Trypanosomes: their genomes and two example of their extreme biology Trypanosoma Images, Stock Photos & Vectors | Shutterstock Organism Relevance Genome size Number of genes predicted Giardia duodenalis (assemblage B) Human pathogen (Giardiasis) 11.7 Mb 4,470 Giardia duodenalis (assemblage A) Human pathogen (Giardiasis) 11.7 Mb 6,470 Leishmania braziliensis Human pathogen (Leishmaniasis) 33 Mb 8,314 Leishmania infantum Human pathogen (Visceral leishmaniasis) 33 Mb 8,195 Leishmania major Human pathogen (Cutaneous leishmaniasis) 32.8 Mb 8,272 Naegleria gruberi Human pathogen (Primary amoebic meningoencephalitis) 41 Mb 15,727 Trichomonas vaginalis Human pathogen (Trichomoniasis) 160 Mb 59,681 Trypanosoma brucei Human pathogen (Sleeping sickness) 26 Mb 9,068 Trypanosoma cruzi Human pathogen (Chagas disease) 34 Mb 22,570 Excavata Entamoeba histolytica Human pathogen (amoebic dysentery) 23.8 Mb 9,938 Excavata Amoebozoa genome of Trypanosoma An external file that holds a picture, illustration, etc. Object name is zmr9990922620001.jpg An external file that holds a picture, illustration, etc. Object name is zmr9990922620001.jpg Po přepisu se vlákno RNA štěpí na mRNA kódující jednotlivé geny. Při vyštěpování těchto mRNA z dlouhého vlákna dojde k přidání 5‘ CAP struktury a 3’polyA sekvence na konce každé mRNA. Tento proces se nazývá trans-splicing „EXCAVATA“: kinetoplastida ›mitochondria, nucleus › ›unique organelle called the kinetoplast ›accumulation of mitochondrial DNA ›kDNA ›an appendix of their single mitochondrion located near the basal body of the flagellum (kinetosome) ›contains a giant network of thousands of small interlocking circular DNAs › › 2. Properties - Trypanosoma brucei 1910-20 Feulgen reaction + „ kinetonucleus“ (J. Kulda a E. Nohýnková „Buňka prvoků“, 2006) „EXCAVATA“: kinetoplastida ›mitochondria, nucleus › ›unique organelle called the kinetoplast ›accumulation of mitochondrial DNA ›kDNA ›an appendix of their single mitochondrion located near the basal body of the flagellum (kinetosome) ›contains a giant network of thousands of small interlocking circular DNAs › › 2. Properties - Trypanosoma brucei Lecture 23: Flagellates - Class Kinetoplastida - Genera Trypanosoma & Leishmania Flashcards | Quizlet ›contains circular DNA in two forms ›maxicircles and minicircles ›10 – 20% cell DNA (trypanoplasms 40%) ›system of circular molecules ›maxicircles: between 20 and 40kb in size, a few dozen identical copies per kinetoplast ›minicircles: between 0.5 and 10kb in size, several thousand copies usually nearly identical in size but heterogeneous in sequence Kinetoplast kDNA network structure. (A) Electron micrograph of the periphery of an isolated kDNA network from T. avium. Loops represent interlocked minicircles (the arrowhead indicates a clear example). Bar, 500 nm. (B) Diagrams showing the organization of minicircles. (I) Segment of an isolated network showing interlocked minicircles in a planar array. (II) Section through a condensed network disk in vivo showing stretched-out minicircles. The double-headed arrow indicates the thickness of the disk, which is about half the circumference of a minicircle. Beyond replication: Division and segregation of mitochondrial DNA in kinetoplastids - ScienceDirect A B https://www.nature.com/scitable/topicpage/kinetoplastids-and-their-networks-of-interlocked-dna-1436 8046/ kinetoplast maxicircles ›encode typical mitochondrial gene products ›e.g. rRNAs and subunits of respiratory chain complexes ›some of the protein-coding genes are encrypted ›to generate functional mRNAs, the cryptic maxicircle transcripts undergo posttranscriptional modification via an intricate RNA editing process ›involves insertion and deletion of uridine residues at specific sites in the transcripts ›the genetic information for editing is provided by guide RNAs (gRNAs) ›mostly encoded by minicircles, although a few are encoded by maxicircles › maxi mini kinetoplast › minicircles ›encoding gRNAs is the only known function ›some organisms that edit extensively (such as Trypanosoma brucei) possess about 200 different minicircle sequence classes in their network to provide sufficient gRNAs maxi mini kinetoplast ›kinetoplasts not forming networks and forming networks › › › › › › › › ›different members of kinetoplastida reveals different structure › Pro-k Mega-k Eu-k Pan-k Poly-k Proposed evolution of kinetoplastids, emphasizing differences in kDNA organization and compaction. kDNA (k) is the structure within the mitochondrial matrix. fl, flagellum; m, mitochondrion; n, nucleus. kDNA in C. helicis is pan-kDNA, that in T. borreli is mega-kDNA, that in D. trypaniformis is poly-kDNA, that in B. saltans is pro-kDNA, and that in T. brucei is a kDNA network. process of network structure formation (molecule „relaxation“ is required!) ISEPpapers 1: Summer 2018 ISEPpapers 1: Summer 2018 ISEPpapers 1: Summer 2018 Closing the gaps in kinetoplast DNA network replication | PNAS kinetoplast: replication of the kDNA network ›replication of the kinetoplast occurs simultaneously to the duplication of the adjacent flagellum and just prior to the nuclear DNA replication › ›minicircles are released from the network into a kinetoflagellar zone (region between the kinetoplast and the mitochondrial membrane) in which they initiate replication › ›after replication the minicircles migrate by unknown mechanisms to the antipodal protein complexes that contain several replication proteins ›an endonuclease, helicase, DNA polymerase, DNA primase, and DNA ligase ›which initiate repair of remaining discontinuities in the newly replicated minicircles ›the minicircles (still containing at least one nick or gap) are then linked to the network periphery where the repair of the remaining minicircle gaps occur when replication is completed ›this process occurs one minicircle at a time, and only a small number of minicircles are unlinked at any given moment ›to keep track of which minicircles have been replicated, upon rejoining to the kDNA network a small gap remains in the nascent minicircles, which identifies them as having already been replicated Closing the gaps in kinetoplast DNA network replication | PNAS kinetoplast: replication of the kDNA network ›this process occurs one minicircle at a time, and only a small number of minicircles are unlinked at any given moment › ›to keep track of which minicircles have been replicated, upon rejoining to the kDNA network a small gap remains in the nascent minicircles, which identifies them as having already been replicate ›after replication the minicircles migrate by unknown mechanisms to the antipodal protein complexes that contain several replication proteins ›an endonuclease, helicase, DNA polymerase, DNA primase, and DNA ligase ›which initiate repair of remaining discontinuities in the newly replicated minicircles ›the minicircles (still containing at least one nick or gap) are then linked to the network periphery where the repair of the remaining minicircle gaps occur when replication is completed ›this process occurs one minicircle at a time, and only a small number of minicircles are unlinked at any given moment ›to keep track of which minicircles have been replicated, upon rejoining to the kDNA network a small gap remains in the nascent minicircles, which identifies them as having already been replicated Closing the gaps in kinetoplast DNA network replication | PNAS kinetoplast: replication of the kDNA network Illustration of kinetoplast rotating during minicircle replication. ›to prevent the build-up of new minicircles, the entire kDNA network will rotate around the central axis of the disk ›the rotation is believed to be directly connected to the replication of the adjacent flagellum ›after replication the minicircles migrate by unknown mechanisms to the antipodal protein complexes that contain several replication proteins ›an endonuclease, helicase, DNA polymerase, DNA primase, and DNA ligase ›which initiate repair of remaining discontinuities in the newly replicated minicircles ›the minicircles (still containing at least one nick or gap) are then linked to the network periphery where the repair of the remaining minicircle gaps occur when replication is completed ›this process occurs one minicircle at a time, and only a small number of minicircles are unlinked at any given moment ›to keep track of which minicircles have been replicated, upon rejoining to the kDNA network a small gap remains in the nascent minicircles, which identifies them as having already been replicated kinetoplast: replication of the kDNA network Kinetoplast replication is linked to nuclear DNA replication and cell division › › ›the exact mechanisms for maxicircle kDNA have yet to be determined in the same detail ›a structure called a nabelschnur (German for "umbilical cord") Kinetoplast - Wikipedia Fig 9 ›Basal body (yellow), flagellum (black line), tripartite attachment complex (TAC) (red), kDNA and nucleus (blue), and TbLAP1 (green) are depicted during the cell cycle of procyclic T. brucei. (A) 1N1K interphase cells bearing one basal body, a flagellum and a single TAC connected with kDNA and TbLAP1. (B) Cells in the earliest stages of division, with basal and pro-basal bodies divided and started to segregate, and TAC division commenced; the growth of the new flagellum becomes evident; kDNA displays an elongated “dumbbell” shape, which reflects its replication; TbLAP1 co-localizes with kDNA. (C) Later stage where kDNA replication is complete and the progeny kDNA networks have divided and appear in a perpendicular position to one another; TbLAP1 is present in the progeny kDNAs and in the nabelschnur; basal and pro-basal bodies proceed with their segregation. (D) Later still, segregation of the newly divided kDNAs continues, together with respective TAC and basal bodies; the nabelschnur begins to fade. (E) Following nuclear DNA replication and segregation, newly divided nuclei appear, as the segregation of kDNA continues. (F) 2K2N cells just prior to cytokinesis, with nuclei completely divided with kDNA and associated structures aligned for the upcoming cell division. ›Stage I ›the kinetoplast has not yet initiated replication ›contains no antipodal protein complexes ›positioned relative to a single flagellar basal body ›Stage II ›the kinetoplast begins to show antipodal protein complexes ›the flagellar basal body begins replication, as does the kinetoplast ›the association of the replicating kinetoplast to the two basal bodies causes it to develop a domed appearance ›Stage III: ›the new flagellum begin to separate and the kinetoplast takes on a bilobed shape ›Stage IV: ›the kinetoplasts appear as separate disks but remain connected by the nabelschnur ›Stage V ›the daughter kinetoplasts are completely separated as the nabelschnur is broken RNA editing ›site-specific posttranscriptional changes in an RNA sequence ›(other than pre-mRNA splicing and 3’-polyadenylation) ›was first described in trypanosomatids ›a widespread phenomenon throughout eukaryotes › Uridine! mitochondriálni DNA (kDNA) transkripce RNA editing translace AAAGUAGAGAACCUGGUAGGU AAAGUAGAUUGUAUACCUGGU RNA editing in Trypanosomes • AAAAAUAAGUAUUUUGAUAUUAUUAAAGUAAAAGAGGAAUUUUGGGCGGAAGAGAAGGAGACAGGAGAGGAAAUGAAGGAGAAAGGUUUUGAGAGGGGG GUUUUUUGAGGGGAGGAAAAAGAAUUUUGAAUUUGAACUAUUUGUUUAAGUUAUGGGAGAGAAGCAAGGAGGAGAAAAGUAGGGGAAUUUUGAGGAGAU UCUUGGGGAGAGGCGGGCGGGCGACGGCGGUUUUGAAAACACCCAUUUUUAGGAGGAUAAGAGGGGAGAAAAGGGGAAAUGGAAUUGGGAAUUGCCUUU GCCAAACUUUUAGAAGAAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAGUUGUGAUUUUGGAGUUAUAGAAUAAGAUCAAAUAAGUUAA UAAUA M K E K G F E R G V F W G E E K E F W I W T I C L S Y G R E A R R R K V G E F W G D S W G E A G G R R R F W K H P F L G G ter E G R K G E M E L G I A F A K L L E E R A G K V R G R R E E R E S C D F G V I E ter D Q I S terter pre-edited • AAAAAUAAGUAUUUUGAUAUUAUUAAAGUAAAAGAGGAAUUUUGGGCGGAAGAGAAGGAGACAGGAGAGGAAAUGAAGGAGAAAGGUUUUGAGAGGGGG 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AuuuG**GuuUGuuA**UUGGAGUUAUAGAAUAAGAUCAAAUAAGUUAAUAAUA editing in progress... edited M F L F F F C D L F W L R L L L C M Y Y C V W S R L C F I V Y F N C L M L I F D F L L F C L F D L Y L F V G L C L F L L L W F M L F N L Y S L I L Y Y C I T Y L N L Y L L F C I V F L L Y I A F L F L F C F L C D F F L F N N L L V G D S F M D V F F I R F L L C F L E C F S L L C R C L S T F L R L F C N L L S S H F L L L M F F D F F Y F I F V F F F W C F L L L I Y F I Y F C V L F L F I I L C V F I F V G F I C R H I T V I Y F L ter M F L F F F C D L F W L R L L L C M Y Y C V W S R L C F I V Y F N C L M L I F D F L L F C L F D L Y L F V G L C L F L L L W F M L F N L Y S L I L Y Y C I T Y L N L Y L L F C I V F L L Y I A F L F L F C F L C D F F L F N N L L V G D S F M D V F F I R F L L C F L E C F S L L C R C L S T F L R L F C N L L S S H F L L L M F F D F F Y F I F V F F F W C F L L L I Y F I Y F C V L F L F I I L C V F I F V G F I C R H I T V I Y F L ter Benne et al. (1986), Bhat et al. (1990) T. brucei A6 RNA editing RNA editing in Trypanosomes What is RNA editing? It is a form of RNA processing required for expression of mitochondrial genes in trypanosomatids. Consider this mitochondrial transcript. It does not translate into anything that would show some sort of homology. Now, editing will remodel this transcript through the insertion and deletion of uridine residues... ...AAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAGUUGUGAUUUUGGAGUUAUAG... |·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC... ...AAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAGUUGUGAUUGGAGUUAUAG... ||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC... ...AAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAGUUGUGuuAUUGGAGUUAUAG... ·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC... ...AGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAGUUGuuUGuuAUUGGAGUUAUAG... ·||·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC... ...AAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAGGuuUGuuAUUGGAGUUAUAG... |·||·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC... ...GAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAuuuGGuuUGuuAUUGGAGUUAUAG... ·||||·||·||·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC... ...GCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG... ||··||||·||·||·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC... ...AGGAAAGGUUAGGGGGAGGAGAGAAGAAAGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG... ··|||··||||·||·||·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC... ...GAAAGGUUAGGGGGAGGAGAGAAGAAAuuGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG... ||···|||··||||·||·||·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC... ...AAAGGUUAGGGGGAGGAGAGAAGAAuAuuGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG... ||||···|||··||||·||·||·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC... ...AGGUUAGGGGGAGGAGAGAAGAuuAuAuuGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG... ||·||||···|||··||||·||·||·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC... ...GUUAGGGGGAGGAGAGAAGuuAuuAuAuuGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG... |·||||·||||···|||··||||·||·||·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC... ...UUAGGGGGAGGAGAGAuAGuuAuuAuAuuGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG... ····|··|·|·|||·||||·||||···|||··||||·||·||·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC... Anchor Information U Tail Editing Block pre-mRNA gRNA Více gRNA je nutných pro editaci jednoho transkriptu RNA editing in Trypanosomes RNA editing in Trypanosomes ›the uridine (U) insertion/deletion editing ›occurs in the kinetoplast ›trans-acting guide RNAs and entails the insertion of hundreds and deletion of dozens of U residues from mitochondrial RNAs to produce mature, translatable mRNAs ›catalytic machinery termed the editosome or RNA editing core complex (RECC) Pre-edited mRNAs are transcribed from mitochondrial maxicircles, while the majority of gRNAs are transcribed from the minicircle component of mitochondrial DNA. The gRNA 5’ anchor region (olive) basepairs with the mRNA and the gRNA information region (yellow) directs the number of U's inserted or deleted. The gRNA 3’ oligo(U) tail stabilizes the gRNA/mRNA interaction. Enzymes contained within the RNA editing core complex (RECC) catalyze mRNA endonucleolytic cleavage at an editing sites, U insertion by a 3’ TUTase, and U deletion by a U-specific exoribonuclease as directed by the sequences of gRNAs. Cleaved mRNAs are resealed by RNA ligases. RNA editing in Trypanosomes Multi-round editing entails sequential utilization of multiple gRNAs. Because the anchor region of a given gRNA basepairs with edited mRNA sequence specified by the prior gRNA, editing progresses in a 3’ to 5’ direction along an mRNA. Multiple black arrowheads symbolize multiple editing sites within an editing block, as defined by the hybridized gRNA. Dashed gRNA labels indicate that they are turned over during/after an editing block has been processed. An editing domain is a stretch of mRNA sequence that requires the gRNA cascade for its processing. An external file that holds a picture, illustration, etc. Object name is nihms-726503-f0003.jpg RNA editing in Trypanosomes Antigenic variation: Trypanosoma Figure 1 from University of Dundee Antigenic variation in African ... Consequences of antigenic variation → prolonging infection Traditional view ›each growth peak contains one variant ›reduction of each growth peak is due to antibodies against each VSG Reality ›each growth peak generally contains many variants › › › › › › › › › ›reduction of each growth peak depends on two factors: ›differentiation to the non-dividing stumpy stage ›anti-VSG antibodies Fig. 2 | The in vivo dynamics of antigenic variation in ... Fig. 2 | The in vivo dynamics of antigenic variation in ... Trypansoma antigenic variation VSG (variant sufrace glycoprotein) TRYPANOSOME'S SURFACE COAT of VSG's is visible as a diffuse, dark layer in an electron micrograph. A cross section of the parasite's body and flagellum. The double membrane just inside the surface coat is the cell membrane. The structure of GPI anchor of VSG from bloodstream form of ... Variable surface glycoprotein (VSG) Glycosylphosphatidylinositol is a phosphoglyceride attached to the C-terminus of a protein during posttranslational modification. The hydrophobic C-terminal sequence is then cleaved off and replaced by the GPI-anchor. GPI is composed of a phosphatidylinositol group linked through a carbohydrate-containing linker (glucosamine and mannose glycosidically bound to the inositol residue) and via an ethanolamine phosphate (EtNP) bridge. The two fatty acids within the hydrophobic phosphatidyl-inositol group anchor the protein to the cell membrane. PROTEIN CHAIN of a typical VSG is composed of about 500 amino acids, The first 20 or so of these, at what is called the N terminal of the protein, constitute a signal peptide, which is cleaved from the protein before the VSG is implanted in the cell membrane. The next 360 amino acids (color) constitute the variable region, which is different in each antigenicalIy distinct VSG. The final 120 amino acids at the C terminal are quite similar in each of two "homology groups" of VSG's. The last 20 amino acids of this region are cleaved from the chain and replaced by a large molecule that anchors the VSG in the cell membrane. VSG'S OF SURFACE COAT may be assembled as indicated in this somewhat speculative drawing. The structure of part of the variable region of one VSG (color) is based on X-ray-crystallographic data (see illustratioll Oil next page); the full extent of the VSG and the location of its neighbors are indicated by the gray cylinders. The variable region is a dimer, or double molecule, that appears to be a bundle of the protein structures called alpha helixes. Carbohydrate molecules flank the bundle. Two more carbohydrates at the base of the VSG may incorporate a small sugar molecule: the cross-reacting determinant. Fatty acids extending from these two carbohydrates appear to anchor the VSG in the membrane. The structure of GPI anchor of VSG from bloodstream form of ... VSG homodimer N C Variable surface glycoprotein (VSG) bloodstream form VSG in the surface coat: PROTECTION Antibody cannot access invariant antigen, only the VSG (this is why coat change is required). … like a dense homogenous forest a dense monolayer of ~5×106 identical VSG dimers VSG in the surface coat: PROTECTION Host antibodies are removed from the surface Engstler et al., Cell Host-Derived Antibodies Are Removed from the Cell Surface of Bloodstream Stage Trypanosoma brucei (A) Schematic representation (drawn to scale) of IgG and IgM molecules bound to the trypanosome variant surface glycoprotein (VSG) coat (left). VSGs are homodimers (monomers in light blue and purple) that are attached to the plasma membrane via GPI anchors (right). (B) Visualization of antibody removal. Cells were surface labeled with blue-fluorescent AMCA-sulfo-NHS and incubated at a density of 108 cells/ml for 10 min on ice with 0.1 mg/ml VSG-specific IgG, resulting in antibody labeling of 0.26% of all VSG dimers on the plasma membrane. Following 0-3 min of incubation at 37C, cells were chemically fixed and permeabilized. Anti-VSG antibodies were detected with species-specific Alexa Fluor 488-conjugated second antibodies (green). Open arrows indicate the position of the flagellar pocket, and filled arrows point to the lysosome. Scale bar, Variable surface glycoprotein (VSG) metacyclic form The crystal structure and localization of Trypanosoma brucei ... Structural model of the T. brucei metacyclic surface glycocalyx displaying the GPI-anchored metacyclic VSG homodimers (mVSG), MISP (metacyclic invariant surface protein) and remains of BARP (brucei alanine-rich protein). Besides antigenic variation and its impact on the host’s antibody response, VSGs affect transmission through metacyclic VSG expression; host range, because they can confer human infectivity to some T. brucei strains; resistance to the drug suramin; nutrient scavenging through the mediation of transferrin binding for iron import; and immune modulation, as they are known to trigger a proinflammatory response whilst suppressing the activation of complement. ANTIGENIC VARIATION IS BASED ON SILENT INFORMATION → A LARGE ARCHIVE OF SILENT VSG GENES https://www.researchgate.net/profile/Gloria_Rudenko/publication/51975122/figure/fig7/AS:32496026758 7594@1454487974000/Genomic-location-of-the-VSG-gene-repertoire-in-Trypanosoma-brucei-a-The-vast-maj ority_W640.jpg https://www.researchgate.net/profile/Gloria_Rudenko/publication/51975122/figure/fig7/AS:32496026758 7594@1454487974000/Genomic-location-of-the-VSG-gene-repertoire-in-Trypanosoma-brucei-a-The-vast-maj ority_W640.jpg Genomic location of the VSG gene repertoire in Trypanosoma brucei. (a) The vast majority of the VSG genes and pseudogenes (more than 1500 in T. brucei) are located in extensive subtelomeric tandem arrays. These haploid regions are attached to the diploid chromosomal cores. The vast majority of the VSGs (more than 90%) are pseudogenes (ψ) (indicated with gray filled boxes), with functional VSGs indicated with filled colored boxes. (b) A subset of the VSGs (more than 200) are located adjacent to the telomere repeats of small chromosomes including an abundant class of minichromosomes (of which there are about 100 in the cell). (c) About 15 VSGs are located adjacent to the telomeres of the VSG ES transcription units (promoters indicated with flags). Only one VSG expression is transcribed at a time (indicated with an arrow). Monoallelic gene expression ›more than 1,000 VSG genes and pseudogenes are packed as gene arrays or located at subtelomeres ›transcription occurs only from specialised subtelomeric transcription units known as Bloodstream Expression Sites, BESs (sometimes called telomeric expression sites, ESs) › ›~15 BESs in a cell ›polymorphic in size and structure ›reveal a surprisingly conserved architecture in the context of extensive recombination › ›in a given cell only one BES is active at any time, and therefore only one VSG protein expressed ›a diverse range of polymorphic genes called Expression Site Associated Genes (ESAGs) ›membrane-associated or membrane-targeted proteins, transmembrane receptors, etc. › › › › › › › › ›polycistronic unit contains a number of ESAGs all expressed along with the active VSG ›BES transcription results from the recruitment of RNA polymerase I (pol I) on a promoter of the ribosomal type, in a non-nucleolar nuclear structure termed the “BES body“ „one gene at a time“ Monoallelic gene expression Bloodstream Expression Sites, BESs • spatial integration of transcription and splicing • VEX2 – sustains exclusive interaction between a single VSG ES and SL-array Spatial integration of transcription and splicing sustains VSG monoallelic expression in T. brucei bloodstream forms. (a) The single active VSG is transcribed within the ESB and establishes a stable interchromosomal interaction with one of the SL-arrays. VEX2 and VEX1 form discrete protein condensates that associate with the active VSG and the SL-array, respectively. VEX2 sustains the exclusive interaction between a single VSG ES and the SL-array, and following its depletion, all VSG ESs can access the SL-arrays and are derepressed. ESB1 is a stage-specific transcriptional activator whose depletion leads to a transcriptional drop at the active ES. Moreover, the active VSG ES resides within a highly SUMOylated focus [138] and TDP1, a high-mobility-group box protein, is enriched at this site and facilitates Pol-I transcription [139]. Only VEX2 and ESB1 are highlighted in the figure because those are the only two factors known to specifically associate with the ESB to date. Two additional nuclear bodies likely to be involved in RNA processing/splicing associate with the ESB and the SL-factory (Cajal and NUFIP bodies). Furthermore, the silent ESs display more peripheral nuclear locations: several repressing factors (red circles) associated with heterochromatin formation (including chromatin remodellers, histone chaperones, histone modifiers and components of the nuclear lamina) and telomere stability sustain their ‘inactive’ state. (b) ESB1 and VEX1/VEX2 are positive and negative regulators of VSG expression, respectively. ESB1 depletion leads to a transcriptional drop at the active ES without any effect on distal VSG loci, whereas VEX depletion, especially VEX2, leads to derepression of silent VSGs. Moreover, VEX2 depletion leads to an increase in the expression of ESAGs from the active ES, though usually much less abundantly than the active VSG. VSG SWITCHING https://microbewiki.kenyon.edu/images/9/91/VSGexpression.jpg ›two main mechanisms are used to change the expressed VSG gene and therefore perform antigenic variation (but there more mechanisms!) ›transcriptional switching between BESs (a process called “in situ activation”), which turns off the active BES and turns on a new one ›homologous recombination (gene conversion or telomere exchange), which replaces the VSG gene in the active BES › Early switches: ›<10% are transcriptional switching among the BES pool ›>90% are duplicative switching from silent archive (mostly from minichromosomes) › › Late switches: ›duplicative switching from silent archive (from array intact genes) ›mosaic gene formation (by partial duplication from array pseudogenes) › › › › transcriptional switching: “in situ activation” ›to silence the active expression site and activate a new one (an in situ switch) ›this method of switching accesses a relatively small pool of ~15 VSG genes ›on and off states differ at the level of transcript elongation › ›recent evidence indicates that the different VSG expression sites each contain genes encoding receptor proteins that are optimized for different hosts ›this could mean that in situ switches are important during the establishment of the Trypanosoma in a new host species › › Mechanisms of VSG switching in Trypanosoma brucei. The large open ... VSG SWITCHING recombination(al) switching ›recombination is central to antigenic variation, allowing the parasite to utilise complete VSG archive, typically by copying (duplication of) a different gene into the active BESs › ›gene conversions or telomere exchange › Mechanisms of VSG switching in Trypanosoma brucei. The large open ... Mechanisms of VSG switching in Trypanosoma brucei. The large open ... gene conversions (array conversions) ›access the largest pool of VSG genes (virtually all of them) ›a silent VSG gene is copied and inserted into the active expression site, replacing the old VSG gene › ›1. array gene conversion ›2. telomere conversion › telomere exchange ›a silent VSG gene at a chromosome end is flipped into the active VSG expression site VSG SWITCHING Segmental VSG conversion: mosaic genes Mechanisms of VSG switching in Trypanosoma brucei. The large open ... ›during chronic infection, novel MOSAIC GENES are expressed ›they are assembled from segments of damaged VSG genes (pseudogenes) Segmental gene conversion of multiple VSG pseudogenes can result in the creation of a new functional chimeric VSG. Three different VSG pseudogenes are indicated above, with disruptions of the ORF indicated with arrow heads and vertical lines. Multiple successive gene conversion reactions can take place, resulting in the creation of a new functional VSG which is a mosaic of segments of the different VSG pseudogenes. https://www.researchgate.net/profile/Gloria_Rudenko/publication/51975122/figure/fig11/AS:3249602717 81905@1454487974914/Segmental-gene-conversion-of-multiple-VSG-pseudogenes-can-result-in-the-creatio n-of-a-new_W640.jpg VSG SWITCHING Most VSG switching is recombinational… ›initially, mosaics were found only late in infection, or in distinct infections › Early switches: ›<10% are transcriptional switching among the BES pool ›>90% are duplicative switching from silent archive (mostly from minichromosomes) › Late switches: ›duplicative switching from silent archive (from array intact genes) ›mosaic gene formation (by partial duplication from array pseudogenes) › › › › VSG SWITCHING Most VSG switching is recombinational… These DNA rearrangements probably are triggered by a DNA double strand break (DSB) in the 70-bp repeats of the BES. ›artificial induction of a DSB triggers recombinational switching ›DSB appear naturally in the 70-bp repeats of the BES ›DSB formation is followed by creation of a gap (which removes the expressed VSG gene) ›the gap requires repair – from a silent gene ›mosaic formation probably occurs differently, by recombination within the VSG coding sequence › › › VSG SWITCHING THE END