MUNI SCI Bi4025en Molecular Biology Mgr. Jiří Kohoutek, Ph.D. 1 Department of Experimental Biology Lecture 8 • Regulation of gene expression in prokaryotes and eukaryotes. 2 Department of Experimental Biology MUNI SCI Content of the Course • 1. Definition and brief history o the molecular biology discipline. • 2. Nucleic acids: primary, secondary and tertiary structure of nucleic acids, conformation of DNA and RNA, different conformations of DNA and their significance for biological systems, genetic information and genetic code. • 3. Molecular structure and replication of prokaryotic and eukaryotic genomes. • 4. Transcription of prokaryotic and eukaryotic genomes, posttranscriptional modifications and processing of RNA, mechanisms of RNA splicing and self-splicing. • 5. Translation of prokaryotic and eukaryotic mRNAs. 3 Department of Experimental Biology MUNI SCI Content of the Course 6. Posttranslational processing of proteins. • 7. Regulation of gene expression in prokaryotes. • 8. . Regulation of gene expression in eukaryotes. • 9. Molecular mechanisms of mutagenesis and recombination. • 10. DNA Repair mechanisms. • 11 Mobile genetic elements, transposons and retrotransposons. • 12. Basic principles of genetic engineering. 4 Department of Experimental Biology MUNI SCI Why to regulate gene expression? • The products of all genome genes are not necessary at every moment of a cell's life - mechanisms to ensure gene expression at the right time and in the right place (spatiotemporal regulation). • Variability of the external environment. Responses to signals from the environment (e.g. temperature, osmotic pressure, nutrient availability,..) and signals from other cells, tissues and organs (e.g. developmental processes, injuries,..). • Variability of the internal environment of the cell during the cell cycle, cell commitment, differentiation and etc. • Gene expression is highly energetic process. 5 Department of Experimental Biology MUNI SCI • Regulation of gene expression in the eukaryotes. 6 Define footer - presentation title / department Eukaryotic gene expression • Eukaryotic gen expression (like us!) can be controlled at various stages, from the availability of DNA to the production of mRNAs to the translation and processing of proteins. • Different genes are regulated at different points, and it's not uncommon for a gene (particularly an important or powerful one) to be regulated at multiple steps. • Chromatin accessibility. • Transcription. • RNA processing. • RNA stability. • Translation. • Protein activity. https://www.khanacademy.org/science/ap-biology/gene-expression-and-7 Department of Experimental Biology regulation/regulation-of-gene-expression-and-cell-specialization/a/overview-of- eukaryotic-gene-regulation MUNI SCI Components of eukaryotic gene expression • Enhancer and Silencer - DNA control element ?™ far from or close a gene or intron. • Activators - bind to enhancers to turn on transcription of a gene o Transcription factors needed for transcription to begin • Repressors - bind to silencers, o Turn off transcription o Block activators from binding to enhancers or their interaction with transcription machinery Repressors ^Activators Chromatin coactivators Ac f\ Ac Ac Ac Ac Me Me Transcriptional I Activators activators \ Ac Closed chromatin Open chromatin Gene "On" 8 Department of Experimental Biology https://www.uwyo.edu > 13-miller-chap-7c-lecture MUNI SCI Components of eukaryotic gene expression • Co-activator and Co-repressor - the distinction between an activator/co-activator and repressor/co-repressor is based on whether or not the protein binds specifically to DNA. • Namely, activators/repressors have DNA binding domains that allow them to bind to DNA. • Co-activators/co-repressors typically don't bind to specific sequences in DNA. • They typically exert their effects on transcription initiation via protein-protein interactions within transcription initiation complexes at promoters, or by modifying histone tails. 9 Department of Experimental Biology https://www.uwyo.edu > 13-miller-chap-7c-lecture MUNI SCI Chromatin accessibility Chromatin accessibility - the structure of chromatin (DNA and its organizing proteins) can be regulated. More open or "relaxed" chromatin makes a gene more available for transcription. Interphase chromatin exists in two different condensation states. Inactive state [Mě| (Měj Active slate Historie acBtyltra esterase Hi ston a deacetylase Histons dem ethy lase ff ff Histone meihyltransferase // / DMA demethylase imethyltrarcslerase Euchromatin Hetero chromatin 1 (.LITI 10 Department of Experimental Biology • Heterochromatin is a condensed form that has a condensation state similar to chromatin found in metaphase chromosomes. Heterochromatin typically is found at centromere and telomere regions, two types: • Facultative • Constitutive • Euchromatin is considerably less condensed. Most transcribed genes are located in regions of euchromatin. https://www.uwyo.edu > 13-miller-chap-7c-lecture MUNI SCI Chromatin accessibility • Transition between state of heterochromatin and euchromatin mediated by various modifications of histones. Heterochromatin (inactive/condensed) Me, I 3 H3 ARTKQTARKSTGGKAPRKQLATKAARKSAPAT 9 Me, I 3 H3 ARTKQTARKSTGGKAPRKQLATKAARKSAPAT 27 Euchromatin (active/open) Me, Ac Ac Ac Ac H3 ARTKQTARKSTGGKAPRKQLATKAARKSAPAT 4 910 14 18 27 Amino acids available for chemical modification Histone tails DNA double helix (end view) (a) Histone tails protrude outward from a nucleosome Unacetylated histones Acetylated histones (b) Acetylation of histone tails promotes loose chromatin structure that permits transcription ©2011 Pearson Edut 11 Department of Experimental Biology https://www.sciencedirect.com/topics/engineering/peptide-bond is MUNI SCI Euchromatin to heterochromatin transition The trimethylation of histone H3 at lysine 9 (H3K9Me3) plays an important role in promoting chromatin condensation to heterochromatin. Trimethylated sites are bound by heterochromatin protein 1 (HP1) which self-associates and oligomerizes resulting in heterochromatin. Heterochromatin condensation is thought to spread laterally between "boundary elements" that mark the ends of transcriptionally active euchromatin. Recruitment of the H3K9 histone methyl transferase (HMT) to HP1 sites promotes heterochromatin spreading by catalyzing H3 methylation. 12 Department of Experimental Biology https://www.uwyo.edu > 13-miller-chap-7c-lecture Histone H3K9 methyl transferase Binding of HP1 chromodomain to H3K9Me3 HP! oligomerization Heterochromatin MUNI SCI Euchromatin to heterochromatin transition Another mechanism involved in heterochromatin formation from euchromatin is direct histone deacetylation. The UME6 repressor binds to URS1 control elements and recruits a co-repressor complex containing SIN3 and RPD3 to these sites. • RPD3 is a histone deacetylase, an enzyme removing acetyl groups from histones in the vicinity of the URS1 sequence. The nucleosomes bound to DNA in this region (which contains a TATA box promoter) subsequently condense, and expression of the gene is repressed. Repressor-directed histone deacetylation Deacetylation of histone RPD3 "H^ N-terminal tails Zl 77— 77 77 1Z ii • • N-terminal • * • • tail 13 Department of Experimental Biology https://www.uwyo.edu > 13-miller-chap-7c-lecture MUNI SCI Heterochromatin to euchromatin transition • In general, the genes can be turned on by histone acetylation and chromatin decondensation. For instance, GCN4 activator first binds to its UAS upstream of the TATA box of a regulated gene and recruits a co-activator complex containing the GCN5. Activator-directed histone hyperacetylation Hyperacetylation of histone GCN5 ^ N-terminal tails Si • The GCN5 acetylates histone N-terminal tails. • Hyperacetylation of histones leads to chromatin decondensation. • General TFs and RNA Pol II are then able to interact with the promoter, and the gene is transcribed. 14 Department of Experimental Biology https://www.uwyo.edu > 13-miller-chap-7c-lecture UNI SCI DNA Methylation • Cytosine methylation in higher eukaryotes: 10-30%. • Mediated by DNA methyltransferases (DNMTs). • Targeted sequence is short: GC in animals and GNC in plants. • DNA methylation typically weakens gene expression. • Genes with continuous transcription mostly do not have GC methylated islands. • Methyl groups protrude into a large DNA groove „ thus preventing proper binding of transcription factors". 15 Department of Experimental Biology MUNI SCI DNA Methylation • The process of DNA methylation involves the transfer of methyl group from S-adenosylmethionine (SAM) to the C-5 position of cytosine, catalyzed by DNA methyltransferases. • DNA methylation is an "epigenetic switch" that regulates the balance between "open" and "closed" form of chromatin by changing the interactions between DNA and protein. "dma M£thylatí&*v nh, (dnmti; NH, J II I H i li ^C. CH I h Cytosine 5' NTetbyl-tytosine Active (open) chromatin Condensed (closed) chromatin '$L'jJ(tht-d r r -Switched Unmet hylated cytokines Methylated cytosines 16 Department of Experimental Biology Pharmacology & Therapeutics (2018), https://doi.Org/10.1016/j.pharmthera.2018.02.006 MUNI SCI Transcription - activity of eukaryotic cell • Transcription is a key regulatory point for many genes. Sets of transcription factor proteins bind to specific DNA sequences in or near a gene and promote or repress its transcription into the RNA. • Unlike prokaryotes, eukaryotic genes are not completely turned on or off, but there is modulation of transcription. • Basal transcription - with the participation of basal TF, transcriptional levels, minimum level of transcription. • Constitutive transcription - with the participation of basal and constitutive TFs, allowing different transcription rates of different gene. o General TF = basal + constitutive, activate operational genes • Induced transcription - transcription regulated by inducible specific TFs which activity is influenced by stimuli from the external or internal environment. o Specific TF= cellular and time-specific regulatory proteins. MUNI 17 Department of Experimental Biology r> r» t Requirements for transcription initiation • Positioning RNA-polymerase in the active state. • Binding of TF to the promoter (with the participation of activators and coactivators, necessary to create a pre-initiation complex. • Binding of specific (inducible) TFs to transcription enhancers with unique response sequences (RE). • TF interaction allowing the transcription promoter and enhancer to interact. • Active RNA polymerase state. , „ ^ , , https://www.khanacademy.orq/science/bioloqy/macromo MUNI 18 Department of Experimental Biology r. ' . .' , . . a. a' r _ _ _ v v a' acids/a/orders-of-protein-structure Q P T Transcription initiation by RNA Pol II • RNA Polymerase II (RNA Pol II) requires general TFs in addition to tissue-specific transcription factors for transcription of most genes in vivo. • General transcription factors, TFs, position RNA Pol II at start sites and assist the enzyme in melting promoter DNA. General TFs are highly conserved across species. The general TFs used at TATA box. • Architectural regulators facilitates DNA looping. Such as, TFIID consists of TBP (TATA box binding protein) and 13 TBP-associated factors (TAFs). ^ , , https://view.officeappsJivexom/op/view.aspx? MUNI 19 Department of Experimental Biology . '. ... .„ ^„ . .' 0n™ft,~,-, ^ r a* scriptionshde.ppt&wdOrigin=BROWSELINK Q p T Characteristics of transcription factors (TF) • Provide a response to various extracellular or intracellular stimuli indicating the need to turn on one or more genes. • Unlike most proteins, they are able to enter the nucleus. • Recognize and bind to specific DNA sequences. • Make contact with the transcription apparatus, either directly or indirectly. 20 Department of Experimental Biology MUNI SCI Characteristics of transcription factors (TF) • DNA-binding domains of several transcription factors employ similar tertiary structure - motif. DNA- binding motifs are evolutionarily conserved AA sequence which have a defined conformation since AA sequence ultimately determines structure. •A given DNA-binding motif can occur in a number of proteins where it carries out the same or similar functions. • For examples of the coiled-coil, EF hand/helix-loop-helix, and zinc-finger motifs. (a) Coiled-coil motif n n (b) EFhand/helix-loop-helix motif (c) Zinc-finger motif Ca2+ Asn 21 Department of Experimental Biology https://view.officeappsJivexom/op/view.aspx?src=http%3A%2F%2Fwww.hixonparvo.info%2FTran scriptionslide.ppt&wdOrigin=BROWSELINK MUNI SCI DNA-binding motifs in TFs • DNA-binding motifs bind specifically to DNA via non-covalent interactions. • Helix - Turn - Helix. The second helix in this motif (the DNA recognition helix) typically binds to a specific sequence of bases in DNA. • Alpha-helices are one of the most common types of DNA-binding sequences. The side-chains of residues within the g-helix often bind to the surfaces of bases exposed in the major groove of double-helical DNA. Binding to phosphates and bases in the minor groove typically is less important. • Helix-turn-helix TFs are common in bacteria. MUNI SCI DNA-binding motifs in TFs Zinc finger motif i-—-1 Zinc finger motif Zinc finger motif Figure 28-12 Lehninger Principles of iiicchc:r:istr/, Seventh Editi © 2017 W. H. Freeman and Company • Zinc finger - most common DNA-binding motif in human and multicellular animal. Two types of zinc finger TFs: • C2H2 zinc finger TFs- • 2 cysteine (Cys) • 2 histidine (His) • residues bind to zinc ions (Zn2+) and the a-helix containing the 2 histidines binds to bases in the major groove. • Most TFs containing this motif are dimeric. Nuclear receptors, which bind steroid hormones and other compounds, contain this motif. Zinc ions are bound to the DNA recognition helix of this motif, which contacts bases in the major groove. 23 Department of Experimental Biology https://www.coursehero.com/Lehninger_Ch28.ppt-28, Regulation of Gene Expression MUNI SCI DNA-binding motifs in TFs • Leucine-zipper TFs • Contain extended a-helices wherein every 7th amino acid is leucine. This periodicity creates a nonpolar face on one side of the helix that is ideal for dimerization with another such protein via a coiled-coil motif. • So-called basic zipper (bZip) TFs have a similar structure except that some leucines are replaced by other nonpolar amino acids. • The N-terminal ends of both leucine-zipper and bZip proteins contain basic amino acids that interact with bases in the major groove. MUNI SCI DNA-binding motifs in TFs • Helix - Loop - Helix TF Another class of TF, the basic helix-loop-helix (bHLH) proteins are similar to bZip proteins, but contain a loop between the DNA recognition helix and the coiled-coil region. bZip and bHLH proteins commonly form heterodimeric TFs. h o I II r—n—c—c—r' I I h ch2 Glutamine (or asparagine) ch2 h o I II r—n—c—c—r I I ... h ch2 Argimne j ch2 ch2 nh I h—n— |-"+vn—h \ N- (d) Thymine = Adenine °"'h-n/*"n Cytosine=Guanine Basic residues Figuře 2B-10 Ich n Inger Prlfítíp *cs of Moc! OIOlJW.H.Fiwmanaid Department of Experimental Biology https://view.officeapps.live.co m/op/view.aspx?src=http%3A%2F%2Fwww.hixonparvo.info°/o2FT ran scriptionslide.ppt&wdOrigin=BROWSELINK https://www.coursehero.com/Lehninger_Ch28.ppt- 28, Regulation of Gene Expression RNA Pol II preinitiation complex formation TATA box TBPO TFIIB TFIIE 26 Department of Experimental Biology • The sequential steps leading to the assembly of the RNA Pol II pre-initiation complex. • 1. TBP binds to the TATA box and bends (DNA looping) DNA near the promoter. • 2. TFIIB binds, and then a complex between Pol II and TFIIF loads onto the promoter. • 3. TFIIF positions the Pol II active site at the mRNA start site and helps maintain chromatin at the promoter in an uncondensed state. • 4. TFIIE then binds creating a TFIIH docking site. MUNI SCI https://view.officeappsJivexom/op/view.aspx?src=http%3A%2F%2Fwww.hixonparvo.info%2FTran scriptionslide.ppt&wdOrigin=BROWSELINK RNA Pol II preinitiation complex formation TFIIH O Preinitiation complex Nascent RNA Release of general factors, except TBP 27 Department of Experimental Biology • 5. With the addition of TFIIH, the assembly of the pre-initiation complex is complete. • 6. Subsequently, one subunit of TFIIH melts DNA at the promoter, obtaining energy by ATP hydrolysis. RNA Pol II then begins transcribing the mRNA. • Another subunit of TFIIH phosphorylates the RNA Pol II CTD, making RNA Pol II highly processive. • Tissue-specific TFs bound to enhancers and promoter-proximal elements also play important roles in transcription initiation in vivo. https://view.officeappsJivexom/op/view.aspx?src=http%3A%2F%2Fwww.hixonparvo.info%2FTran MUNI scriptionslide.ppt&wdOrigin=BROWSELINK SCI Elongating Pol II with phosphorylatecl CTD Transcription - elongation phase • Right after transcription initiation the RNA polymerase II is paused typically 30-50 nucleotides after transcription initiation site. WELF Negative transcription elongation factors DSIF, NELF. • RNA Pol II undergone promoter escape and contains phosphorylation of serine 5 (Ser5) in the C-terminal domain (CTD). 55^ QAflA iiiiiiütui DPE.Í PB ON Kinase 28 Department of Experimental Biology Cell, 2011, Levine M., May 13, 145 Recruitment of P-TEFb (positive transcription elongation factor b) causes phosphorylation of Ser2 in the CTD, resulting in RNA Pol II transcription elongation. MUNI SCI Transcription - elongation phase Posttranslational modification, phosphorylation in particular, of CTD of RNA Pol II corresponds with specific phase of transcription. Other modification of CTD of RNA Pol II. o Acetylation o Proline isomerization. We talk about CTD code. Phosphorylation state Proline isomerization state Department of Experimental Biology Trends Genet. 2012 Jul;28(7):333-41. c o c E o o CD YSPTSPS none • YSPTSPS S2 • YSPTSPS T4 • YSPTSPS S5 • YSPTSPS S7 • • YSPTSPS S2,T4 • • YSPTSPS S2, S5 • • YSPTSPS S2, S7 YSPTSPS T4.S5 • • YSPTSPS T4.S7 • • YSPTSPS S5,S7 • mm YSPTSPS S2,T4, S5 • • • YSPTSPS S2,T4, S7 • • • YSPTSPS S2, S5, S7 • YSPTSPS T4, S5, S7 • •• • YSPTSPS S2,T4, S5, S7 XCO c £ o o YSPTSPS c c cis, els YSPTSPS ^ T els, trans YSPfTS^S trans, els YSPTSPS trans, trans X 52 repeats in mammals [minus the changes in the non-consensus repeats (Figure 2)] X 26 repeats in yeast TRENDS in Genetics MUNI SCI Transcription - elongation phase • Modification of CTD of RNA Pol II coincides with various steps in transcription. o DNAand chromatin remodeling. o Histone modifications. o DNA processing. o Transcription termination and polyadenylation. GO Protein-coding genes (b) snRNA genes sn/snoRNA genes (yeast and mammals) (mammals) (yeast) Start _ Start Initiation ( y Mediator (1 MDa) Initiation ___ ,--- - TBP YSPTSPS C__ _3 —»- TBP _) —*~ TBP Histone modification. ( \ Capping enzyme \ bpÖ ( 'i ) /^„H hiln^-l LJ^.j-,i \ Histone YSPTSPS YSPTSPS RNA 5' end capping \_JVL_-Jv/L/ (^9*1 i MCG1, MCG1 ) YSPTSPS^S modification, RNA 5' end { \ Capping enzyme VJ?L_nJ~*~ - RPAP2 /integratorJ K /RPAP2N ^L_^J$Ls . Integrator YSPTSPS (Int4) YSPTSPS^S { I ! Histone i modification, splicing, elongation E YSPTSPS i ) ---- j IS) y > Pin1/Ess1, - VQDTQDQ Scp1, Prp40, CA150, i isrisra RecQ5, U2AF65, Set2, ■ ^-Ssd1, Hrr25, Yra1 1 J >■ Spt6, Npl3 YSPTSPS RNA 3 end CZ) ssu72 YSPTSRS (Z*) Nrd1 YSPTSPS processing and termination pc111 7 YSF?TSI?S / C&Z) Rtt103 / YSPTSPS RNA 3' end processing and termination r ^tegrata^L|X I /^PAP2 \ J / YSPTSPSYSPTSPS / L->-int11 dj) Ssu?2 YSPTSRS GH) ^ Pct11 YSPTSPS \ El id End TRENDS in Genetics 30 Department of Experimental Biology Trends Genet. 2012 Jul;28(7):333-41. MUNI SCI Enhancers and Activators • Distal control DNA elements, which are called enhancers, may be far away from a gene or even located in an intron. • Enhancers can be thousands of nucleotides away from the TATA box of the promoter. • Can be bound by activators. •Activators have two domains DNA-binding, protein-binding, and/or signal molecule-binding domains coupled with transcription activating domain. • Facilitate a sequence of protein-protein interactions that result in transcription of a given gene. • Some regulate a few genes; some regulate many hundreds of genes. https://www.camphillsd.k12.pa.us/shortchap18.ppt |J 31 Department of Experimental Biology https://ww2.chemistry.gatech.edu/Lehninger_Ch28.ppt Q n T Enhancers and Activators \ dna: Activators Promoter Gene Enhancer Distal control element TATA box General transcription '/^ O factors Group of mediator proteins RNA polymerase II RNA polymerase II ©20n Pearson Education, Inc. 32 Department of Experimental Biology Transcription^ ^ initiation complex RNA synthesis https://www.camphillsd.k12.pa.us/ short chap 18.ppt MUNI SCI Chromatin remodeling complexes • CRE - cAMP response element. • CREB - (cAMP response element-binding protein/CRE-binding protein), o Specific transcription factor. o Transcription of genes regulated by CREB: somatostatin, c-fos, tyrosine hydroxylase, neuropeptides, enkephalin, genes involved in circadian rhythm control, and more. • CBP-CREB binding protein, o Histon acetyltransferase. o Co-activator CREB. o Many other transcription factors (c-myb, c-fos, p53, E2F, NF-kB,...). 33 Department of Experimental Biology MUNI SCI Chromatin remodeling complexes mRNA Core histones Repressed chromatin Decreased transcription Inflammatory gene silencing Active chromatin Increased transcription Inflammatory gene activation 34 Department of Experimental Biology European Respiratory Journal 2005 25: 552-563 MUNI SCI Chromatin remodeling complexes Gene repression DNA Histone acetylation Gene transcription Nuclepsome HATs. cbp p300, rnA polymerase II i v_/r\ij QIC. COACTIVATORS Transcription factor r Histone deacetylation HDAC1-11 NCoR, NuRD, mSin3, etc. COREPRESSORS /\A/V mRNA Acetylation of lysines Inflammatory protein 35 Define footer - presentation title / department European Respiratory Journal 2005 25: 552-563 MUNI SCI Mediator complex Mediator is an ~30-subunit multiprotein complex. Mediator functions as a molecular bridge between RNA Pol II and transcription factors bound to enhancers and promoter proximal transcription control sequences. Mediator influences all stages of transcription - from initiation to elongation. Non-coding RNA interacts directly with Mediator to influence transcription. CihE Mec!4 Medl Med5 |cdk| CytC Med 12 Med 13 Med2E Media fei^T ^ Medl7 Mcdll Med22 McdíŮ McdĚl Media MedĽ vicdm Medii MM3 Med iE. tail |middle| GENE "OFF" Diagram of mediator with a CDK kinase domain Condensed chromatin General transcription factors RNA polymerase 36 Department of Experimental Biology PNAS, 2012, 109 (48) 19519-19520 https://en.wi kipedia.org/wi ki/Mediator_(coactivator) MUNI SCI Mediator complex neighboring genes using a cis-mediated mechanism. 37 Department of Experimental Biology Trends in Biochemical Sciences, Nov 2013, Vol. 3, Is. 11, P531-537 MUNI SCI RNA processing Splicing, capping, and addition of a poly-A tail to an RNA molecule can be regulated. Different mRNAs may be made from the same pre-mRNA by alternative splicing. pre-mRNA spliced mRNA Department of Experimental Biology https://courses.lumenlearning.com/wm-biology1/chapter/reading-post-translational control-of-gene-expression/ Alternative splicing • Alternative RNA splicing is a mechanism that allows different protein forms to be produced from one gene when different combinations of introns, and sometimes exons, are removed from the transcript. • It is a common mechanism of gene regulation in eukarvotes; according to one estimate, 70 percent of genes in humans are expressed as multiple proteins through alternative splicing. • The cause of many genetic diseases is improper alternative splicing rather than mutations in a sequence. Exon skipping Alternative 5' donor sites Alternative 3' acceptor sites Intron retention 39 Department of Experimental Biology https://courses.lumenlearning.com/wm-biology1/chapter/reading-post-translational-control-of-gene-expression/ MUNI SCI Splicing Produces Related but Distinct Protein Isoforms FQFR2 gene Exonsi 12 14 1618 Ext«rior Cell membrane Cytoplasm Fibroblast growth factor receptor 2 First isoform Fibroblast growth factor receptor 2 Second isoform FlftiF*«-l9 mtfodttt m to Omak Ano^n. lenth tdrtion ■ Jill / W M ftttmtn mů Company Department of Experimental Biology https://slideplayer.com/slide/5746723/ UNI SCI RNA stability • RNA stability. The lifetime of an mRNA molecule in the cytosol affects how many proteins can be made from it. • Capping. • Degradation of mRNA. • Small regulatory RNAs called miRNAs can bind to target mRNAs and cause them to be chopped up. • Small nuclear RNA - 7SK RNA, IMsnRNA. • Long noncoding RNA. MUNI 41 Department of Experimental Biology https://open.lib.umn.edu/evolutionbiology/chapter/5-8-using-the-genetic-code-2/ r» t O U 1 Regulation of 5 - CAP g Standard mode of translation f Pioneer round of translation NPC lmp-p e mRN A export c Histone pre-mRNA '/ /dna a Transcription elongation I Pre-mRNA processing The 5' cap has four main functions: • Regulation of nuclear export of RNA is regulated by the cap binding complex (CBC), which binds to 7-methylguanylate-capped RNA. The CBC is then recognized by the nuclear pore complex and the mRNA exported. • Prevention of degradation by exonucleases. • Promotion of translation. • Promotion of 5' proximal intron excision. 42 Department of Experimental Biology https://wou.edu/chemistry/courses/online-chemistry-textbooks/ch450-and-ch451 -biochemistry-defining-life-at-the-molecular-level/chapter-10-transcription-and-rna-processing/ MUNI SCI Degradation of RNA The first step of untranslated mRNA degradation is deadenylation, removal of polyA tail. Deadenylated mRNAs are degraded in the 3'-to-5' direction by the exosome complex or, mainly, are transferred to P-bodies for decapping. eiF4G mRNA Pabl Ti7Gppp H. < t. AAAAA7 F4E %, ' elF4G m7Gppp. elF4E ati 3ati Pop2 Caf^ .AAA Ccr4 f Cafi^o Noti/2/3/4/5 Dhhi m7Gp6p Lsmi-7 Dcpi/2 AA^4 rn7Gppp exosome Edci/2 X • In P-bodies the m7Gppp cap is removed by Dcp1/2 - dipeptydil carboxypeptidase 1 and 2. • RNA without cap is degraded in the 5'-to-3' direction by the Xrn1-5'-3'exoribonuclease 1. Xrni Lsmi 43 Department of Experimental Biology FEMS Yeast Research, Volume 18, Issue 6, September 2018, foy050, MUNI SCI MicroRNA miRNA • 18-25 nt, single-strand molecules. • Post-transcriptionally regulate gene expression. • 2694 human miRNAs (miRBASE in 22, March 2018). • Evolutionarily conserved and transcribed by RNA Pol II. • 1-2 % of the genome. • Genes for miRNAs on all human chromosomes except Y. • Regulation of the expression of up to 50% of protein-coding genes. • One miRNA can regulate tens to hundreds of target mRNAs. MUNI 44 Department of Experimental Biology r> r» t Biogenesis of miRNA A miRNA is first transcribed from the mir gene as a long RNA molecule being modified at the 5' end by Cap and polyA at the 3' end. It is called pri-miRNA. The pri.-miRNA forms base pairs with itself and folds over to make a hairpin. Next, the hairpin is chopped up by Drosha enzymes, releasing a small double-stranded fragment of about 22 nucleotides recognized as pre-miRNA. Biosynthetic pathway of microRNA 45 Department of Experimental Biology MUNI SCI Biogenesis of miRNA • The pre-miRNA is exported form the nucelus by Exprotin-5 complex. • In the cytoplasm pre-miRNA i processed by Dicer to dsRNA. • The dsRNA is transfered to the RISC -RNA-induced silencing complex and one of the strands is degraded and the other one is the mature miRNA targeting particular sequence. • RISC with miRNA initiates o mRNA cleavage. o Translation repression. Biosynthetic pathway of microRNA 46 Department of Experimental Biology MUNI SCI Function of miRNA Physiological function o Proliferation, o Differentiation, o Apoptosis and etc. Tumorigenesis. Other miRNAs miß-30c, -103, -2ĎQ, -107, -182, -129 chrl Other mfRNAs? Ge no toxic stress chrll Apoptosis Cell cycle arrest Senescence Bel 2 \ _mm iR-34's CdkA WET 47 Department of Experimental Biology https://cen.acs.org/articles/89/web/2011/04/Pyrrolysine-Synthesis-Revealed.html MUNI SCI Small nuclear RNA Small nuclear RNA (snRNA) is one of the small RNA with an average size of 150 nt. Eukaryotic genomes code for a variety of non-coding RNAs. snRNA is a class of highly abundant RNA, localized in the nucleus with important functions in intron splicing and other RNA processing. Ul snRNA ( B2 RNA / -. 75KSF1RNA H Promoter Promoter ^ _JJ > Initiation * Droximal * Elongation t Termination activation r pausing ******* IKI U |\| 48 Define footer - presentation title / department Trends in Genetics, Mar 2021, Vol. 37, Is. 3., P279-291. SCI Long noncoding RNA - IncRNA • Longer than 200 nt; encoded by genes on different chromosomes (in non-coding regions, even in gene introns). • Usually transcribed RNA pol II or RNA pol III, often have a cap at the 5'-end and a poly(A) at the 3'-end, subject to splicing; do not have ORF, cannot be translated. • Often tissue-specific and developmental stages specific. • Some IncRNA found in specific DNA locus. • Changes in the expression of IncRNAs associated with various diseases (cancer, Alzheimer's, atherosclerosis), can also serve as markers. • Present in body fluids, possibility of non-invasive diagnostics. • In humans about 50,000 -100,000 genes for IncRNA, up to 270,000 different transcripts IncRNAs. 49 Department of Experimental Biology MUNI SCI Effect of IncRNA on gene expression I'rnltm g%--, ( \ ( hnuir:ilin reitiťrtJťUlLR ,,,,t^JL_ V TianstripliüT fiClot I ncK\A I rjirtfi plirmjl -■hi---!'..... Trid\íip!itiTiůl| ^ u [tri1 ■■ I a H ^ P TraiHliitfuii i nil Million OsiHlťiiitJ tllMlulnlinn e-1 SiMflijIdilili» Liil-KNA roicm r " £~ lltgrjidiirimi 50 Department of Experimental Biology J Cell Mol Med. 2017 Dec;21(12):3120-3140. MUNI SCI Effect of IncRNA on gene expression LncRNAXist regulation network of genetic interactions. Xi t T Nuceloscm« pOKSmtí I H3K27me3 H2AK119* I it1.Cpi/ — i Maintaining XCI Nucleus HRNHEJ BRCA1 DSU Cytoplasm BARD Scaffold for proteins microRNA decoy GUARDIN 51 Department of Experimental Biology Front. Cell Dev. Biol., 10 June 2021 • Cytoplasm - sponge - stabilizes TRF2 mRNA • Nucleus - enables interaction of BRCA1 and BARD to help recruit DSB machinery. MUNI SCI Regulation of translation Translation of mRNA involves many "helper" proteins, which make sure the ribosome is correctly positioned. Eukaryotic initiation factor-2 (elF-2) binds to a part of the ribosome called the small subunit. When elF-2 is phosphorylated, it's turned "off,, -it undergoes a shape change and can no longer play its role in initiation, so translation cannot begin. When elF-2 is not phosphorylated, in contrast, it's "on" and can carry out its role in initiation, allowing translation to proceed. elF2 Ribosome small (40S) subunit elF2 Ribosome small (40S) subunit When elF2 is phosphorylated, translation is blocked. — No Translation When elF2 is not phosphorylated, translation occurs. Translation occurs In this way, phosphorylation of elF-2 acts as a switch, turning translation on or off. 52 Department of Experimental Biology MUNI SCI Signaling pathways in regulation of translation 5' UTRs Extremely short 5' UTR and/or TISU element Cyclins, BCL-xL, MCL-1,VEGF,c-MYC Components of the ETC complexes Skp2, pl20ctn, BRCA1/2,MRE11 8CL-2, c-MYC, Cydins 53 Department of Experimental Biology • The mTOR and MAPK pathways affect the translatome by modulating the expression of specific subsets of mRNAs. • Phosphorylation of the 4E-BPs by mTOR leads to their dissociation from elF4E, which stimulates the interaction of elF4E with elF4G and assembly of the elF4F complex. • Phosphorylation of elF4E also seems to bolster the translation of mRNAs encoding proteins involved in tumor dissemination. • Also elF4A promotes the translation of mRNAs with G/C-rich 5' UTR sequences, such as the 12-nucleotide guanine quartet (CGG)4 motif, which can form RNA G-quadruplex structures. MUNI https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5974435/ O 0 J. Signaling pathways in Inflammatory cytokines Environ mental stress Growth factors regulation of translation The Ras/ERK and p38MAPK pathways are activated by a wide range of stimuli, including cytokines, growth factors, and diverse environmental stresses. MNK interacts with elF4G and phosphorylates elF4E on Ser209, a site that increases its oncogenic potential and facilitates the translation of specific mRNAs. RSK phosphorylates rpS6, elF4B, PDCD4, and eEF2K, which are important regulators of translation. ERK and RSK also collaborate in the regulation of ribosome biogenesis by promoting TIF-1A phosphorylation. 54 Department of Experimental Biology https://www.cell.com/fulltext/S0960-9822(02)01135-1 MUNI SCI THANK YOU FOR YOUR ATTENTION. 55 Department of Experimental Biology https://steemit.eom/science/@pjheinz/control-of-gene-expression-part-ii MUNI SCI