No description available.

We developed a new program called GeneTack for ab initio frameshift detection in intronless protein-coding nucleotide sequences. The GeneTack program uses a hidden Markov model (HMM) of a genomic sequence with possibly frameshifted protein-coding regions. The Viterbi algorithm nds the maximum likelihood path that discriminates between true adjacent genes and a single gene with a frameshift. We tested GeneTack as well as two other earlier developed programs FrameD and FSFind on 17 prokaryotic genomes with frameshifts introduced randomly into known genes. We observed that the average frameshift prediction accuracy of GeneTack, in terms of (Sn+Sp)/2 values, was higher by a signicant margin than the accuracy of the other two programs. GeneTack was used to screen 1,106 complete prokaryotic genomes and 206,991 genes with frameshifts (fs-genes) were identifed. Our goal was to determine if a frameshift transition was due to (i) a sequencing error, (ii) an indel mutation or (iii) a recoding event. We grouped 102,731 genes with frameshifts (fs-genes) into 19,430 clusters based on sequence similarity between their protein products (fs-proteins), conservation of predicted frameshift position, and its direction. While fs-genes in 2,810 clusters were classied as conserved pseudogenes and fs-genes in 1,200 clusters were classied as hypothetical pseudogenes, 5,632 fs-genes from 239 clusters pos- sessing conserved motifs near frameshifts were predicted to be recoding candidates. Experiments were performed for sequences derived from 20 out of the 239 clusters; programmed ribosomal frameshifting with eciency higher than 10% was observed for four clusters. GeneTack was also applied to 1,165,799 mRNAs from 100 eukaryotic species and 45,295 frameshifts were identied. A clustering approach similar to the one used for prokaryotic fs-genes allowed us to group 12,103 fs-genes into 4,087 clusters. Known programmed frameshift genes were among the obtained clusters. Several clusters may correspond to new examples of dual coding genes. We developed a web interface to browse a database containing all the fs-genes predicted by GeneTack in prokaryotic genomes and eukaryotic mRNA sequences. The fs-genes can be retrieved by similarity search to a given query sequence, by fs- gene cluster browsing, etc. Clusters of fs-genes are characterized with respect to their likely origin, such as pseudogenization, phase variation, programmed frameshifts etc. All the tools and the database of fs-genes are available at the GeneTack web site http://topaz.gatech.edu/GeneTack/

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abstract: Immunotherapy has been revitalized with the advent of immune checkpoint blockade treatments, and neo-antigens are the targets of immune system in cancer patients who respond to the treatments. The cancer vaccine field is focused on using neo-antigens from unique point mutations of genomic sequence in the cancer patient for making personalized cancer vaccines. However, we choose a different path to find frameshift neo-antigens at the mRNA level and develop broadly effective cancer vaccines based on frameshift antigens. In this dissertation, I have summarized and characterized all the potential frameshift antigens from microsatellite regions in human, dog and mouse. A list of frameshift antigens was validated by PCR in tumor samples and the mutation rate was calculated for one candidate – SEC62. I develop a method to screen the antibody response against frameshift antigens in human and dog cancer patients by using frameshift peptide arrays. Frameshift antigens selected by positive antibody response in cancer patients or by MHC predictions show protection in different mouse tumor models. A dog version of the cancer vaccine based on frameshift antigens was developed and tested in a small safety trial. The results demonstrate that the vaccine is safe and it can induce strong B and T cell immune responses. Further, I built the human exon junction frameshift database which includes all possible frameshift antigens from mis-splicing events in exon junctions, and I develop a method to find potential frameshift antigens from large cancer immunosignature dataset with these databases. In addition, I test the idea of ‘early cancer diagnosis, early treatment’ in a transgenic mouse cancer model. The results show that ii early treatment gives significantly better protection than late treatment and the correct time point for treatment is crucial to give the best clinical benefit. A model for early treatment is developed with these results. Frameshift neo-antigens from microsatellite regions and mis-splicing events are abundant at mRNA level and they are better antigens than neo-antigens from point mutations in the genomic sequences of cancer patients in terms of high immunogenicity, low probability to cause autoimmune diseases and low cost to develop a broadly effective vaccine. This dissertation demonstrates the feasibility of using frameshift antigens for cancer vaccine development. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2018

HIV-1 co-opts host cell proteins at every step of its replication cycle to ensure proper replication. Our work identified that the HIV-1 genomic RNA is not a substrate for nonsense-mediated mRNA decay (NMD) even though it has multiple open reading frames as well as a long 3'UTR. We demonstrate that Up-frameshift protein 1 (UPF1) is involved in HIV-1 genomic RNA stability such that overexpression of UPF1 increases HIV-1 genomic RNA levels and Gag translation. Moreover, the role of UPF1 in HIV-1 is NMD-independent, is observed in both nuclear and cytoplasmic compartments and does not require binding to UPF2. Furthermore, the shuttling function of UPF1 is required for HIV-1 genomic RNA export since a UPF1 nuclear export mutant sequesters the genomic RNA in the nucleus and a nuclear localization mutant does not immunoprecipitate with the HIV-1 genomic RNA. UPF1's role in HIV-1 genomic RNA export is observed in both Rev-dependent and -independent conditions. In addition, UPF1 is found in complex with Rev, CRM1, Nup62 and DDX3, cellular proteins with already characterized roles in HIV-1 genomic RNA export. Lastly, we also identified UPF2 as a negative regulator, such that its binding to UPF1 results in the nuclear sequestration of the HIV-1 genomic RNA. We have identified a possible mechanism to explain how HIV-1 escapes the RNA quality control mechanism of NMD by co-opting UPF1 function for efficient HIV-1 genomic RNA export, stability and translation. / Le VIH-1 requiert plusieurs protéines cellulaires à chaque étape de son cycle de réplication pour assurer une réplication efficace. Notre travail a mené à l'identification que l'ARN génomique du VIH-1 n'est pas un substrat pour la dégradation des ARNm aberrants (NMD), même si elle a plusieurs cadres de lecture ainsi qu'une longue 3'UTR. Nous avons démontré que UPF1 est impliqué dans la stabilité de l'ARN génomique du VIH-1 car la surexpression de UPF1 a engendré une augmentation des niveaux d'ARN du VIH-1 et de Gag. Par ailleurs, le rôle de UPF1 est distinct de son rôle dans le mécanisme NMD, il est observé dans les compartiments nucléaires et cytoplasmiques et ne nécessite pas sa liaison à UPF2. De plus, la fonction navette de UPF1 est requise pour assurer l'exportation de l'ARN génomique du VIH-1 car le mutant NES d'UPF1 bloque son export et le mutant NLS n'immunoprécipite pas avec celui-ci. Ce nouveau rôle d'UPF1 est observé dans la présence et l'absence de Rev. De plus, UPF1 se trouve en complexe avec Rev, CRM1, Nup62 et DDX3, les protéines cellulaires déjà caractérisées comme étant impliquées dans l'exportation de l'ARN génomique du VIH-1. Enfin, nous avons également identifié UPF2 comme étant un régulateur négatif, de telle sorte que sa liaison à UPF1 engendre un blocage nucléaire de l'ARN génomique du VIH-1. Nous avons identifié un mécanisme possible démontrant comment le VIH-1 s'évade du mécanisme NMD en cooptant UPF1 pour faciliter l'exportation, la stabilité et la traduction de l'ARN génomique du VIH-1.

Spel kan användas för att förmedla budskap, lärdomar och åsikter. I detta examensarbete skapades ett digitalt kortspel som med hjälp av procedurell retorik och ett skifte av det ramverk som spelet upplevs igenom söker att förmedla ett budskap. Spelets mekanik skapades för att uppmuntra spelarna till en viss typ av beteende och därigenom utveckla en strategi för att på bästa möjliga sätt vinna spelet. När sedan skiftet av ramverket skedde så avslöjades ny information som fick spelarna att känna en medbrottslighet och därför ifrågasätta sitt tidigare beteende. Trots att spelets regler och mål inte ändrades så kunde det genom observationer iakttas att spelarnas beteende ändrades och att nya implicita mål skapades av spelarna själva. I de uppföljande intervjuerna så fick deltagarna svara på frågor om bland annat den upplevelse de haft och vad de trodde att spelets budskap var.

Protein synthesis, translation, performed by the ribosome, is a fundamental process of life and one of the main targets of antibacterial drugs. This thesis provides structural and biochemical understanding of three aspects of bacterial translation. Elongation factor G (EF-G) is the target for the antibiotic fusidic acid (FA). FA binds to EF-G only on the ribosome after GTP hydrolysis and prevents EF-G dissociation from the ribosome. Point mutations in EF-G can lead to FA resistance but are often accompanied by a fitness cost in terms of slower growth of the bacteria. Secondary mutations can compensate for this fitness cost while resistance is maintained. Here we present the crystal structure of the clinical FA drug target, Staphylococcus aureus EF-G, together with the mapping and analysis of all known FA-resistance mutations in EF-G. We also present crystal structures of the FA-resistant mutant F88L, the FA-hypersensitive mutant M16I and the FA-resistant but fitness-compensated double mutant F88L/M16I. Analysis of mutant structures together with biochemical data allowed us to propose that fitness loss and compensation are caused by effects on the conformational dynamics of EF-G on the ribosome. Aminoglycosides are another group of antibiotics that target the decoding region of the 30S ribosomal subunit. Resistance to aminoglycosides can be acquired by inactivation of the drugs via enzymatic modification. Here, we present the first crystal structure an aminoglycoside 3’’ adenyltransferase, AadA from Salmonella enterica. AadA displays two domains and unlike related structures most likely functions as a monomer. Frameshifts are deviations the standard three-base reading frame of translation. -1 frameshifting can be caused by normal tRNASer3 at GCA alanine codons and tRNAThr3 at CCA/CCG proline codons. This process has been proposed to involve doublet decoding using non-standard codon-anticodon interactions. In our study, we showed by equilibrium binding that these tRNAs bind with low micromolar Kd to the frameshift codons. Our results support the doublet-decoding model and show that non-standard anticodon loop structures need to be adopted for the frameshifts to happen. These findings provide new insights in antibiotic resistance and reading-frame maintenance and will contribute to a better understanding of the translation elongation process.

Studies of ribosomal reading frame maintenance are often based on frameshift mutation suppression experiments. In this thesis, suppression of a frameshift mutation in Salmonella enterica serovar Typhimurium by a tRNA and a ribosomal protein are described. The +1 frameshift mutation hisC3072 (that contains an extra G in a run of Gs) is corrected by mutations in the argU gene coding for the minor tRNAArgmnm5UCU. The altered tRNAArgmnm5UCU has a decreased stability and reduced aminoacylation due to changed secondary and/or tertiary structure. Protein sequencing revealed that during the translation of the GAA-AGA frameshifting site the altered tRNAArgmnm5UCU reads the AGA codon inefficiently. This induces a ribosomal pause, allowing the tRNAGlumnm5s2UUC residing in the ribosomal P-site to slip forward one nucleotide. The same frameshift mutation (hisC3072) was also suppressed by defects in the large ribosomal subunit protein L9. Single base substitutions, truncations, and absence of this protein induced ribosome slippage. Mutated ribosome could shift to the overlapping codon in the +1 frame, or bypass to a codon further downstream in the +1 frame. The signal for stimulation of slippage and function of L9 needs to be investigated. During the search for suppressors of the hisD3749 frameshift mutation, a spontaneous mutant was isolated in the iscU gene that contained greatly decreased levels of the thiolated tRNA modifications ms2io6A and s2C. The iscU gene belongs to the iscR-iscSUA-hscBA-fdx operon coding for proteins involved in the assembly of [Fe-S] clusters. As has been shown earlier, IscS influences the synthesis of all thiolated nucleosides in tRNA by mobilizing sulfur from cysteine. In this thesis, it is demonstrated that IscU, HscA, and Fdx proteins are required for the synthesis of the tRNA modifications ms2io6A and s2C but are dispensable for the synthesis of s4U and (c)mnm5s2U. Based on these results it is proposed that two distinct pathways exist in the formation of thiolated nucleosides in tRNA: one is an [Fe-S] cluster-dependent pathway for the synthesis of ms2io6A and s2C and the other is an [Fe-S] cluster-independent pathway for the synthesis of s4U and (c)mnm5s2U. MiaB is a [Fe-S] protein required for the introduction of sulfur in ms2io6A. TtcA is proposed to be involved in the synthesis of s2C. This protein contains a CXXC conserved motif essential for cytidine thiolation that, together with an additional CXXC motif in the C-terminus may serve as an [Fe-S] cluster ligation site.

No description available.

No description available.