Transcription activation in multicellular organisms. .iam.Keeqan@ceitec.muni.cz Eukaryotic total RNA. Ribosomal RNAs and tRNAs are major bands, mRNA is a smear on denaturing gel stained with Ethidium Bromide. Figure 1. Intact vs. Degraded RNA. Two ug of degraded total RNA and intact total RNA were run beside Annbion's RNA Millennium Markers™ on a 1.5% denaturing agarose gel. The 1SS and 2SS ribosomal RNA bands are clearly visible in the intact RNA sample. The degraded RNA appears as a lower molecular weight smear. ľ0000'SI0r3U3Sj/68££'0I :j°P ™oij psjdBpy VNduM 1=1 VNyus H VNJdOUS H VNyou|J8M;o H OOOOOOOO o/0\ uv\\i sssq VNbPJP 1=1 1=1 }S!X 1SZ VNU!W 1=1 VNyous VNyus 1=1 1=1 VNßdi =] [=1 30000 OOOOO ooooo OOOOO ooooo ooooo ooooo ooooo ooooo ooooo ooooo ooooo ooooo ooooo ooooo ooooo ooooo ooooo ooooo sa|n39|oiu jo jaquinu Áq y^H VNyus □ VNyous h •••••••••• VNÍdULj □ •••••••••• isz 1=1 OOOOOOOOOO VNyou|j9Luo 1=1 OOOOOOOOOO VNiPJP 1=1 ########## VNdlLU 1=1 • •#•••< m h ••••ooo< }S!X VNidlw 1=1 1=1 VNÍdOU| jámo =] 1SZ 1=1 1=1 VNyous 1=1 VN^w =1 VNidl 1=1 VNyJ m OOOOOOOOOO OOOOOOOOOO OOOOOOOOOO OOOOOOOOOO OOOOOOOOOO OOOOOOOOOO OOOOOOOOOO OOOOOOOOOO OOOOOOOOOO OOOOOOOOOO ssbui Áq VXTH • Eukaryotic RNA Polymerases Three nuclear RNA polymerases (E. coli has only one). RNA pol I (in nucleolus) ^ transcribes rRNA genes -^50-70 % cell's RNA synthesis resistant to > 500 \ig/m\ a-amanitin, an octapeptide from Amanita phalloides (Death Cap Mushroom) that grows near Oak trees. RNA pol II (in nucleoplasm) transcribes all protein-encoding genes <& most small nuclear RNAs -> 20 - 40% cell's RNA synthesis inhibited by low ~ 0.03 |ug/ml a-amanitin RNA pol III (in nucleoplasm) transcribes 55, tRNA genes <& some small nuclear RNAs -> < 10% cell's RNA synthesis inhibited by 20 |ug/ml a-amanitin in animal cells resistant to a-amanitin in yeast and insects 20.2 Eukaryotic RNA Polymerases Consist of Many Subunits 25 Related to bacteria! sub-unit ji' Binds DNA Has CTD = (YSPTSPS)n [yeast rs - 26; mouse n = 52] Related to bacterial subunit (3 Binds nucleotides Related to bacterial subunit a -Common to all three polymerases -Common to atl three polymerases Common to all three polymerases Figure 20.02: Some subunits are common to all classes of eukaryotic RNA polymerases and some are related to bacterial RNA polymerase. All eukaryotic RNA polymerases have ~12 subunits and are complexes of -500 kD. Some subunits are common to all three RNA polymerases. The largest subunit in RNA polymerase II has a CTD (carboxy-terminal domain) consisting of multiple repeats of a heptamer. Eukaryotic RNA polymerases are similar to that of E. coli but have 12 subunits. E. catf&ore Eukaryotic RNA polymerase RNA polymerases I ll in CTD p] and /Mike subunits a subumts • • • 1 • • • 0 0 0 > o o o J L_ ■ □ □ □ J +5 +4 ♦7 \ Common subunits 1 Additional +7 > en zym ^specific J subunits Transcription of protein-coding mRNAs by RNA polymerase II. Defining RNA polymerase II promoters. 20.5 The Start Point for RNA Polymerase II • RNA polymerase II requires general transcription factors (called TFNX) to initiate transcription. • RNA polymerase II promoters frequently have a short conserved sequence Py2CAPy5 (the initiator Inr) at the start point. • The TATA box is a common component of RNA polymerase II promoters and consists of an A-T-rich octamer located ~25 bp upstream of the start point. TATAA ......N20...... YYCAYYYYY......N54......AG AC Figure 20.07: A minimal pol II promoter i_____i i__i i_T_i TATA box Iní DPE may nave a TATA box ~25 bp upstream of Core promoter w the Inr. containing TATA ^ TATA-1 ess ^ core promoter 20.5 The Start Point for RNA Polymerase II • The downstream promoter element (DPE) is a common component of RNA polymerase II promoters that do not contain a TATA box (TATA-less promoters). • A core promoter for RNA polymerase II includes the Inr and, commonly, either a TATA box or a DPE. - It may also contain other minor elements. 20.6 General transcription factors (GTFs) TBP in TFIID Is a Universal Factor Pol III promoters ■ TBP TFimB Pol I promoters SI 1 RNA polymerase I Pol II promoters TBP TATA-binding protein (TBP) is a component of each of the different positioning factors required for each type of RNA polymerase to bind its promoter. The factor for RNA polymerase II is TFMD, which consists of TBP and ~14 TAFs, (TBP-associated factors) with a total mass -800 kD. Figure 20.08: RNA polymerases are positioned at al promoters by a factor that contains TBP. 20.6 TBP Is a Universal Factor TBP binds to the TATA box in the minor groove of DNA. TBP forms a saddle around the DNA and bends it by ~80°. Figure 20.09: A view in cross-section shows that TBP surrounds DNA from the side of the narrow groove. Photo courtesy of Stephen K. Burley 20.7 The Basal Apparatus Assembles at the Promoter • The upstream elements and the factors that bind to them increase the frequency of initiation. • Binding of TFND to the TATA box or Inr is the first step in initiation. 20.7 The Basal Apparatus Assembles at the Promoter (a TAF5 TAF 6 TAF 9 TAF 1 TAF 10 TF|iD TAF 2 TF„E RNA Pol Ha Ha B RNA Pol llo Ps Ps' /tbp\ RNA Pol llo Figure 20.11: An initiation complex assembles at promoters for RNA polymerase II by an ordered sequence of association with transcription factors. Adapted from M. E. Maxon, J. A. Goodrich, and R. Tijan, Genes Dev. 8 (1994): 515-524. 20.8 Initiation Is Followed by Promoter Clearance and Elongation • TFNB, TFNE, and TFMH are required to melt DNA to allow polymerase movement. • Phosphorylation of the CTD is required for promoter clearance and elongation to begin. • Further phosphorylation of the CTD is required at some promoters to end pausing and abortive initiation. Regulatory elements controlling RNA Eoi- II transcription in yeast and higher eukaryotes \ Silencer [ CO _ OAS ľ í^H TATA Core promoter +1 Distal hancgr iNsj~ insulator Nŕ TATA h ■Lam Proximal promoter +1 elements Core promoter Downstream i enhancer! • Promoters • Proximal regulatory elements • Enhancers Specific DNA sequence recognition by DNA-binding activator or repressor proteins is the key to differential gene expression. Deletion analysis of yeast GAL1 upstream region defined an Upstream Activating Site (UASG). a 275 bp ^^^^^^ | GAL 0 UASg Mig1 _____^wri^^^^^^E j |^____ 1 2 3 Four copies of a nearly symmetric 17 bp GAL4 site. GALl-lacZ and GALlO-lacZ fusions used for deletion analysis. UASG had many properties of the Enhancer defined in SV40. Showing that GAL4 binds the GAL UAS. Methods for identifying and characterising sequence-specific DNA- binding proteins. DNase I protection assay to precisely define binding sites of sequence-specific DNA binding proteins and to measure DNA- binding affinity, (DNase I Footprinting) The GAL4-binding site is CGG-N(11)-CCG. Regulatory elements controlling RNA Eoi. II transcription in yeast and higher eukaryotes \ Silencer [ CO _ OAS ľ í^H TATA Core promoter +1 Distal hancgr iNsj~ insulator Nŕ TATA h ■Lam Proximal promoter +1 elements Core promoter Downstream i enhancer! • Promoters • Proximal regulatory elements • Enhancers 25.7 Response Elements Are Recognized by Activators • Response elements may be located in promoters or enhancers. Proximal regulatory elements were targets of the earliest studies. Many response elements are found in the MT gene Response elements GRE E-box BLE MRE V R L BLE THE -260 -240 -220 -200 Protein binding SJS1Ä USF Al receptor 180 -160 -140 -120 -100 /"3 MTF1 AP2 AP1 BLE = basal level element GRE = glucocorticoid response element MRE = metal response element TRE = TPA response element MRE GC MRE TATA MTF1 Figure 25.11 Many gene regulators from mammalian cells were identified and purified using the Bandshift assay or Gel mobility shift assay. (also Gel Retardation assay, Electrophoretic Mobility Shift Assay (EMSA)). DNA fragmQnt DNA fragment + DNA-binding protein ) frGG DNA bound DNA freG DNA BOX 16-1 FIGURE 2 Gel mobility shift assay. The principle of the mobility shift assay is shown schematically. A protein is mixed with radiolabeled probe DNA containing a binding site for that protein. The mixture is resolved by acrylarnide gel electrophoresis and visualized using autoradiography. DNA not mixed with protein runs as a single band corresponding to the size of the DNA fragment (left lane). In the mixture with the protein, a proportion of the DNA molecules (but not all of them at the concentrations used) binds the DNA molecule. Thus, in the right-hand lane, there is a band corresponding to free DNA, and another corresponding to the DNA fragment in complex with the protein. Gel is non-denaturing and buffer is mild to avoid disrupting complex. Gel matrix 'traps' protein DNA complex keeping components together to prevent disassociation. Advantages of the bandshift assay. • DNasel footprinting required pure proteins but bandshift assays could detect different scarce DNA-binding proteins even in crude nuclear extracts. • If the probe DNA fragment comes from an enhancer then a point mutation that inactivates the enhancer should also prevent DNA binding by the key regulatory protein in the extract. (Some proteins bind DNA ends or other sites in the probe DNA) • The specific bandshift provides a convenient assay to purify the regulatory protein. DNA-affinity chromatography using a specific oligonucleotide target sequence can purify the binding protein in one step. bud DNA "T GC box UNA Lolljlllll V I Add mixture ät High salt l.iiir. r I'Lltill^lSf!! Figure 6.25 Purification of Sp1 by DNA-afflnity chromatography Oligonucleotide attached to affinity column contains repeats of a DNA binding site for Sp 1 prote (5'-GGGCGGG-3')N A double-stranded -ligo--.ijcleotiae containing repeated GC box sequences is bound to agarose beads, which are poured into a column A mixture of cell proteins containing Sp1 is then applied to the column; because Sp I specifically binds to the GC box oligonucleotide, it is retained on the column while other proteins flow through. Washing the column with high salt buffer then dissociates Sp1 from the GC box DNA, yielding purified Sp1. From: Regulation of Transcription in Eukaryotes Cf.u The Cell; A Molecular Approach. 2nd edition. Cooper GM. Sunderland (MA): Sinauer Associates; 2000. Copyright ©20Q0, Geoffrey M Cooper. NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health. Examples of important transcription regulators identified after purification from nuclear extracts. • Fos/jim, API. oncoproteins studied by Tom Curran and others. Steve MacKnight showed these proteins contain a leucine zipper. • Nf-kappaB (Baltimore lab, MIT) Nuclear factor binding the immunoglobulin kappa B enhancer. Important protein in innate immunity and response to viral infection. • SRF (Maniatis lab and Richard Treisman in London) Serum response factor activates genes involved in growth of cells in tissue culture. • SP1 (Tjian lab) The simplest sequence-specific DNA recognition mechanism of all! CAAT/Enhancer binding protein (C/EBP). 21. Leucine zipper (2dgc) 22. Helix-loop-helix (1am9) The mechanism of transcription activation by the gene-specific regulators. • The primary idea is the same as for GAL4 in yeast. • Recruitment of transcription inititation factors and RNA polymerase to the promoter by DNA loop formation between the enhancer and the promoter. Mediator. In vitro transcription extracts were developed in which Pol II and the General Transcription Factors responded with increased transcription when GAL4 or another activator was added. This allowed further purifications to isolate additional components needed to mediate the activation. Depending on what activator was used this purified coactivators or mediator complex. Some argue that the complex of Pol II, GTFs and Mediator can exist as a 'holoenzyme' that recruits to promoters as in E. coli. Fig. 1 8. Cryo-EM structure of an RNA polymerase ll-Mediator complex. The pol II structure was docked in the central density, and is shown in a similar direction of view and color scheme as Fig. 5. 25.2 Gene specific transcription factors (Activators/Coactivators) are distinct from the General Transcription factors . Several types of factors affect transcription Enhancer RNA polymerase and basal factors bind at promoter Activators bind at promoter Activators bind to distal sites in promoter or to enhancers Coactivators connect activators to basal factors Coactivators/ regulators act on local structure of gene The basal apparatus determines the startpoint for transcription. Activators determine the frequency of transcription. • Activators work by making protein-protein contacts with the basal factors. Activators may work via coactivators. Some components of the transcriptional apparatus work by changing chromatin structure. Coactivator p300/CBP (CREB-binding protein) is a histone acetylase targeting H4. Figure 25.2 Several factors in the basal apparatus are targets with which activators or coactivators interact. Many activators contact different TAFs. Acidic activators contact TFrUR Figure 25.8 ctivators contact the basal apparatus Activator contacts TAF in TFMD Activator contacts TFMB An activator that does not have an activating domain may work by binding a coactivator that has an activating domain. Strong viral activators like Herpesvirus VP 16 and Adenovirus El A are recruited by binding other proteins. An activator may use a coactivator Coactivator Figure 25.7 The IFN-p enhanceosome complex. Vo N , Goodman R H J. Biol. Chem. 2001;276:13505-13508 ©2001 by American Society for Biochemistry and Molecular Biology Transcription activation in chromatin. • Getting transcription initiation and later regulated transcription on chromatin templates required extra factors that operate on nucleosomes. • Constructs with enhancers reintroduced in mammalian chromosomes were often silenced. This turned out later to be due to lack of regulatory elements like the globin gene Locus Control Region (LCR). • Only certain sequence-specific DNA binding proteins can still bind their sites if histones are added first (pioneer transcription factors). Chromatin immunoprecipitation for genome-wide location analysis of chromosomal proteins in living cells 1. Formaldehyde cross link DNA to transcription factors. 2. Lyse cells and fragment cliromatin by sonication 3. Immimopre cipitation Hp" Factor X bound to target gene Unbound fragments are Fragrn e nt s b o und to anti-X antib o di e s was ^e ^ a way ' . Purify on beads 4. Reverse cross-links, extract DNA 5. Analyse which DNA fragments are enriched in the immunoprecipitated fraction. 6. Best done now by Next Generation sequencing (ChlP-Seq) 7. Compare reads of immunoprecipitated sequences back to the genome sequence to locate w the protein was bound. Mapping Polycomb-repressed domains in the bithorax complex using in vivo formaldehyde cross-linked chromatin. Orlando V, Paro R. Cell. 1993 Dec 17;75(6): 1187-98. There is a contrast between a sequence-specific DNA-binding protein and the DNA packaging nucleosome. Histone amino acid side chains do not enter the major groove and 'read' the DNA sequence. ... but nucleosomes are very crowded along the DNA. Tight wrapping of DNA in nucleosomes means they have positional preferences to bind over more bendable A-T rich sequences first. Very G-C sequences are covered last or not at all. Most yeast promoters are relatively "nucleosome free55 .. .many mammalian promoters also.... Figure 2 | Nucleosomal Landscape of yeast genes. The consensus distribution of nucleosomes (grey ovals) around all yeast genes is shown, aligned by the beginning and end of every gene. The resulting two plots were fused in the genie region. The peaks and valleys represent similar positioning relative to the transcription start site (TSS).The arrow under the green circle near the 5' nucleosome-free region (NFR) represents the TSS. The green —blue shading in the plot represents the transitions observed in nucleosome composition and phasing (green represents high H2A.Z levels, acetylation, H3K4 methyLation and phasing, whereas blue represents low levels of these modifications). The red circle indicates transcriptional termination within the 3r NFR. Figure is reproduced, with permission, from REF. 20 © (2008) Cold Spring Harbor Laboratory Press. Relatively nucleosome-free regions, DNAse hypersensitive sites and specific histone modifications help identify enhancers in chromatin. • Chromatin immunoprecipitation methods are easier to use with abundant nucleosomes than with scarce sequence-specific DNA-binding proteins. • Modified histones associated with active (H3K4 Mel9Me3) and inactive (H3K27 Me3) genes have been mapped extensively. This is a proxy for mapping the regulatory proteins themselves and allows likely enhancers and promoters to be identified. • Promoters and enhancers also tend to be nucleosome-free. Histone modifications associated with ACTIVE promoters, polll and DNAsel sensitivity. (H3K4 Mel,Me3) Histone modifications associated with a silent region (H3K27 Me3). RNA-seq = reads of cDNA sequence showing where transcripts are coming from. CTCF = insulator binding protein, blocks ! effects of distant j enhancers, thought to separate regions of gene control SNPs = single nucleotide polymorphisms between human individuals (3X106 per person!). Some of these cause disease - how do we identify which ones? RNA-seq H3K4mel H'3K4meJ H307me3 Chr 13 0.5617- 0.003 20- 1 20- 1-20- 20- 1-20- H3K36meB 100 kb |-29,650.000 | 29,700.000 | 29750.000 | 29.800.000 | KATNAU ll I. I - ■■ ......± I. J,.. .. . J.h A, . J. I .[ 1------ I ..J ..JL.lJi^.1.. -I-I..^. .1. 1 I 1 RNAPII Digital DNase I Conservation SNPs 100- 1 dil.^X_Lai 1 1 ..... 1 ■' ■i. .. -i ... . i^. - - i... |j..L ^.1—..i ...j.l.h-.I^lL - j. ^ -■ - - "i -■ .J . I ■ JLii -0.3 iiBiiniRm miin^HMiiim u« ■■iihihii Transgenic mouse — Skin fibroblasts Retroviral transfection (0ct3/4, Sox2, K1Í4, c-Myc) Antibiotic selection Growth in culture — iPS cell line The Yamanaka experiment. Four factors for induced pluripotent stem cells (iPS cells) Normal mouse Examples of transcription factors being turned on and acting sequentially and combinatorial!)^ to establish positional coordinates across the developing body and to control specification of (250?) cell types at the correct positions A hierarchy of sequence-specific DNA-binding proteins control Drosophila embryonic segmentation. ■rVf, EN 30974992 After the segment specification stage the segment identities are set by homeotic genes encoding homeodomain transcription factors. Sex combs reduced (Str) An tetwapetiia (Antp) Vtirabillitimz (Ubx) Gene regulatory networks (GRNs) with different types for feedback and feedforward effects. Pioneered by Eric Davidson for mesoderm specification in sea urchins Temporal specification by homeodomain transcription factors turned on in sequentially born neuroblasts. (Chris Doe, Richard Mann and others in Drosophila, Oliver Hobert in worms.) Specification of vertebrate motorneurons by homeodomains (Tom Jessell and Silvia Arber). Pre-mRNA processing in eukaryotes. - Ribozymes and the RNA world- Lecture outline • Eukaryotic mRNA stability, 5' cap and polyA addition and splicing. • Mechanism of pre-mRNA splicing and alternative splicing. RNA editing. • Self-splicing Group II introns, RNA catalysis, Ribozymes and the The RNA World. Capping and polyA tailing of pre- mRNAs. Tra n sc ri pt 1 o n trans lati on deg ra da ti on 0 min Transcription begins ppp 5' end is triphosphate 0.5 min Ribosomes begin translation 1.5 min Degradation begins at 5' end 2.0 min RNA polymerase terminates at 3J end 3.0 min Degradation continues, ribosomes complete transla slation The life cycle of a bacterial mRNA. Bacterial mRNA is unstable and has a half-life of only a few minutes. Transcription in both bacteria and eukaryotes is not very fast at 40 nucleotides/sec, 2.4 kb/min. Most bacterial mRNAs can be transcribed in a few minutes whereas a human transcript of 100 kb would take 40 minutes. Figure 7.14 • hnRNA/pre-mRNA processing A typical eukaryotic mRNA: 5'cap............... nontranslated leader <300nt coding ......................AAAAAAAAAn long 3' trailer n =100-200 often >1000nt Pre-mRNA 5' cap addition and 3' polyadenylation and splicing are completed before export from the nucleus. Eukaryotic mRNA is modified and exported <1 min Transcription starts: 5' end is modified 6 min 3' end of mRNA is released by cleavage A/ PPP 20 min 3' end is poly adenylate d AAAAAA 25 min mRNA is transported to cytoplasm NUCLEUS CYTOPLASM AAAAAA > 240 min Ribosomes translate mRNA AAAAAA Figure 7.17 The 5' End of Eukaryotic mRNA Is Capped to protect it against 5' exonucleases. A 5' cap is formed by adding a G to the terminal base of the transcript via a 5-5' link. The capping enzyme is guanylyl transferase. 1-3 methyl groups are added to the base of the new terminal guanosine 2 methyl-riboses near cap. Base modification m6A in 5' cap and also inside mRNAs at RRACH consensus, especially around stop codon. uii!;ijj.ijij.,i=uiJ.!:ii:|t,MJ.[ijiajj,ra Present in all caps O CH3 can be methylated in cap 1 I , ro o o CHo-O-P-O -P-O-P-O-CH c CH Present in cap 1- I CH3 O-P-O-CH O Present in cap 2- CH3 Figure 7.18 3' end cleavage and polyadenylation Template DNA RNA polymerase 5'i II AAUAAAI Cap RNA (a) 5'i Enzyme complex 11 AAUAAAI (b) endonuclease 5'i 11AAUAAAI hOH(3' ) ATP (c) polyadenylate pp. polymerase L 1 5'i :3' iiAAiiAAAi h- AAA(A)n, - OH( 3') The cleavage and polyadenylation specificity factor (CPSF) recognizes AAUAAA (AtwoU, Athree). Cleavage factors (CFI and CFII), endonuclease cleaves RNA 30 bases downstream. Poly(A) polymerase add -200 A residues processively to the 3' end. The polyA tail stabilizes mRNA, facilitates translation. • Poly A tail is >200 nucleotides long and binds many copies of Poly A-binding protein (PABP). • PABP stabilizes the mRNA against degradation. • PABP binds eIF4G (eukaryotic initiation factor 4G) and facilitates translation of message by the ribosome. • 5' and 3' ends are thought to contact one another by forming a loop of the mRNA. Removal of cap and polyA tails are required for regulated degradation of mRNA. Decapping leads to 5'-3' degradation Cap Dcp1 Figure 7.24 Poly(A}/PABP AAAAAAAAAAAA 1 Deadenyiation Ccr4 Decapping 5-3' degradation Degradation of yeast mRNA requires removal of the 5' cap and the 3' poly(A). One yeast pathway involves exonucleolytic degradation from 5'—>3'. • Another yeast mRNA degradation pathway uses a complex of several exonucleases that work in the 3'—»5f direction The exosome. • The deadenylase of animal cells may bind directly to the 5' cap. The 3-5 pathway has three stages Deadenylation ^^^^^^^^^^^^m AAAAAAAAA AA^H Endonucleolytic degradation 3'-5' exonucleolytic degradation Figure 7.25 The mechanism of eukaryotic pre- mRNA splicing. Figure 26.2 Pre-mRNA splicing. c mRNA is 1 Exon 1 Intron 1 Exon 2 Intron 2 Exon 3 Intron 3 Exon 4 Intron 4 Exon 5 Cap at 5r end exon intron Transcription Poty(A) at 3' end End modification Splicing exon Exon intron junctions are broken Exons are joined ™ NUCLEUS Translation CYTOPLASM Consensus 5' and 3' splice sites. The GU-AG rule Intron-exon boundaries have short consensus sequences in the intron Left (i >')site Right (3') site Exon A G G U AAGU .............12PyN C A G ™M 64 73 100 100 62 68 84 63 * 65 100 100 N Exon '-1-1 intron Protein coding sequence must be 'in frame' across the splice junction. Remember GU...AG are splice junction sequences. Mnemonic; GULAG maybe? Intron sequences are generally not conserved even between closely related genes. Figure 26.3 & EM heteroduplex analysis of Adenovirus RNA transcripts annealed to Adenovirus DNA showed formation of D (displacement) loops. interpretation of the EM image Annealing RNA from virus-infected cells with viral DNA revealed the existence of seven iolmns-transcribed regions of the DNA removed from the mature mRNA. Splicing must preserve the open reading frame. • 5' and 3' splice site have to be in the same reading frame. • Reading frames are 1, after first base of codon, 2 after second base and 3 after third base, ie. between codons • GU..AG consensus are the ends of the intron to be removed, not in the actual coding sequence. Splice Junctions Are Read in Pairs All 5' splice sites are functionally equivalent, and all 3' splice sites are functionally equivalent. Usually draw exons as boxes to emphasise them with splices joining box corners and introns as lines between the boxes. Gene rearrangements within big introns have a good 1/3 chance to produce fusion proteins. (Exon shuffling in evolution). Correct splicing removes three Introns by pa if wise recognition of the [unctions _GU AG Pairing of wrong junctions would remove exons Splicing proceeds through a Jariat 5, 5' site 3' site GU UACUAAC AG PV80 N PVM P*87 PU75 A P*95 Animal consensus Cut at 5' site and form lariat by 5—2' bond connecting the intron 5'-G to the 2' of A at the branch site UACUAACAG 2' Cut at 3' site and join exons; intron released as lariat u =„Q§ .3r ^ I5'— 3r 5':-3' UACUAACAG 2' 5' =3' Debranch intron 5' GU V_ UACUAACAG 3' Alkaline lysis of ssRNA. The reactive T OH groups on riboses can also mediate complete cleavage of 5'-3' phosphate bonds in single stranded RNA under alkaline conditions Figure 26.7 Splicing is catalysed by a series of five small nuclear ribonucleoprotein particles (snRNPs, 'SNURPs'). Conserved small structured RNAs with Sm and other proteins bound. SNURPs assemble sequentially on pre-mRNA to form a spliceosome. Cryo-EM picture (Stark et al. Nature 409, 539) 5' Splice Site Recognition Ul snRNP 3' Splice Site Recognition U2AF branch point 3' Splice Site Recognition U2AF Splice Site Recognition branch point U2AF The U4/U5/U6 tri snRNP arrives last and base pairs to U2 3' LJ6 snRNA A ACAGAGAUGAUC- C J ACUAG A^y U2 snRNA 3'—I Exon 2 |— G A UGUAGUA ACAUCUA V 3 5J—I Exon 1 |—G U 3' Precursor mRNA Conformation changes bring splice junctions together. Patel and Steitz, Nature Reviews Molecular Cell Biology (2001) 4, 960-970 Pre-mRNA RNA processing is coupled to transcription. o o © i > o 6 Mediator -1—^ 9 Splicing Is Connected to Export and efficient translation of mRNA Splicing is required for mRNA export Exon Intron Exon Splicing m Protein binds splicing complex Protein remains at exon-exon junction i Complex (EJC) assembles at exon-exon junction EJC binds proteins involved in RNA export, localization, decay Figure 26.16 Alternative splicing is an important strategy to increase the number of gene products produced from a single gene. Modes of alternative splicing. b Exon skipping/Inclusion Alternative 3' splice sites Alternative 5' splice sites Mutually exclusive e^ons Inlron retention 1 Constitutive axon I I Alternatively spliosd axon Carteeni L. Chew SL. Krainer Alternative splicing in Drosophila Dscam . genomic DNA and Q pre-mRNA mRNA exon 4 exon 6 12 alternatives 48 alternatives i-[ r i i i 4 6 exon 9 33 alternatives i-1 exon 17 2 alternatives n I ~n r 17 M ii £13-11 Copyright© 2004 Pearson Education, Inc., publishing as Benjamin Cummings Inclusion of alternative exons may be promoted by exonic splicing enhancers that bind serine/arginine-rich (SR) RNA-binding proteins. Exon Heterogeneous ribonuclear proteins (hnRNP proteins) coat pre-mRNAs and mRNAs and often inhibit splicing events that SR proteins promote. ' Ying-yang' balance of these effects on many splicing events. Splicing complexes Can be Formed by Intron Definition or Exon Definition • The direct, intron definition, way of forming an E complex at short introns is for: - Ul snRNP to bind at the 5' splice site - U2AF to bind at a pyrimidine tract between the branch site and the 3' splice site • Another possibility, exon definition, for very long introns is for the complex to form between: - U2AF at the pyrimidine tract - Ul snRNP at a downstream 5' splice site The importance of alternative splicing It is estimated that 95% of human genes are alternatively spliced from new sequencing information. Alternative splicing creates protein diversity 26,000 human protein-encoding genes, yet 90,000 proteins Aberrant splicing is a frequent effect of human disease gene mutations. Base deamination RNA editing also occurs site-specifically in some transcripts and changes codon meaning. Adenosine deaminases acting on RNA (ADARs) edit 4% of Drosophila transcripts (972 sites), and -250 known sites in human transcripts, including those encoding brain ion-channel subunits, particularly GluR B, the dominant subunit of AMPA class glutamate receptors. APOBECs are C to U deaminases first identified in RNA ediding but more important as DNA editors. Activation induced deaminase (AID) edits DNA in immunoglobulin genes to initiate Somatic Hypermutation of antibody variable regions. APOBEC3G edits HIV and interferes with virus replication. RNA catalysis, Ribozymes and the RNA World. The RNA World. Self-splicing introns are small transposable elements that remove themselves cleanly from the transcripts they are inserted in. Group I intron splicing in 26S rRNA of Tetrahymena is catalysed by RNA in the absence of protein (Tom Cech). Upstream exon 5 Intron Spliced exons -OH Downstream exon 414 399 o 395 RNA Lad axon_Right exon Spliced exons Ccpynghi ..