Model of a transcription unit RNA polymerase 3. Termination 2. Elongation 1. Bind to promoter DNA double helix Attached RNA transcript Transcription unit Start signal Termination signal 6. Regulation of gene expression in eukaryotes nuclear receptors (cell signaling) Regulation is mediated by 1) interactions of regulatory proteins with regulatory sequences on DNA 2) ncRNA Six steps of information transfer in eukaryotes that constitute potential regulatory points of gene expression Plasma membrane DNA Primary RNA transcript mRNA Transcriptional control mRNA Interactive mRNAProtein Active/ Inactive Protein Processing transcript Nuclear envelope Transport control mRNA stability control Translational control by ribosome selection Protein posttranslation control 1 2 3 45 6 Copyright © mot5111318 Levels of gene expression control 0. Chromatin 1. Where and how often is a given gene transcribed (transcriptional control) 2. How the primary transcript is spliced (posttranscriptional-spliced control) 3. Selection of RNAs to be transported from the nucleus to the cytoplasm (control of RNA transport) 4. Selection of mRNAs to be translated on ribosomes (translational control) 5. Selective destabilization of certain mRNAs in the cytoplasm (mRNA degradation) 6. Selective activation, inactivation and compartmentalization of specific proteins after they have been synthesized (protein activity control - posttranslational control, transport) DNA segments that can modulate transcription by binding gene regulatory proteins Upstream enhancer Downstream enhancer Core promoter Gene sequence CAAT TATA 30 bp ~ 80 bp +1 transcription Regulatory protein DNA binding site Polymerase Unmasked DNA sites Protein/protein interactions +1 +1 +1 A B Copyright © motifolio.com5111320 Regulation is mediated by 1) interactions of regulatory proteins with regulatory sequences on DNA 2) ncRNA Protein-DNA interactions - proteins interact with sugar phosphate skeleton (phosphate) or through grooves with bases - Sequences not sequence specific (skeleton - histones; structurally specific - HMG proteins) or sequentially specific (skeleton + grooves combination: BglII (AGATCT) and BamHI (GGATCC) contact the same bases and They "read" the curvature of the surroundings The shape and charge specificity of DNA determines the types of DNA binding domain DNA…) Types of interactions Phosphates can interact with salt bridges • Arg and Lys - saline • salt bridges • (positive charges Arg and Lys • creates a bond with the negative • phosphate group charge) • Electrostatic charge / surface indicates protein binding capabilities • salt bridges - between phosphates and + charged AK side chains (Lys, Arg, His) • hydrogen bonds - between phosphates, sugars, bases in NK and peptide bond or hydrophilic AK side chains • stacking - between aromatic amino acids (Trp, Tyr, Phe, His) and bases • hydrophobic interactions between bases in NK and nonpolar side chains of AK Hydrogen bonds • sequence-specific protein contacts the base ("direct" • readout) - through a large or small groove - a large groove is • more accessible - hydrogen bonds (donor vs electron acceptor related to recognition and helium - pre interaction and helix with DNA ex. many direct interactions between AK side chains and NA bases. Uncommon interactions: O6 or N7 guan atoms… Side chains Arg, Lys, Gln, Asn, Ser Less often: N6 or N7 adenine atoms Exceptionally: Pyrimidines often occurs: more H-bonds to 1 base water-mediated H-bond between protein and NA Binding of proteins to DNA via hydrogen bonds • The large groove has the size corresponding to the dimensions and the helix • and has exposed H-linking groups • Ade residues C-6 (NH2) and N-7 may form specific ones • hydrogen bonds with Gln and Asn • Gua can form specific hydrogen bonds with Arg • Strong binding, sequence specific - affinity nM - uM • Weak binding, structural specific - affinity uM - mM Protein motifs interacting with DNA Rooman, Marianne and Wintjens, René (March 2015) Protein–DNA Interactions. In: eLS. John Wiley & Sons, Ltd: Chichester. DOI: 10.1002/9780470015902.a0001348. pub3 Motivy- Transkripční faktory –zipper typ GCN4 a AP1 a c-Myc Helix-otáčka-helix – HTH – Winged helix – TALE General transcription factors-motive HTH Zinc finger DNA-interacting p53 protein Rooman, Marianne and Wintjens, René (March 2015) Protein–DNA Interactions. In: eLS. John Wiley & Sons, Ltd: Chichester. DOI: 10.1002/9780470015902.a0001348.pub3 Loop-sheet-helix - loops coming out of main core domain - protrudes b-sheet and α-helix - 3 Cys and 1His coordinate Zn helix in a large groove and loop in a small groove - Activation of transcription through acidic TA domain - core / DNA-binding domain p53 transcription factor important for cell cycle regulation, apoptosis and repair of the damaged DNA (tumor suppressor) Motives receptors for hormons,loop-sheet-helix, GAL4 15 Regulation at the transcriptional level Basic regulation of transcription (common to all genes) Regulation by components of the " basal transcription complex " (RNA polymerase binding to the TATA box, TATA binding proteins and other "basal" transcription factors binding to the RNA polymerase or in the promoter region) Genes regulated only in this way: Constitutively expressed genes Specific effects on gene expression: Through regulatory sequences in DNA and specific transcription factors. basal transcription complex Scheme of the activity of a master gene Master gene regulatory protein mRNA mRNA Protein 1 Protein 3 Gene 1 Gene 2 Gene 3 Gene 4 On Off On Off No protein 2 No protein 4 Binding to DNA elements Copyright © motifolio.com5111321 17 transcription factors Animal Genetics - Gene exoression. [online]. [cit. 2014-08-15]. Dostupné z: http://web2.mendelu.cz/af_291_projekty2/vseo/stranka.php?kod=307 Necessary to initiate transcription They usually induce transcription, exceptionally they can inhibit it Their various combinations bind to the promoter before the RNA polymerase is attached General transcription factors in all or most cell types necessary to induce transcription basal TF-low activity, minimal cell requirements most common: TFIIA, TFIIB, TFIID (includes a subunit called TATA binding protein (TBP) - binds specifically to the TATA box sequence), TFIIE, TFIIF and TFIIH) constitutive TF - increase the basal activity of the cell according to the cell type, the basic requirements of the cell (present (and active) in the cell at all times - general transcription factors, Sp1, NF1, CCAAT) - special transcription factors They apply to inducible transcription - only in cells of certain tissues and certain situations (example p53) https://employees.csbsju.edu/hjakubowski/classes/ch331/bind/eukar ypromNat0703e.htm Figure 1 Comparison of a simple eukaryotic promoter and extensively diversified metazoan regulatory modules. a, Simple eukaryotic transcriptional unit. A simple core promoter (TATA), upstream activator sequence (UAS) and silencer element spaced within 100–200 bp of the TATA box that is typically found in unicellular eukaryotes. b, Complex metazoan transcriptional control modules. A complex arrangement of multiple clustered enhancer modules interspersed with silencer and insulator elements which can be located 10–50 kb either upstream or downstream of a composite core promoter containing TATA box (TATA), Initiator sequences (INR), and downstream promoter elements (DPE). 18 Terminology Enhancers - regulatory sequences in DNA that bind transactivators Transactivators bind coactivators Silencers - regulatory sequences that bind the corepressor Hormones bind to the intracellular receptor, which binds to the hormone response element These terms are still used. The terms are gradually being replaced: regulatory sequences in DNA (enhancer, silencer, hormone response element) specific transcription factors (different from basal transcription factors) mediator proteins -coactivators •Transcription factors are proteins that help turn specific genes "on" or "off" by binding to nearby DNA. •Transcription factors that are activators boost a gene's transcription. Repressors decrease transcription. •Groups of transcription factor binding sites called enhancers and silencers can turn a gene on/off in specific parts of the body. •Transcription factors allow cells to perform logic operations and combine different sources of information to "decide" whether to express a gene. ➢Components of the eukaryotic promoter: 5_regulace_2020FaF 19 http://www.cbs.dt u.dk/dtucourse/co okbooks/dave/Lek t03bkg.html • Basal transcription factors (basal, constitutive) Special transcription factors 1.Constitutive - present (and active) in the cell at all times - general transcription factors, Sp1, NF1, CCAAT 2.Conditionally active their activation required https://employees.csbsju.edu/hjakubowski/classes/ch331/bind/olbindtransciption.html 5_regulace_2020FaF 20 Mechanical division Transcription factors of the general transcription complex (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH - are ubiquitous and react with the promoter (often TATA box) of structural genes, important in the development of vertebrates and invertebrates Upstream transcription factors (UTF) upstream - towards the 5´ part, proteins that bind to the regulatory part of the RNA polymerase I promoter at position -110 to -180, the presence is not necessary to initiate transcription, but multiplies its efficiency (it can also repressive) Inducible transcription factors - same as UTF, but need to be activated or inhibited 5_regulace_2020FaF 21 A. “The eukaryotic transcription apparatus can be divided into three sets, which include the RNA polymerase II core complex and related general transcription factors (TFIIA, -B, -D, -E, -F and -H), multi-subunit cofactors (mediator, CRSP, TRAP, ARC / DRIP, etc.) and various chromatin modifying or remodeling complexes (SWI / SNF, PBAF, ACF, NURF and RSF). B. Metazoa organisms have developed multiple gene-selective and tissue-specific TFIID-like assemblies using alternative TAFs (TBP [TATA Binding Protein] -related factors such as ovarian-specific TAF105), as well as TRF (TBP[TATA Binding Protein-associated factors] related factors, such as is TRF2 in Drosophila and mice), which mediate the formation of specialized RNA polymerase initiation complexes that direct the transcription of tissue-specific and gene-selective expression programs. "(Natural link in the picture above.)"" ➢Methods of activation of transcription factors: ➢ligand-induced conformation change (signal, eg hormone) ➢conformational change after removal of the inhibitory protein ➢conformational transition induced by phosphorylation ➢phosphorylation by protein kinase ➢phosphatase dephosphorylation ➢stabilization of the active conformation of the transcription factor against its degradation 5_regulace_2020FaF 22 Gene regulation by members of the nuclear receptor superfamily A. Glucocorticoid receptor Cortisol Hsp70 Hsp90 Inactive receptor IP Transcription activated Plasma membrane Nucleus Copyright © motifolio.com5111362 The glucocorticoid receptor (GR, or GCR), also known as NR3C1 (nuclear receptors of subfamily 3, group C, member 1), is a receptor to which cortisol and other glucocorticoids bind. GR is expressed in almost every cell in the body and regulates genes that control development, metabolism and the immune response. Because the receptor gene is expressed in several forms, it has many different (pleiotropic) effects in different parts of the body. When glucocorticoids bind to GR, its primary mechanism of action is the regulation of gene transcription. The unbound receptor resides in the cytosol of the cell. After the receptor is bound to the glucocorticoid, the receptor-glucocorticoid complex can proceed in either of two ways. The activated GR complex regulates the expression of antiinflammatory proteins in the nucleus or suppresses the expression of proinflammatory proteins in the cytosol (by preventing the translocation of other transcription factors from the cytosol to the nucleus). In humans, the GR protein is encoded by the NR3C1 gene, which is located on chromosome 5 (5q31). Glucocorticoid receptor - https://en.qaz.wiki/wiki/Glucocorticoid_recepto r Gene regulation by members of the nuclear receptor superfamily B. Estrogen receptor Transcription activated Estrogen Receptor dimer Hsp90 Inactive receptor HAT Coactivator Plasma membrane Nucleus HAT – histone acetyltransferase Copyright © motifolio.com .[2] Estrogen receptors (ERs) are steroid receptors present in the cell nucleus [1] of vertebrates to which estrogen binds. Humans and other mammals have two types of estrogen receptors, the estrogen receptor α (ERα, also ESR1) and the estrogen receptor β (ERβ, also ESR2). Both receptors can form homodimers as well as common heterodimers. However, the GPER receptor, which is a special G protein-coupled receptor, also responds to estrogen. [2] All of these types of receptors also occur in other vertebrates, including fish. [2] Estrogen receptors allow the detection of estrogen at specific sites in the vertebrate body. At rest, they are usually found in the cytosol, while upon binding to the ligand (estrogen), they are activated, dimerized, and enter the cell nucleus. There it binds to DNA sequences known as estrogen responsive units (EREs). The binding is also affected by other co-regulators (coactivators and corepressors). [3] Due to its receptors, estrogen controls reproduction, both the development of the reproductive system and reproductive behavior. The best known, however, is the influence on the development of female (female) genitals. Furthermore has several functions not related to reproduction, e.g. affects bone density and strength, blood lipid levels, fat storage, and management of water with salts, as well as some higher brain functions (memory effect). However, it probably also affects the development of parts of the male reproductive system, such as sperm maturation. [2] Gene regulation by members of the nuclear receptor superfamily C. Thyroid receptor Transcription activated HAT Coactivator HDAC Thyroid Plasma membrane Nucleus Absence of hormone Corepressor Thyroid hormone receptor Transcription repressed HDAC – histone deacetylase HAT – histone acetyltransferase Copyright © motifolio.com 5111364 The thyroid hormone receptor (TR)[1] is a type of nuclear receptor that is activated by binding thyroid hormone.[2] TRs act as transcription factors, ultimately affecting the regulation of gene transcription and translation. These receptors also have nongenomic effects that lead to second messenger activation, and corresponding cellular response.[3] Thyroid hormone receptors regulate gene expression by binding to hormone response elements (HREs) in DNA either as monomers, heterodimers with other nuclear receptors, or homodimers.[4] Dimerizing with different nuclear receptors leads to the regulation of different genes. THR commonly interacts with the retinoid X receptor (RXR), a nuclear retinoic acid receptor.[9] TR/RXR heterodimers are the most transcriptionally active form of TR.[10] Regulation after transcription Alternative splicing, miRNAs and siRNAs, translation initiation factors, & protein modifications. • Even after a gene has been transcribed, gene expression can still be regulated at various stages. • Some transcripts can undergo alternative splicing, making different mRNAs and proteins from the same RNA transcript. • Some mRNAs are targeted by microRNAs, small regulator RNAs that can cause an mRNA to be chopped up or block translation. • A protein's activity may be regulated after translation, for example, through removal of amino acids or addition of chemical groups. 5_regulace_2020FaF 26 Regulation of RNA level rare in bacteria, common in higher organisms RNA processing- alternative splicing mRNA stability - for many genes, RNA interference affects life span or translation rate translation - regulatory proteins bind to mRNA and / or the ribosome and affect the translation rate control options: mRNA degradation rate control (mRNA stability) converting the non-translatable mRNA into a form that can be translated translational control by regulatory proteins binding of antisense RNA to mRNA 27 Regulation of gene expression by transcriptional modification Alternative splicing and variation of thepolyadenylation site at the 3 'end causes a single gene to produce different proteins RNA editing In some cases, the RNA may be edited after transcription. The primary transcript (hnRNA) is identical, after transcription there is a base exchange or nucleotide addition (deletion) Gen apoB produkuje v játrech protein obsahující 4563 AK Tentýž gen v enterocytech produkuje apoB obsahující jen 2152 AK Konverze C(cytosin) na U (uracil) deaminací v RNA transkriptu generuje stop-kodón v intestinální mRNA. Tak protein produkovaný v enterocytu má pouze 48 % délky proteinu hepatálního Syntéza apoB v hepatocytech a enterocytech (je součástí chylomikronů a VLDL) RNA level regulation • RNA stability • mRNA has a short half-life, upon degradation it undergoes ribonuclease degradation • sensitivity to RNases depends on the secondary structure • this may be affected by regulatory signals that induce the binding of regulatory proteins to RNA 5_regulace_2020FaF 28 • Translation regulation • the ribosome binding site (RBS) on the mRNA may be hidden by the secondary structure • cleavage of a portion of the mRNA by RNase III restores RBS accessibility 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 General scheme of messenger RNA decay pathways. 8_MB-2017 (A) mRNAs containing an AU-rich element (ARE) in their 3' UTR undergo rapid AREmediated 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 5_regulace_2020FaF 31 TEST Regulatory mechanisms mediated by transcription factors by RNA 8_MB-2017 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 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 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 RNA interference - RNAi • sequence-specific gene silencing mechanism triggered by double stranded RNA, on the post-transcriptional 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 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 Cenorhabditis elegans Discovery of RNA interference (1998) - silencing of gene expression with dsRNA 8_MB-2017 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 39 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 40 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 41 miRNA biogenesis 8_MB-2017 42 8_MB-2017 43 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 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 44 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 sRNA mode of action also depends on complementarity - incomplementarity in cleavage site prevents RNase activity 8_MB-2017 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 47 8_MB-2017 48 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 (RNA-induced 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 microRNA 5_regulace_2020FaF 49 small non-coding RNA of 18-25 nucleotides in size negative regulatory expression genes that degrade target mRNA or block its translation microRNAs arise from primary pri-miRNA transcripts that are relatively large (even several kb) pri-miRNAs are treated in the nucleus with Drosha RNAase and protein Pasha binding dsRNA to pre-miRNA about 70 nucleotides long with imperfect hair structure pre-miRNAs are exported to the cytoplasm by Exportin 5 and digested with Dicer nuclease to final 22 kb miRNA duplexes The miRNA binds to the RISC, one fiber degrades and the other mediates the degradation or translation inhibition of the respective mRNA 8_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 8_MB-2017 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 MicroRNAs as tumor suppressors or oncogenes 8_MB-2017 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 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 repeat-spacer unit at the leader end of the CRISPR locus. (B) Immunity process: The CRISPR repeat-spacer array is transcribed into a pre-crRNA 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 CRISPR/C as9 • 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 • 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 - nonspecific 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 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 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] 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