IV + secondary structures of viral RNAs (!) mRNA fragment after Ago cleavage (secondarysiRNA ) 8_MB-2017 23 24 / 728_MB-2017 24 Matzke MA, Matzke AJM – This figure is adapted from one by Matzke MA, Matzke AJM (2004) Planting the Seeds of a New Paradigm. PLoS Biol 2(5): e133 doi:10.1371/journal.pbio.0020133. Overview of RNA interference. The dicer enzymes produce siRNA from double-stranded RNA and mature miRNA from precursor miRNA. miRNA or siRNA is bound to an argonaute enzyme and an effector complex is formed, either a RISC (RNAinduced silencing complex) or RITS (RNAinduced transcriptional silencing) complex. RITS affects the rate of transcription by histone and DNA methylation, whereas RISC degrades mRNA to prevent it from being translated. Overview of RNA interference 25 / 72 RNA-directed DNA methylation (in detail) - methylation (by DRM2) occurs only under interaction of AGO4(6,9)-siRNA with RNA Pol V (large subunit C-term domain) and RNA-RNA complementarity Pol IV a V – RNA polymerases RDR2 - RNA dep.RNA polymerase DCL3 – dicer-like protein AGO4 – ARGONAUTE DRM2 – de novo metyltransferase DRD1 – chromatin remodelling protein SHH1 – dual histon-code reading (H3K9me2, H3K4) Saze et al. 2012 SHH1 SHH1 Zhang et al. 2013 8_MB-2017 26 / 72 RNA-directed DNA methylation – why so complicated and energy consuming? - de novo methylation of TE in new insertion sites - transmission of info from histons (CMT2/3) less reliable (less dense nucleosomes) - PTGS – TGS transition SHH1 SHH1 Zhang et al. 2013 Zemach et al. 2013 8_MB-2017 27 / 72 Secondary siRNA formation - target RNA (mRNA, TAS transcripts) - cleaved by Ago + primary sRNA (miRNA or siRNA) - RDR6 – complementary strand synthesis: dsRNA  DCL2(4)  secondary siRNA Function of secondary siRNA - signal amplification - formation of siRNA from neighbor seq. (transitivity – new targets) ta-siRNAs (miRNA na TAS) (trans-acting siRNA – widening of miRNA targets) 8_MB-2017 28 / 72 - overexpression of pigment gene (enzyme for pigment synthesis) caused loss of pigmentation in flower sectors Cosupression in Petunia - occurrence of aberrant transcripts due to overexpression - formation of dsRNA from aberrant transcripts by RdRP (RDR6) - formation of siRNAs that silence both transgene and internal gene dsRNA 8_MB-2017 28 29 / 72 Interfering RNA The phenomenon of RNA interference collides with the concept of transcription factors Although transcription factors start their own transcription, but interfering RNAi decide which transcripts will be used 8_MB-2017 30 / 72 How was the mechanism of RNAi created? dsRNA?  that's a virus! Primitive immune system If a cell detects dsRNA, considers it as a virus - which must be destroyed! Protection of cells against viral infection 8_MB-2017 31 / 72 This was followed by adaptation of RNA interference Has RNAi been an evolutionary phenomenon leading to multicellular? The resulting mechanism of defense against viruses was subsequently adapted for eukaryotic cells, where it serves as a regulatory mechanism for the rapid locking of translation at the current "unnecessary" transcripts This allows ontogeny 8_MB-2017 32 / 72 How are miRNAs formed? - I Discovered only in plants, animals and fungi pri-miRNA (hundreds to thousand bp)  mostly transcription from areas where there are not structural genes  But even within introns and exons  coordinated transcription hnRNA 8_MB-2017 33 / 72 How are miRNAs formed? - II adjustment to dsRNA = miRNA/miRNA* (21 bp) pri-miRNA (100-1000 bp) cleavage of edges and formation of pre-miRNA (70 bp, hairpin) RNA polymerase II RNase III - DROSHA export to cytoplasm RNase III - DICER degradation of miRNA* action miRNA8_MB-2017 34 / 72 Path of RNAi from signal to action Denli AM and Hannon GJ, Trends in Biochemical Sciences, 2003 Complex DICER Complex Argonaute transgennic dsRNAprecursor miRNA In vitro synthesis of dsRNA viral dsRNA transposonal dsRNA heterochromatin dsRNA formation of hetero-chromatin domains cleavage mRNA inhibition of translation degradation of mRNA 8_MB-2017 35 / 72 Site of action of interfering RNA siRNA metylation of sequences in promoter Transcription siRNA, miRNA, piRNA degradation mRNA inhibition of adjustments of mRNA blocking of translation activation of interferon Posttranscriptional processes 8_MB-2017 36 / 72 Mechanism of RNA interference - I Resulting molecule of siRNA or miRNA is incorporated in „RNA-induced silencing complex“ (RISC) 8_MB-2017 37 / 72 Mechanism of RNA interference - II Based on homology of siRNA or miRNA for mRNA, RISC complex causes degradation of this mRNA 8_MB-2017 38 / 72 Structure of RNAi and its target site interaction of miRNA-155 with target sequence gene for miRNA-155 (pre-miRNA-155) mature miRNA-155 8_MB-2017 39 / 72 Fundamental differences between the siRNA and miRNA •Both types regulate expression  siRNA originated in dsRNA  siRNA is often associated with a foreign RNA (usually viral), and is 100% complementary  miRNA comes from molecules of ssRNA, which forms a dsRNA hairpin structures  miRNAs regulate post-transcriptional gene expression 8_MB-2017 40 / 72 Other differences between the siRNA and miRNA siRNA  dsRNA  protection against viruses and transposons  protection against overproduction  cause degradation of target molecules  Absolute complementarity with the target sequence miRNA  ssRNA (hairpins)  regulation of ontogeny and development processes  do not cause degradation, just translation blockade  complementarity to the target sequence is not absolute  formed by activity of RNA polymerase II8_MB-2017 41 / 72 Differences in miRNA biogenesis plants 8_MB-2017 animals TEST 42 / 72 Differences between plant and animal miRNA Plants Animals source intergenic regions intergenic regions, introns miRNA clusters rare common mechanism mRNA cleavage translation repression target site on mRNA ORF 3´-terminus 8_MB-2017 TEST 43 / 72 RNA interference overview 8_MB-2017 TEST 44 / 728_MB-2017 RNA interference based on enzyme degradation or translation inhibition of specific mRNA Drosha (RnasaIII) Pasha (protein) Exportin 5 (transporter) Dicer (RNasaIII) RISC (multiprotein complex) TEST 45 / 728_MB-2017 46 / 72 siRNA and miRNA utilisation:  iRNA usage does not fall under GMO  Yet usage of cassettes producing iRNA does! 1) gene analysis 2) gene therapies 3) anti-viral vaccines 4) transgenic organisms that have transiently inhibited selected genes 8_MB-2017 47 / 72 RNAi therapeutic applications - I 1) RNAi as antivirotics, should block expression of viral genes and viral genome replication. Most anticipated RNAi therapy target is HIV. 2) RNAi induced epigenetic changes on local chromatin structure could specifically control gene expression. 3) It is assumed that 35% to 70% of human genes are transcribed into hnRNA transcripts that later undergo alternative splicing. Defects in alternative splicing can lead to severe diseases. RNAi could be used to block these defective alternatively spliced molecules. 8_MB-2017 48 / 72 RNAi therapeutic applications - II 4) RNAi can target genes involved in metabolic diseases. For example the central role in insuline resistance in diabetes mellitus II is due to defects in gene expression. 5) Gene „knock-outs“ in pathogen genomes can be used as a research tool to gain a better understanding over pathogenic modes of action and therefore aid in developing effective countermeasures. 8_MB-2017 49 / 72 piRNA properties • described in animals • form complexes with Piwi proteins (piwi proteins - regulatory proteins responsible for maintaining incomplete differentiation in stem cells and maintaining the stability of cell division rates in germ line cells) • affect ontogenesis (sperm is not produced without piRNA transcription) • do they transport miRNA to target sequences?  26 to 31 bp  in silico analysis described 52 934 possible piRNA molecules in mice, 52 099 in human and 47 024 for rats  originate from only a handful of intergenic clusters as ssRNA piRNA = Piwi-interacting RNA 8_MB-2017 50 / 72 Piwi-interacting RNA (piRNA) is the largest class of small non-coding RNA molecules expressed in animal cells. piRNAs form RNA-protein complexes through interactions with piwi proteins. These piRNA complexes have been linked to both epigenetic and post-transcriptional gene silencing of retrotransposons and other genetic elements in germ line cells, particularly those in spermatogenesis. They are distinct from microRNA (miRNA) in size (26– 31 nt rather than 21–24 nt), lack of sequence conservation, and increased complexity. It remains unclear how piRNAs are generated, but potential methods have been suggested, and it is certain their biogenesis pathway is distinct from miRNA and siRNA, while rasiRNAs (repeat associated small interfering) are a piRNA subspecies. 8_MB-2017 51 / 72 RNA stability - mRNA has a short half-life, it is readily degraded by ribonucleases - mRNA secondary structure is a key component in RNAse sensitivity - mRNA secondary structure can be altered by protein binding – regulation signals 8_MB-2017 52 / 72 General scheme of messenger RNA decay pathways. 8_MB-2017 (A) mRNAs containing an AU-rich element (ARE) in their 3' UTR undergo rapid ARE-mediated mRNA decay (AMD) in resting cells. Concealing ARE sequence from AMD induces gene expression. (B) Quality control mechanisms. mRNAs that contain a premature termination codon (PTC) are recognized and specifically degraded by the nonsense-mediated mRNA decay (NMD) pathway. (C) The basic mRNA decay machinery in the cytoplasm initially removes the poly(A) tail through the activity of deadenylating enzymes. Subsequently, the mRNA can be further degraded from the 3' end by a complex of 3'–5' exonucleases known as the exosome. Alternatively, the mRNA is decapped at the 5' end, and the 5'–3' exonuclease Xrn1 proceeds to degrade the body of the mRNA. 3'-5' decay 53 / 72 RNAa (RNA activation) molecules activate genes! • described in 2006 • RNAa mode of action – protect ARE sequences • RNAi shutdown genes for 5 to 7 days, RNAa activates genes for 13 days 8_MB-2017 • The molecular mechanism of RNAa is not fully understood. • Similar to RNAi, it has been shown that mammalian RNAa requires members of the Ago clade of Argonaute proteins, particularly Ago2, but possesses kinetics distinct from RNAi. • In contrast to RNAi, promoter-targeted agRNAs induce prolonged activation of gene expression associated with epigenetic changes 54 / 72 RNAa therapies? 1) RNAa can be used on its own 2) We still have to consider that the treatment can deliver complex effects. For example small interfering RNAi can act not only as a negative but at the same time as a positive regulator → inhibiting one gene can indirectly activate another. Such side effects are to be expected also with RNAa. 8_MB-2017 55 / 72 encoding genes represent less than 2% of the total genome sequence vs. at least 90% of the human genome is actively transcribed the more complex organism, the more it comprises non-coding RNAs World of noncoding RNAs Recent evidence suggests that the non-coding RNAs (ncRNAs) may play major biological roles in cellular development, physiology and pathologies. NcRNAs could be grouped into two major classes based on the transcript size: small ncRNAs and long ncRNAs. 8_MB-2017 56 / 72 Sana et al, J Transl Med, 2012 A new classes of non-coding RNAs 8_MB-2017 57 / 72 HISTORY 1993 Ambros, Ruvkun – discovery of miRNA lin-4 1998 Fire, Mello – RNA interference 1999 Tuschl, Zamore, Bartel, Sharp -RNAi 21-23 fragments 2000-2001 Hannon -Ago2, Dicer 2002 Zamore RNAi and miRNA effector share its orbit 2002 Croce, Calin miR-15,miR-16 in CLL 2004 Croce 50% miRNA genes on chromosome fragile sites 2006 Croce Deregulation of miRNAs in tumor tissue … 2007 The first original work on a topic of miRNAs in oncology in the Czech Republic  Cell, 1993 Nature, 1998 PNAS, 2004 8_MB-2017 58 / 728_MB-2017 microRNA = 43832 microRNA and cancer = 19433 19.10.2015 23.29 59 / 72 MicroRNAs as tumor suppressors or oncogenes 8_MB-2017 60 / 72 MicroRNAs as tumor suppressors or oncogenes Lujambio, Nature 2012 Kong et al, Lancet Oncology 2012 8_MB-2017 61 / 72 Kasinsky, Slack, Nature Reviews Cancer, 2012 MicroRNAs as oncogenes or TS depending on the context 8_MB-2017 62 / 72 • https://www.youtube.com/watch?v=Vh3-NHdjnyQ • microRNA siRNA • https://www.youtube.com/watch?v=5YsTW5i0Xro • https://www.youtube.com/watch?v=cK-OGB1_ELE 8_MB-2017 62 63 / 72 RNA interference in bacteria and archae? • First mentioned in 2007 Sheilagh Molloy (2007): First evidence of prokaryotic RNAi? Nature Reviews Microbiology 5, 329.8_MB-2017 64 / 72 CRISPR system In 2008, it was described RNAi analogous system designed to the degradation of viral NA It uses internal "virus" sequences inserted in the inverted repeats (CRISPR) CRISPR = clusters of regularly interspaced short palindromic repeats After transcription of this sequence leads to their progressive cleavage by Cas proteins The resulting products interfere with the nucleic acid of the entering virus • Each of repeats followed by short segments called Spacer DNA, obtained during previous meetings with relevant bacterial viruses or plasmids. Brouns et al. (2008): Small CRISPR RNAs Guide Antiviral Defense in Prokaryotes, Science 321, 960-964 8_MB-2017 65 / 72 Structure of CRISPR Towards the end of 2008, the CRISPR reported in about 40% of early the sequenced bacteria and archaea All repeats contain a short length of 24-48 nucleotides and space of approximately the same length Marraffini a Sontheimer (2008): Science 322, 1843 – 1845 Edgar (2007): BMC Bioinformatics 8:18 repetitions spacers 8_MB-2017 CRISPR History of viral infections 66 / 72 P. Horvath et al., Science 327, 167-170 (2010) 8_MB-2017 CRISPR / Cas system is a prokaryotic immune system, providing resistance against foreign genetic elements such as plasmids or phages, [3], [4], and therefore constitutes a form of acquired immunity. Spacer DNA of these exogenous genetic elements detected and deactivated in a manner analogous to the mechanism of RNA interference in eukaryotic organisms. [5] CRISPR loci have been found in about 40% bacteria and archaea in 90% [6]. CRISPR interference technology has enormous potential for the application, including altering the human germ line, animals (and other organisms) or modification genes food crops. Delivering a protein and appropriate guidance Cas9 RNA into the cell genome of the target organism can be cut open at any desired point. [7] [8] [9] CRISPRy in connection with specific endonucleases, intended for editing the genome or targeted regulation of genes have already been tested in a variety of organisms [10]. From an ethical point of view seems to be especially worrisome possibility to edit the human germ line. [11] Fig. 3. CRISPR interference. The CRISPR/Cas systems may target either DNA or RNA to interfere with viruses, plasmids, prophages, or other chromosomally encoded sequences. 67 / 72 Horvath, 2010 Science Overview of the CRISPR/Cas mechanism of action. (A) Immunization process: After insertion of exogenous DNA from viruses or plasmids, a Cas complex recognizes foreign DNA and integrates a novel repeatspacer unit at the leader end of the CRISPR locus. (B) Immunity process: The CRISPR repeat-spacer array is transcribed into a precrRNA that is processed into mature crRNAs, which are subsequently used as a guide by a Cas complex to interfere with the corresponding invading nucleic acid. Repeats are represented as diamonds, spacers as rectangles, and the CRISPR leader is labeled L.8_MB-2017 68 / 72 CRISPR restricts horizontal transfer of DNA from Staphylococcus Spacer in CRISPR encodes crRNA CrRNA sequence is homologous to the gene nickase that occurs in almost all conjugative plasmids in Staphylococcus Marraffini a Sontheimer (2008): Science 322, 1843 - 1845 Binding of crRNA to nickase preventing conjugation and plasmid transformation Interference occurs at the level crRNA-DNA rather than mRNA-crRNA CRISPR prevents the spread of antibiotic resistance 8_MB-2017 69 / 72 • CRISPR/Cas9 • CRISPR-associated protein 9 – nuclease from Streptococcus pyogenes • adaptive immunity against bacterial viruses (generally foreign DNA) • RGN – RNA-guided nuclease • sequence specificity is determined by the interactions of DNA-RNA • Bacteria – incorporation of foreign DNA into the CRISPR repeats in the genome • These subsequently transcribed into RNA (crRNA) • crRNA – protospacer –fragment of foreign DNA repeat - CRISPR 8_MB-2017 https://www.youtube.com/watch?v=MnYpp mstxIs What is CRISPR? 70 / 72 • crRNA then hybridized with CRISPR transactivating RNA (tracrRNA) • -This complex interacts with the RNA nuclease Cas9 • - protoscpacer RNA directs the entire complex to the foreign DNA (sequence complementarity) • - resulting ribonucleoprotein complex cleaves the foreign complementary DNA 8_MB-2017 71 / 72 Mechanism of action A) Nuclease Cas9 recognizes a target sequence through crRNA and tracrRNA B) The fission process to participate in a total of five Cas proteins; Cas3 is a nuclease and helicase Brouns, SJJ (2012): Science 337: 808-8098_MB-2017 72 / 728_MB-2017 73 / 72 Year 2013 - enzymes involved in CRISPR mechanism are used as the latest achievement in the preparation of GMO in vitro mutagenesis 8_MB-2017 74 / 72 CRISPR/Cas9 • the whole system is modified for targeted mutagenesis • - vector - gRNA = crRNA tracr + RNA • • part gRNA and 20nt complementary section to the target site in the genomic DNA • - + Coexpression of Cas9 nuclease (even the same vector) 8_MB-2017 75 / 72 • PAM – protospacer adjacent motif • sequence in the vicinity of gDNA • required for efficient cleavage by Cas9 nuclease • the original system "NGG" (but the development of systems with other sequences) • according to the system target sequence must be in the N 20 -GG 8_MB-2017 gDNA libraries available for various organisms - beware of non-specific binding non-specific cleavage - use several different gDNA - reversion of the phenotype by increasing the expression - introduction of mutation - Various companies - different platforms (modifications of the original system) - available software can be used to design 76 / 72 A. Wild-type Cas9 nuclease site specifically cleaves double-stranded DNA activating double-strand break repair machinery. In the absence of a homologous repair template non-homologous end joining can result in indels disrupting the target sequence. Alternatively, precise mutations and knock-ins can be made by providing a homologous repair template and exploiting the homology directed repair pathway. B. Mutated Cas9 makes a site specific single-strand nick. Two sgRNA can be used to introduce a staggered double-stranded break which can then undergo homology directed repair. C. Nuclease-deficient Cas9 can be fused with various effector domains allowing specific localization. For example, transcriptional activators, repressors, and fluorescent proteins.8_MB-2017 77 / 72 PASRs – 12/2008 PaRNA = promotor associated RNA transcriptionally active region is between nucleosomes • transcription of hnRNA • short RNA generated by transcription in both directions Buratowski (2008): Science 322, 1804-18058_MB-2017 78 / 72 paRNA • paRNA is actually simple antisense RNA • occurs concurrently with transcription of hnRNA it is synthesized by RNA polymerase II 8_MB-2017 Their expression is often coordinated with that of neighboring protein-coding genes, and in many cases, related transcripts can influence each other at one step or another during their biogenesis. 79 / 72 2009 lncRNA – long non-coding RNA lncRNA jsou předpovězené B-Okazakiho fragmenty 8_MB-2017 Long non-coding RNAs (long ncRNAs, lncRNA) are non-protein coding transcripts longer than 200 nucleotides.[1] This somewhat arbitrary limit distinguishes long ncRNAs from small regulatory RNAs such as microRNAs (miRNAs), short interfering RNAs (siRNAs), Piwiinteracting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), and other short RNAs.[2] 80 / 72 Long ncRNAs in the regulation of gene transcription 8_MB-2017 In eukaryotes, RNA transcription is a tightly regulated process. NcRNAs can target different aspects of this process, targeting transcriptional activators or repressors, different components of the transcription reaction including RNA polymerase (RNAP) II and even the DNA duplex to regulate gene transcription and expression (Goodrich 2006). In combination these ncRNAs may comprise a regulatory network that, including transcription factors, finely control gene expression in complex eukaryotes. Long ncRNAs in genespecific transcription 81 / 72 LncRNAs in the inactivation of the X chromosome J T Lee (2012): Science 2012;338:1435-1439 Xist lncRNA formed transcription inactivation center (Xic) inactive X chromosome (Xi) Xist RNA covers the entire chromosome expression and sleep patterns of modifications of histones and DNA 8_MB-2017 82 / 72 The core area Xic and its lncRNA J T Lee (2012): Science 2012;338:1435-1439 Tsix = antisense transcript= negative regulator of Xist Jpx = positive regulator Xist 8_MB-2017 83 / 72 Targeting Xist requires three dimensions Dimond A a Fraser P (2013): Science 2013; 341:720-721 Primary binding site for Xist located near the site of transcription locus Xist Xist does not bind to specific sequences (sequential dependency), but in those places where they happen to appear after transcription (spatial dependence) Only then effect of Xist spread over the genome further 8_MB-2017 84 / 72 Interaction lncRNA-protein for initiation XCI J T Lee (2012): Science 2012;338:1435-1439 Tsix preventing connection PRC2-RepA to chromatin and prevents X chromosome inactivation If Tsix does not form, complex PRC2-RepA binds to chromatin PRC2 methylates future Xi Expression of Xist RNA that binds to PRC2 PRC2-Xist extends along the future Xi and methylate an entire chromosome 8_MB-2017 85 / 72 LncRNAs connects epigenetic complexes to chromatin ... J T Lee (2012): Science 2012;338:1435-1439 … allowing allele and locus-specific regulation Emerging lncRNA binds to the complex epigenetic (eg. PRC2) Along with him is bound to chromatin through DNA binding factors (e.g. YY1 for Xist RNA) Epigenetic modifications are put to sleep gene LncRNA rapid degradation prevents its diffusion to other loci 8_MB-2017 86 / 72 Principles of regulation of miRNA and TF O. Hobert Science 319, 1785 -1786 (2008) 8_MB-2017 87 / 72 Regulatory network of miR- 124 E. V. Makeyev et al., Science 319, 1789 -1790 (2008) 8_MB-2017 88 / 72 • https://www.youtube.com/watc h?v=2pp17E4E-O8&t=4s 8_MB-2017 88