MUNI SCI Bi4025en Molecular Biology Mgr. Jiří Kohoutek, Ph.D. 1 Department of Experimental Biology Lecture 11 • Mobile genetic elements, transposons and retrotransposons 2 Department of Experimental Biology Maize - Zea Mays One of the world's most important crops. The natives of the Americans cultivated many different and differently colored varieties, attributing aesthetic and religious significance to colors. Scientific significance: color patterns are the result of a phenomenon called transposition. • Barbara McClintock was awarded by Nobel Prize in Physiology or Medicine 1983. • Careful observation of plants in the field. | • Changes in inheritance ratios and traits such as grain color. MUNI 3 Department of Experimental Biology _ _ T O 0 J. Barbara McClintock • Transposable elements were discovered by Barbara McClintock during experiments conducted in 1944 on maize. • Since they appeared to influence phenotypic traits, she named them controlling elements. • However, the idea that a gene could "jump" from place to place was contrary to Mendel's laws. • Her presentation at the 1951 Cold Spring Harbor Symposium was not understood and at least not very well received. She had no better luck with her follow-up publications. • Her discovery was brought back to life after discovery of insertion sequences (IS) in bacteria by Szybalski's group in the early 1970s. 4 Department of Experimental Biology Methods in Molecular Biology, vol. 1910, https://doi.org/10.1007/978-1-4939-9074-0_6 MUNI SCI C - value paradox Rejecting the idea of an static genome meant changing the paradigm of genetics. Part of the new view was the acceptance that genomes are largely made up of repeating sections of DNA (repetitive DNA). * ProOrytte • EuUryotc DNA Viru» 10'3 Mudpuppy 10'2 10-' 10° 10' 102 103 10'' Genome size (Megabases) Human f \ \ .-..rkHNA □ • C-value paradox in Eukaryotes: increase of genome size depends of the increase of non-coding elements. • The human genome, for example, comprises less than 2% protein-coding regions. 5 Department of Experimental Biology https://www.sciencedirect.com/topics/neuroscience/c-value The Role of Non-coding Structural Information in Phylogeny, Evolution and Disease, DOI: 10.13140/RG.2.1.3803.6322 MUNI SCI C - value paradox A Large proportion of DNA Is noncoding. Most of the DNA of bacteria and yeasts encodes RNAs or proteins, but a large percentage of the DNA of multicellular species is noncoding. m c . CD O c c ~o o o £ o c rt Interspersed alaflients L R TSC» 12 Department of Experimental Biology Genomics Inform. 2014 Sep; 12(3): 98-104. 1 Composite SINE tran&poscni MUNI SCI Transposable elements (TEs) in prokaryotes Prokaryotes - two types of DNA Transposons: IS elements (Insertion sequences). Tn elements (Transposons). Insertion sequence 5' 3' ATCCGGT... ...ACCGGAT TAGGCCA... ...TGGCCTA 3 5 Inverted repeat Transposase gene Inverted repeat Transposon _a_ Insertion sequence Antibiotic resistance gene Insertion sequence /-A-* Inverted repeats Transposase gene L*>Pti*pA& iTCft Leucaiwn !ne I'uH jhog m l"ea-5»;nU»-|«(ninlJuiMt-ing) AI i^fi-i maw: 13 Department of Experimental Biology 3 5' MUNI SCI Transposable elements (TEs) in prokaryotes • Tn elements (Transposons): • Nonreplicative Tn „cut and paste": o Set aside from the original location and integrated into the new one. o Key enzyme - transposase. o Prokaryotes and Eukaryotes. • Replicative Tn „copy and paste": o They are replicated during transposition (one copy remains in the original location, the other appears in the new location). o Key enzymes - transposase and resolvase. o Prokaryotes. Donor DN'A Replicative Nonreplicative transposition transposition 14 Department of Experimental Biology J. KS I AM Vol.17, No.2, 87-102, 2013 MUNI SCI Insertion sequence (IS) elements Integration of an IS element may: Disrupt coding sequences or regulatory regions. Alter expression of nearby genes. Cause deletions and inversions in adjacent DNA. Result in crossing-over. IraflBcnbed in carlai ce* typea, prison product ů-actwe Piaiem product nol functional TrariEcrlptton activated in othaf cell types No *rod 15 Department of Experimental Biology https://www.bx.psu.edu/~ross/workmg/TranspositionCh9.htm MUNI SCI Insertion sequence (IS) elements • Simplest type of transposable element found in bacterial chromosomes and plasmids. • Encode gene (transposase) for mobilization and insertion. • Range in size from 768 bp to 5 kb. • IS1 first identified in E. coli's galactose operon is 768 bp long and is present with 4-19 copies in the E. coli chromosome. • Ends of all known IS elements show inverted terminal repeats (ITRs). Insertion sequence. IS? Transposase gene 5' GGTGÍTG CTG CCA Ail" TACT GAT 3' CC1C7«GMGIiTIG«TGAC7A 5' 5' ATCAftUACTT 3' UGTmiCAACCTCAGTUfGG 5' MUNI 16 Define footer-presentation title / department https://education.sakshi.com/en/csir-net/study-material/cell-biology/transposons-67119 O 0 J. Transposition of insertion sequence (IS) elements • Original copy remains in place; new copy inserts randomly. • Transposition requires transposase, coded by the IS element. • IS element otherwise uses host enzymes for replication. • Transposition initiates when transposase recognizes ITRs. • Site of integration = target site. • Staggered cuts are made in DNAat target site by transposase, IS element inserts, DNA polymerase and ligase fill the gaps (critically, transposase behaves like a restriction enzyme). • Small direct repeats (~5 bp) flanking the target site are created. 17 Department of Experimental Biology MUNI SCI Transposition of insertion sequence (IS) elements IS 5 ACAGTTCAG 3 JGTCAAGTC. 1-1-1 IK CTGAACTGT 3 GACTTGACA 5 Insertion of IS element into chromosomal DNA IK Chromosomal 5' DNA 3' Target site Cut TCGAT AGCTA Cut* (— Inserted IS element —1 5 [TCGAT ACAGTTCAG TGTCAAGTC CTGAACTGT I 3' 3' GACTTGACA AGCTA 5' 5' 3 IK Host DNA Gaps filled by DNA polymerase. DNA ligase IR New DNA TCGAT ACAGTTCAG CTGAACTGT TCGAT 3' AGCTA TGTCAAGTC GACTTGACA AGCTA 5' New DNA IK IK Host DNA Duplicated target site sequence 18 Department of Experimental Biology MUNI SCI Transposons (Tn) • Similar to IS elements but are more complex structurally and carry additional genes. • Two types of transposons: Composite Trans poson Trans poson TnlO ' 1 1 f IS10 Tetracycline resistance IS10 —i Simple Transposon Transpose n Tn3 f ^ 1 — 1 IR Transposase Resolvase | ^mp Resistance IR l https://wwwTesearchgate.net/publication/281264950_The_Dynamic_Genome_A_Gate |J 19 Department of Experimental Biology way_of_Evolution O U i. Assembled transposons Consequence of the occurrence of two IS elements in close proximity to each other. A pair of IS-elements will provide mobility to the intermediate region of DNA. End structures of IS-elements preserved. o Carry e.g. Antibiotic resistance: o Kanamycin o Gentamycin o Ampicillin o Tetracycline o Chloramphenicol o Streptomycíne 20 Department of Experimental Biology Tn9 =2500 np _A_ gen carrf 768 np 768 np 23-np obrácené koncové repetice (a) 1329 np (0 Tn5 =5700 np _A_ gen ble' gen karí I gen str 1533 np ^ a t íl a | 1533 np ISJ IS? IS50L 9-np — obrácené — koncové repetice (b) TnIO -9300 np _/\_ gen teť A_ 1329 np 22-np — obrácené — koncové repetice MUNI SCI Composite transposons - Tn10 • Carry genes, for example gene for an antibiotic resistence, flanked on both sides by IS elements. • Tn10 is 9.3 kb and includes: o 6.5 kb of central DNA (includes a gene for tetracycline resistance, o 1.4 kb inverted IS elements. • IS elements supply transposase and ITR recognition signals. I 1,400 bp ->• iSlOL \ / Inverted repeats of IS element -1- I_ Transposon, TnlO 9,300 bp 6,500 bp Tetracycline resistance gene (TcR) 1,400 bp 1 ISWR \ / Inverted repeats of IS element Inverted IS elements 21 Department of Experimental Biology MUNI SCI Composite transposons - Tn10 • IS10 sequences at the ends are not identical. • One IS encodes functional transposase. • The other IS mutant (often the difference of a single nucleotide pair). Transposase Functional IS 10 not functional JetR transposase 1 1 ISlOleft IS 10 right j Y TnlO 9300 bp • 22 Department of Experimental Biology MUNI SCI Non composite transposons - Tn3 • Carry genes, e.g. gene for antibiotic resistence, but do not terminate with IS elements. • Ends are non-IS element repeated sequences. • It is a replicative transposon. • Tn3 is 5 kb composed of: • 38-bp ITRs • 3 genes: o bla ((3-lactamase) o tnpA (transposase) o tnpB (resolvase, which functions in recombination). Transposon, Tn3 Left inverted repeat (38 bp) ^-lactamase Resolvase Right inverted repeat (38 bp) mRNAs 23 Department of Experimental Biology MUNI SCI Transposition of transposons • All transposons use a common mechanism in which staggered nicks are made in target DNA, the transposon is joined to the protruding ends, and the gaps are filled. • Similar to that of IS elements - duplication of short sequence at ends of target sites occurs. • Cointegration = movement of a transposon from one genome (e.g., plasmid) to another (e.g., chromosome) integrates transposon to both genomes (duplication). • Transposition replicative (duplication) or non-replicative (transposon lost from original site). • Result in same types of mutations as IS elements: insertions, deletions, changes in gene expression, or duplication. • Crossing-over occurs when donor DNA with transposable element fuses with recipient DNA. 24 Department of Experimental Biology MUNI SCI Non-replicative transposon • Non-Replicative transposon leaves its original place and move to the another location in the genome - "Cut and Paste". • This type of mechanism requires only a transposase. • The insertion elements and composite transposons like Tn5 and Tn10 use this mechanism. • Non-replicative transposons leave a break in the donor molecule which is lethal to the cell unless it is repaired. 25 Department of Experimental Biology MUNI SCI Mechanism of non-replicative transposon transposition • Transposon 5 - Tn5. • Transposition initiated by Transpossase (Tnp) binding to the transposon specific Mosaic ends (MEs), inverted repeats, and the formation of a synaptic complex (SC) by a process called synapsis. • The SC contains Transposase dimer and two MEs. • Catalytic cleavage occurs when an activated H20 coordinated by Mg2+ nicks the transferred DNA strand on both sides of the transposon, through a nucleophilic attack, forming a 3'- hydroxyl group. • The free 3'-hydroyxl group acts as a nucleophile and cleaves the non-transferred DNA strand (NT), forming a hairpin. ME ME Tnp Monomer ME K H H ■3'(NT) ■5'(TS) Transferred strand nicking ? ME ■ 3' (NT) ■5'(TS) Hairpin formation H H ■ 3' (NT) ■5'(TS) Hairpin resolution ■3'(NT) Synapsis -I Cleavage J + Target DNA | Target Capture Strand Transfer 9-bp repeat • J 26 Department of Experimental Biology Journal of Bacteriology, Volume 190, Issue 4, 15 February 2008, Pages 1484-1487. MUNI SCI Mechanism of non-replicative transposon transposition A second activated water molecule resolves the hairpin, resulting in a double-stranded DNA cleavage product. The post-cleavage synaptic complex is now free to bind to target DNA through target capture. The 3'-hydroxyl group of the transposon end attacks the phosphodiester backbone of target DNA during strand transfer. A 9-bp duplication in the target results, due to the staggered strand transfer reactions followed by DNA repair by host enzymes. ME ME Tnp Monomer ^ -J Synapsis 5'- 3'- ME K H H ■ 3' (NT) ■5'(TS) Transferred strand nicking ? ME ■ 3' (NT) ■5'(TS) Hairpin formation H H ■ 3' (NT) ■5'(TS) Hairpin resolution ■3'(NT) -I Cleavage J + Target DNA | Target Capture Strand Transfer 9-bp repeat • J 21 Department of Experimental Biology Journal of Bacteriology, Volume 190, Issue 4, 15 February 2008, Pages 1484-1487. MUNI SCI Replicative transposon • Replicative transposon is first replicated and then one of the copy will move to the another location in the genome. Thus, the transposon will remain on its original position - "Copy and Paste". • Replicative transposition involves two types of enzymatic activity: o Transposase that acts on the ends of the original transposon. o Resolvase that acts on the duplicated copies. Replicative transposition occurs through a cointegrate formation, which is produced by fusion of two replicons, one originally possessing a transposon, the other lacking it: the co-integrate has copies of the transposon present at both junctions of the replicons. • Resolution occurs by a homologous recombination mediated by resolvase enzyme between the two copies of the transposon in a co-integrate leading to the donor and target replicons, each with a copy of the transposon. 28 Department of Experimental Biology MUNI SCI Mechanism of replicative transposon transposition Donor plasmid Recipient plasmid o O The transposase encoded by Tn3 catalyzes the formation of a cointegrate between the donor and recipient plasmids. During this process, Tn3 is replicated so there is a copy ot the element at each junction in the cointegrate. Q Resolvase produced by the tnpR gene resolves the cointegrate by mediating recombination between the two Tn3 elements. o Q Donor and recipient Plasmids separate, each with a copy of Tn3. ■ FIGURE 17.4 Transposition of Tn3 via the formation of a cointegrate 29 Department of Experimental Biology Non-composite transposons (Tn3) is a replicative transposons that undergoes transposition in two stage process. In the first stage, two plasmid - (one containing Tn3 transposons; donor plasmid) and the other recipient plasmid undergoes fusion catalyzed by transposase enzymes giving rise to a structure called cointegrate. https://www.onlinebiologynotes.com/transposable-elements-characteristics-and-mechanisms-of-transposition/ MUNI SCI Mechanism of replicative transposon transposition Donor plasmid Recipient plasmid o O The transposase encoded by Tn3 catalyzes the formation of a cointegrate between the donor and recipient plasmids. During this process, Tn3 is replicated so there is a copy ot the element at each junction in the cointegrate. Q Resolvase produced by the tnpR gene resolves the cointegrate by mediating recombination between the two Tn3 elements. o Q Donor and recipient Plasmids separate, each with a copy of Tn3. ■ FIGURE 17.4 Transposition of Tn3 via the formation of a cointegrate 30 Department of Experimental Biology During the formation cointegrate, Tn3 is replicated, and one copy is inserted at each point where the two plasmids have fused. In the second stage of transposition, the tnpR-encoded enzyme resolvase which mediates a site-specific recombination between the two Tn3 copies at the resolution site, and when it is completed, cointegrate is resolved into its two constituent plasmids, each with a copy of Tn3. https://www.onlinebiologynotes.com/transposable-elements-characteristics-and-mechanisms-of-transposition/ MUNI SCI Transpoable elements (TEs) in eukaryotes • Eukaryotic genomes have many copies of transposons (-45% of the human genome). • Transposition accompanies insertion in a genome site and some (not all) insertions can cause changes in gene expression, its regulation and products and can lead to speciation or diseases. • The insertion sites are random, although some sites (called hot spots) are preferred to others. 31 Department of Experimental Biology MUNI SCI Classification eukaryotic transpoable elements (TEs) Schematic and examples showing the key features and relationships between TE classes, subclasses, superfamilies, and families. 32 Department of Experimental Biology Transposition intermediate RT ■RNA U Integration intermediate and mechanism TPRT circDNA? dsDNA Phylogeny 7I\ /!\ 01 a. LINE SINE PLE Tyl/copia Ty3/gypsy ERV Phylogeny J_ E LINE-1 R2 Alu B2 Penelope Athena Tyl copia Ty3 gypsy HERV-H HERV-K Genome Biology, 2018, volume 19, Article number: 199. DNA- *-1 I circDNA dsDNA circDNA? hAT Tc1 /mariner Ac/Ds Tct hobo mos1 dsDNA? MUNI SCI General properties of plant DNA transposons • Possess ITR sequences and generate short repeats at target sites. • May activate or repress target genes, cause chromosome mutations, and disrupt genes. • Two types: • Autonomous elements transpose themselves; possess transposition gene. • Nonautonomous elements do not transpose themselves; lack transposition gene and rely on presence of another Tn. • McClintock demonstrated purple spots in otherwise white corn (Zea mays) kernels are results of both these types of transposable elements. 33 Department of Experimental Biology MUNI SCI General properties of plant DNA transposons ĎSOtO hncnCúuCMon. lne 34 Department of Experimental Biology MUNI SCI McClintock's discovery of transposon in corn • c/c = white kernels and CI- = purple kernels. • Kernel color alleles/traits are "unstable". • If reversion of c to C occurs in a cell, cell will produce purple pigment and a spot. • Earlier in development reversion occurs, the larger the spot. • McClintock concluded "c" allele results from a non-autonomous transposon called "Ds" inserted into the "C" gene (Ds = dissociation). • Autonomous transposon "Ac" (Ac = activator) controls "Ds" transposon. MUNI 35 Department of Experimental Biology https://www.slideshare.net/DeepakKumarGupta2/general-aspect-of-neoplasia SCI McClintock's discovery of transposon in corn • Ac-element -activator: • Consisting of 4563 bp restricted by inverted repeats 11 bp long and 8 bp long straight repeats (they are formed by duplication at the target point and are not an integral part of the element). • Contains a gene for transposase. • Ac element is autonomous. 4.6 kb CAGGGATGAAA TTTCATCCCTA 36 Department of Experimental Biology MUNI SCI McClintock's discovery of transposon in corn • Ds-elements -dissociator: • Structurally heterogeneous - have the same terminal repeats as Ac-elements, internal sequences are different. • For their transposition they need a transposase of an Ac-element. • This transposase is therefore a transacting protein. • Ds element is nonautonomous. 37 Department of Experimental Biology MUNI SCI Transposon effect on corn kernel color • A corn plant with a genotype of c/c will have colorless kernels, while a genotype of CI- will have purple ones (C gene produces the purple pigment) • The Ac-Ds transposon system controls the distribution of color in a kernel. Ds (dissociation) elements are nonautonomous. Ac (activator) elements are autonomous. • If a reversion occurs in a cell (Ds is removed from the mutant c gene), that cell will produce a purple pigment (c —► C). In the case of the figure, the reversion appears to be late, so the kernel is mostly colorless. • Ac/Ds are developmental^ regulated. 38 Department of Experimental Biology https://slideplayer.com/slide/13044605/ a) Purple kernels J M .._.//- //__| C _ Norma! C gene expressing pigment product b) Colorless kernels Activates Ds transposition Ds can transpose into C r _| Ac ; r c Ds Disrupted (mutant) c gene c) Spotted kernels Activates Ds transposition out of C in a few cells during kernel development Reversion of c mutatio ion or ion to C I J3s Mutant c gene Normal C gene MUNI SCI Transposon effect on corn kernel color Donor site Replicated Ac element in donor site a) Transposition to an already-replicated recipient site Recipient site Donor site Vacated donor site No net increase in number of Ac elements b) Transposition to an unreplicated recipient site Recipient site Vacated donor site Net increase in number of Ac elements 39 Department of Experimental Biology Ac transposes is active only during chromosome replication, employing a conservative mechanism. • Two possible results of Ac transposition, depending on whether the target DNA has replicated. • A) If transposition takes place into an already replicated recipient site, there is no increase in the number of Ac elements. • B) If transposition takes place into an unreplicated recipient site, there is a net increase in the number of Ac elements. • Ds replication is the same (but driven by an Ac element). MUNI SCI Transposable element in Drosophila • It is estimated that 15% of Drosophila genome is mobile. • Crossing certain strains of drosophila produces hybrids characterized by a set of aberrant characteristics, including numerous mutations, chromosome breaks and sterility. • This syndrome of abnormalities was named dysgenesis of hybrids (from Greek "deterioration of quality"). • Strains of drosophila can be divided into two types - M and P - depending on whether or not their crossing leads to dysgenic hybrids. • Only the crossing between M and P strains leads to the emergence of dysgenic species, where the male comes from the P strain, the female from the M strain. MUNI 40 Department of Experimental Biology r> r» t Transposable element in Drosophila • Dysgenesis of hybrids is mediated by presence of P element. • P-elements • Are transposable elements that carry genes for transposase activity that cause the elements to move, and repressor (piwi-interacting RNA) activity that prevents expression of transposase. • P elements vary in length from 500 - 2,900 bp. • P elements code a repressor present in the cytoplasm, which makes them stable in the P strain (but unstable when crossed to the wild type female; female lacks repressor in cytoplasm). Drosophila P element 2.9-kb central sequence; transcribed left to right ! 1 2 4 / 31-bp inverted repeat Intron 1 Intron 2 Intron 3 31-bp inverted repeat 41 Department of Experimental Biology Coding region of central sequence includes a transposase. After transcription and polyadenylation, coding sequences 1 to 4 are spliced in different combinations to produce different polypeptides. MUNI SCI P-element-mediated hybrid dysgenesis in Drosophila P elements are repressed P elements are activated Hybrid dysgenesis occurs when haploid genome of male (P strain) possesses -40 P elements/genome. In a cross between a P-element-carrying female and a laboratory male, repressors in the maternally - derived cytoplasm repress expression of the maternally -inherited P elements. The resulting offspring show the wild-type phenotype. In a cross between a P-element-carrying male and a laboratory female, repressors are absent in the maternally - derived cytoplasm. The two zygotes are chromosomally identical but cytoplasmically different. In the right-hand cross, P elements are activated and undergo transposition in the genome, causing release of mutator activity and a variety of dysgenic phenotypes in the offspring. 42 Department of Experimental Biology https://www.mun.ca/biology/scarr/P-element_hybrid_dysgenesis.htm MUNI SCI Repression of hybrid dysgenesis in Drosophila • Drosophila can prevent the effects of P-elements by RNA-interference using piRNA (derived from the P-elements themselves). • piRNA (PlWI-interacting RNA) is a type of small RNA that can provide targeted degradation of the mRNAP element. • piRNA(length 26-31 nts) form complexes with a specific group of proteins called "piwiproteins" (P-Element Induced Wimpy Testis). • Female P-strains form piRNAa and pass them on to their offspring through cytoplasms. • piRNA restrict P-element aktivity in the embryonic line. • Preventing hybrid dysgenesis. • Maternal transmission of repressing piRNA explains why the offspring of crossing P-females with M-males and P-females with P-males is not dysgenic. MUNI 43 Department of Experimental Biology _ _ _ Repression of hybrid dysgenesis in Drosophila piRNA prevents hybrid dysgenesis only if it is present in the cytoplasm of the egg • A. Dysgenic cross: the crossing between M females (without P element) and P males (with P element) produces sterile offspring since the active transposition of P element disrupts genome and induces gonadal atrophy. • B. Reciprocal cross: the crossing between M males and P females produces fertile offspring since the maternally inherited piRNAs repress activities of P elements in the offspring. piRNA, P-element-induced wimpy testis (PlWI)-interacting RNA. A Dysgenic cross M strain P strain B Reciprocal cross M strain P strain as 9 Fertilized eggs and early embryo í piRNA I F, c-very Sterile X Atrophic rif. WJ5 9 Fertile 44 Department of Experimental Biology Genomics, Proteomics & Bioinformatics, Volume 15, Issue 3, June 2017, Pages 164-176. piRNA Normal MUNI SCI Repression of hybrid dysgenesis in Drosophila • P elements can be manipulated experimentally to introduce genes into the germ line of fruit fly embryos: o The wt+ rosy gene was cloned and microinjected into mutant fly embryos, o Descendants had normal eye color (red). Embryo from rc-i/ mutant r element with inserted my* Recombinant plasm id is cloned In F. arfi and mwroinjoctod into PriK<>/i/ij/ii embryos Mcropipette 1 >».>.«'.i DNA Transposition of P element introduces r,Ky * gone into r>riK.>/>/nJj genome 45 Department of Experimental Biology Targot-srto duplication Descendants had normaJ eye color MUNI SCI Retroelements • Require reverse transcription of RNA into DNA —> this reversal flow of genetic information led to the definition "retro", that is, "reverse" (lat.). • Retroviruses - their genome consists of single-stranded RNA, which is converted into double-stranded DNA after infection of the host cell, with the participation of in reverse transcriptase they are able to leave the cell. • Retrotransposons - limited to their own genome, o Elements similar to forms of DNA retroviruses. o Mobile genetic elements which DNA is formed by reverse transcription of polyadenylated RNA. 46 Department of Experimental Biology MUNI SCI Retroviruses RNA viruses. Discovered in connection with the development of certain tumours in chickens, cats and mice (Peyton Rous; 1966 NC). In 1970: objev reverzní transkriptázy (David Baltimore, Howard Temin; 1975 NC). HIV (Human Immunodeficiency Virus) causing AIDS in humans (Acquired Immune Deficiency Syndrome). Prototypical retrovirus, life cycle and genome structure studied in detail. STRUCTURE OF THE HUMAN Department of Experimental Biology https://www.istockphoto.com/cs/vektor/diagram-viru-hiv-vektor-gm500426119-42895954 MUNI SCI HIV genome and structure • Formed by two identical single-strand RNA molecules; inside the viral particle is found along with several proteins including two reverse transcriptase molecules. • Slightly larger than 10 kb. • Contains several genes: • Gag - viral particle proteins. • Pol - reverse transcriptase and integrase. • Env - glycoproteins of the viral lipid envelope (gp120; gene for virulence). • Reverse transcriptase converts a single-stranded RNA into a double-stranded DNAand it is randomly incorporated into the chromosome of infected cells, which contains many copies of viral genomes. 48 Department of Experimental Biology https://biosci.mcdb.ucsb.edu/immunology/lmmunodeficiencies/HIV-structure.htm MUNI SCI HIV replicative cycle Latent infection Active infection (T) Transcription of proviral DNA Synthesis of viral components (^3) Assembly of viruses (4) Budding of viruses from the host cell 49 Department of Experimental Biology ® Attachment Fmiori inhiljitnrs https://www.alamy.com/stock-photo-latent-and-active-infection-by-hiv-49485818.html Nature Reviews Microbiology, 2012, Volume 10, pages279-290. Natur« Reviews | Microbiology MUNI SCI HIV reverse transcription (A) The RNA genome of a retrovirus (light blue) with a tRNA primer base paired near the 5' end. R U5 ipbs (B) RT has initiated reverse transcription, generating minus-strand DNA (dark blue), and a the RNase H activity of RT has degraded the RNA template (dashed line). b R U5 jjDbs_gag_ (C) Minus-strand transfer has occurred between c I pbs gag the R sequences at both ends of the genome (see text), allowing minus-strand DNA synthesis D bs gag to continue (D), accompanied by RNA < degradation. (D) A purine-rich sequence (ppt), adjacent to U3, is resistant to RNase H cleavage and serves as the primer for the synthesis of plus-strand DNA. 50 Department of Experimental Biology Cold Spring Harb Perspect Med. 2012 Oct; 2(10): a006882. Biology 2012, 1(3), 521-541; https://doi.org/10.3390/biology1030521 HIV reverse transcription (E) Plus-strand synthesis continues until the first 18 nucleotides of the tRNA are copied, allowing RNase H cleavage to remove the tRNA primer. Most retroviruses remove the entire tRNA; the RNase H of HIV-1 RT leaves the rA from the 3' end of the tRNA attached to minus-strand DNA. (F) Removal of the tRNA primer sets the stage for the second (plus-strand) transfer. (G) Extension of the plus and minus strands leads to the synthesis of the complete double-stranded linear viral DNA. I Pbs gag U3 R U5 i pbs gag U3 RU5 .pbs gag LTR 51 Department of Experimental Biology Cold Spring Harb Perspect Med. 2012 Oct; 2(10): a006882. Biology 2012, 1(3), 521-541; https://doi.org/10.3390/biology1030521 I U3i R I U5 i HIV transcription initiation - replication genomic DNA I U3i R i US i Transcription promoter _ooooo.® "IT Poly A signal Cffi R US Central polypurine tract (cPPT) -*- initiation of plus-strand DNA synthesis: Polypu rlne tract [PPT) I ^ I ^ lllllllülfllflJ.lHllH genomic RNA i i Trans-acting responsive element (TAR) %\ L tRNA primer binding site (PBS) dimerizatlon (□is) packaging (PS I) ^ SĽť O Translations • Monopartite, linear, dimeric, ssRNA(+) genome of 9,75 kb, with a 5'-cap and a 3'poly-A tail. • There are two long terminal repeats (LTRs) of about 600 nt long at the 5' and 3' ends. • The LTRs contain the U3, R, and U5 regions. • There are also a primer binding site (PBS) at the 5?end and a polypurine tract (PPT) at the 3end. TŘĚŠ" 52 Department of Experimental Biology https://viralzone.expasy.org/5183 MUNI SCI HIV transcription - splicing Unspliced full length mRNAwill serve as genomic RNAto be packaged into virions or used as a template for translation of gag and gag-(pro)pol (1 ribosomal frameshift) polyproteins. The uncompletely spliced mRNAs encode env that is cleaved into SU and TM envelope proteins, and the accessory proteins vif, vpu, and vpr. Completely spliced mRNAs encode Rev, Tat and Nef accessory proteins. Rev escorts unspliced and uncompletely spliced RNAs out of the nucleus of infected cells. :-R 1 5' 5' 5' 5' 5' E POL GAG Ribawrna! frameshift 5' c 5' tat vpu vpr_ ENV rev nef -■Po» 9 ff 1 feu MCA HC PR HT \ J IN j tat genomic RNA Alror .-r iTIltUtKM : 53 Department of Experimental Biology https://viralzone.expasy.org/5183 MUNI SCI Tumors must acquire additional four hallmarks capabilities PLoS Pathog., 2013 Mar;9(3):e1003241. 54 Department of Experimental Biology MUNI SCI Retroelements - Retrotransposons • Retrotransposons that replicate through an RNA intermediate and a reverse transcription step, the so-called copy-and-paste transposons, and comprises two main families: • LTR retrotransposons - contain long terminal repeat - LTR; sometimes specific subclass Endogenous retroviruses (ERVs). • Non-LTR retrotransposons - without LTR. • Retrosequences - without LTR, without reverse transcriptase and integrase. Reverse transcripts of structural genes - edited transcripts without introns, with attached poly (A). o Retrogenes - functional retrosequences of the original gene coding identical protein. o Retropseudogenes - non-functional forms of genes /eg. Alu-sequence in humans (7SL RNA, 300 pb, in humans 500,000 times copied). MUNI 55 Department of Experimental Biology r> r» t LTR and Non-LTR Retrotransposons LTR retrotransposons occurs in cytoplasmic virus-like particles and leads to the formation of extrachromosomal double-stranded DNA (dsDNA), which is imported into the nucleus before integrating into a new locus. Non-LTR retrotransposons initiate reverse transcription directly at the target locus after cleaving genomic DNA, a process known as 'target-primed reverse transcription'. GAGcapsid + _ TE DNA — TE RNA • Transposase/integrase # Reverse transcriptase GAG protein LTR/Retrovirus {e.q.,ERVl) Non-LTR (e.g..U) Transcription 1 I RNA intermediate -J o -~~~~~ DD(E/D) type Target-primed integration reverse transcription No change No change Um Wells JN.FeschotteC. 2020 ilM Annu. Rev. Genet. 54:539 61 56 Department of Experimental Biology Annu. Rev. Genet. 2020. 54:539-61. MUNI SCI Retrotransposons • LTR retrotransposons occurs in cytoplasmic virus-like particles and leads to the formation of extrachromosomal double-stranded DNA(dsDNA), which is imported into the nucleus before integrating into a new locus. o The coding region usually contains only two genes: gag and pol, they do not have env, i.e. virulence factor, o Ty-elements in yeast (6.3 kb). o Copia-elements a Gypsy-elements in Drosophila (5 kb) • Non-LTR retrotransposons initiate reverse transcription directly at the target locus after cleaving genomic DNA, a process known as 'target-primed reverse transcription'. o F, Ga l-elements in Drosophila. o Short sequences SINE (short interspersed nuclear elements) - 500 bp, 105copies, derived from genes for small RNAs, including tRNA (pseudogenes). o Long sequences LINE (long interspersed nuclear elements) - 6.5 kb, 10 000 - 50 000 copies in mammals. MUNI 57 Department of Experimental Biology r> r» t Distribution fo transposable elements in eukaryotes Genome size 58 Department of Experimental Biology ' I " ■ 1 I ' ' ■ 1 [ 1 1 ■ ' ! ■ ■ " I 0 250 SOO 750 1.000 U50 1.500 1.750 2.000 My« Annu. Rev. Genet. 2020. 54:539-61. MUNI SCI Retrotransposons • Autonomous ERVs and LINEs. The L1 is the only LINE known to be actively mobile in mammals. • Nonautonomous - Alu and SVA, are dependent on L1 for their mobility. Processed pseudogenes are spliced mRNAs copied and inserted in the genome by the Lis. • Gag, group-specific antigen (capsid proteins). • Pol, polymerase. • Env, envelope. • LTR, long terminal repeat. • Prt, protease • INT, integrase domain. • RT, reverse transcriptase domain. • TSD, target site duplication. • EN, endonuclease domain. 59 Department of Experimental Biology Int J Evol Biol. 2013;2013:424726 Retrotransposons AUTONOMOUS a) LTR LTR RT INT LTR L>^^^^[_Gag Pol Env TSD PrtJ-ERV (7-9 kb) TSD b) Non-LTR 5'UTR EN RT C 3' UTR [^^■HHIIM ORF1 ■ ORF2 ^■■AoD TSD LINE1 (6 kb) TSO NONAUTONOMOUS dTa b |a„D TSD Left monomer Right monomer SINE - Alu (300 bp) TSD CCCTCTn - (37-51 bp)n DISK* Alu-likc VNTR SINE-R TSD SVA (< 3 kb) TSD SUTR 3' JTB DJ Exon2 Exon3 Exon4 "lAnD- TSD Processed pseudogenc TSD MUNI SCI Retrotransposons in Yeast • The most highly characterized Ty1 element is Ty1-H3, which was isolated following its retrotransposition into plasmid DNA. Resembles a primitive retrovirus. • Approximately 35 copies per haploid yeast genome. • 5918 base pairs (bp) in length with 334 bp direct repeats, or LTRs, at each end, with 5 bp duplication when incorporated. • Ty1 - element has two genes - TyA and 7~yB, which are homologous to the Gag and Pol retrovirus genes. • Products of TyA and TyB form virus-like particles in the cytoplasm. 60 Department of Experimental Biology MUNI SCI Retrotransposons in Yeast • LTRs - boxed arrowheads. • Functional domains of Pol that synthesized as part of the Gag-Pol polyprotein are posttranslationally cleaved by PR (protease) into separate proteins include. • RT-RH (reverse transcriptase-RNase H). • IN (integrase). • The retroviral envelope gene (ENV) is not present in Ty1. LTR POL/TYB Ty1 ►JGyAG/TY? I LTR PR IN RT-RH 61 Department of Experimental Biology Microbiol Spectr. 2015 Apr 1; 3(2): 1-35. MUNI SCI Mechanism of retrotransposition of Ty element ATy1 element in the host genome is transcribed and the Ty1 RNA is exported to the cytoplasm. The RNA is translated into Gag and Gag-Pol proteins, and associates with these proteins to form Ty1 RNPs, also known as retrosomes. Ty1 RNPs give rise to VLPs that encapsidate a dimer of Ty1 RNA and tRNAiMet. Within the VLP, Gag and Pol proteins are cleaved by PR to form mature Gag, PR, IN and RT proteins. Following VLP maturation, Ty1 RNA is reverse transcribed into cDNA by RT using tRNAiMet as a primer. The cDNA is bound by IN to form the pre-integation complex, which is imported into the nucleus. IN integrates Ty1 cDNA into the yeast genome. At the insertion site of DNA there is duplication of 5 nucleotides similar to bacterial transposons. MUNI Department of Experimental Biology r> r» t Mechanism of retrotransposition of Ty element • Transcription and Ty1 RNA is exported to the cytoplasm. • The RNA is translated into Gag and Gag-Pol. • Formation of Ty1 RNPs retrosomes. • VLP assembly - encapsidation a dimerofTyl RNA and tRNAiMet. • Cleavage of Gag-Pol into Gag, PR, IN and RT. • Following VLP maturation -reverse transcription using tRNAiMet as a primer. • IN integrates Ty1 cDNA into the yeast genome. Translation RNA export V/WNVAAA W\/\ Gag Pol \ VLP assembly tRNAiMet ^ VLP maturation Reverse transcription 63 Department of Experimental Biology Microbiol Spectr. 2015 Apr 1; 3(2): 1-35. MUNI SCI Retrotransposons in Drosophila melanogaster • LTR retrotransposons: o Copia elements - produce a large amount of RNA (hence the name), structurally similar to Ty1-yeast elements, o Gypsy elements - larger than Copia, they also contain a gene similar to the Env gene in retroviruses. o Copia and Gypsy forms virus-like particles in drosophila cells; however, only particles containing gypsy RNA can pass through the cell membrane. • Non-LTR transposons: o The F, G and l-elements - they do not have LTR, at the ends they have sequences formed by reverse transcription of poly(A). o HeT-Aand TART (telomere-associated retrotransposon). 64 Department of Experimental Biology Oncotarget, February 2019 10(12). MUNI SCI Retrotransposons in Drosophila melanogaster Gypsy 5'LTR TG... *4>u3|r1u5<[ CA ACC TGG... pol —I— TSD PBS PR RT RH INT Í Frame shift ? Copia 5' LTR TG... ^DU31r|U5^ CA ACC TGG pol TSD PBS OS ENV? PR INT RT RH t Frame shift ? (A|G), 3'LTR TG... ...CA PPT -DU3|r|U5