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Characterization of sncRNAs in response to HIV
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Os pequenos RNAs não codificantes (sncRNAs) são RNAs que não codificam para proteínas que variam em tamanho de 18 até 200 nucleótidos e são responsáveis pela regulação génica ao nível transcricional e pós transcricional, processamento e modificação de RNA. Em estudos anteriormente feitos no laboratório., novos pequenos RNAs (sRNAs) associados a retrotransposões, foram identificados de dados de sequenciação obtidos de RNA de células T naïve humanas não estimuladas e estimuladas via recetor de células T (TCR) e não infetadas e infetadas com HIV-1 ou HIV-2. Estes sRNAs aparentavam ter novas categorias funcionais, distintas dos micro RNAs (miRNAs), com características que sugerem o potencial para representar um novo mecanismo de defesa do hospedeiro contra a infeção por HIV. Distinta dos miRNAs, sncRNAs com 22 nucleótidos em extensão que são responsáveis por impedir a produção de proteína através de ligação e depois por clivagem do RNA mensageiro (mRNA) ou repressão de tradução, estes novos pequenos sRNAs são mais curtos e com função ou funções desconhecidas. O objetivo era testar as alterações de expressão nestes novos sRNAs, três sRNAs, e caracterizar o seu impacto funcional na interação entre células hospedeiras e o vírus do HIV. Para esse propósito, modelos biológicos foram usados, uma linha celular com o nome J-Lat e células primárias, usadas para a construção de bibliotecas de sequenciação. A linha celular J-Lat tem integrada um proviorus HIV, mas transcricionalmente latente que pode ser ativado. A indução do NF-κB através da estimulação do fator de necrose tumoral alfa (TNF-α) leva à produção de transcritos virais, tornando a linha celular J-Lat adequada para ser um modelo biológico para este estudo. Tendo isso em conta, as células primárias de células não infetadas e infetadas, também servem como modelo biológico. As sequências dos sRNAs alvo com 15 e 17 nucleótidos em extensão provou ser difícil para deteção por métodos de PCR padrão, pois o método tem de ser específico na deteção de sequências curtas em extensão. A transcrição reversa com um método RT-PCR com uma transcrição reversa conhecida por cauda de Poly A, provou ser confiável para a deteção de alvos curtos em extensão enquanto ao mesmo tempo ser específica. Condições apropriadas para a reativação do provirus HIV latente foram estabelecidas. As J-Lat foram colocadas em cultura e estimuladas com TNF-α para levar à produção de transcritos virais. Visualização por microscopia de fluorescência da sequência codificante da GFP no genoma viral, mais a deteção de transcritos virais por RT-PCR, confirmam a reativação do vírus latente. O testar de alterações de expressão por cauda de Poly A e PCR de RNA de células tratadas com TNF-α e células controlo, produziram resultados inconsistentes. Alvos sRNAs foram detetados, mas sem a observação ao longo das amostras de expressão diferencial. Foi então usado RNA das células primárias dos dados de sequenciação para verificar, se o fenómeno poderia ser só observado em células T primárias. Infelizmente, os mesmos resultados foram obtidos com os alvos sRNAs detetados, mas outra vez sem expressão diferencial. Análise bioinformática foi o próximo passo executado, não apenas para tentar verificar as alterações de expressão, mas também para tentar determinar a origem dos alvos sRNAs. Os mesmos dados de sequenciação usados para a identificação dos novos sRNAs foram usados para a análise. Os resultados mostraram que houve um aumento de expressão das sequências nos dados de RNA de células não infetadas e células infetadas, confirmando as suposições iniciais. O aumento em expressão foi mais alto em correspondências exatas para os alvos sRNAs do que em sequências mais longas que as contenham, o que indica que os alvos sRNAs são independentes dessas sequências mais longas. Na determinação da origem, duas das sequências foram associadas a tRNALys3, o primer para a transcrição reversa do HIV-1 e a outra sequência a um RNA ribossomal, 18S rRNA, e dois retrotransposões, THE1C e MSTA. ‘Repeat 1’ e ‘Repeat 2’ alinham com o tRNALys3, com um “mismatch” e com o PBS sncRNA em 12 nucleótidos dos seus 18 totais. A posição “mismatch” é localizada na posição 58, onde existe uma modificação, A58. Na transcrição reversa do RNA para sequenciação, essa posição é propensa a levar a “mismatches”. Essa mesma localização é a onde o nucleótido é diferente na ‘Repeat 1’ e ‘Repeat 2’. Desse modo, isso leva-nos a crer que as duas sequências são uma. A sequencia genómica sRNA foi procurada em conjunto com o PBS sncRNA. Os resultados revelaram que a quantidade de PBS sncRNA presente nos dados de sequenciação são 7 até 32 vezes menos nas bibliotecas de células não infetadas e 32 até 156 vezes menos nas bibliotecas de células infetadas, do que das sequências da ‘Repeat 1’, ‘Repeat 2’ e sequência genómica sRNA. Portanto, a sequência genómica sRNA é uma nova sequência PBS sncRNA. Investigação adicional sugere que o sRNA podem bem fazer parte de uma classe recente de sRNA, fragmentos derivados de tRNA (tRFs). São sncRNAs que são derivados de tRNAs e são usualmente metades de extremidades 5’ ou 3’ de um tRNA, mas podem também existir metades do meio do tRNA. A nossa sequência parece fazer parte de uma nova subcategoria destas metades. The tRFs têm sido associados a muitos processos como silenciamento de RNA, cancro, ou, resposta a infeções virais. Em relação à ‘Repeat 3’, nós determinamos a posição relativa da sequência no contexto da estrutura secundária reportada do 18S rRNA. A posição é localizada na base de um “stem-loop” que é compatível com o caminho de processamento do miRNA canónico. Estudos reportam a existência de miRNAs derivados de rRNA em humanos e ratos. Alguns destes miRNA derivados de rRNA têm sido associados a piRNAs e podem servir de pequenas RNAs guia. As dificuldades na deteção da expressão diferencial dos alvos sRNAs podem ser devidas a especificidade dos tRNAs e rRNAs. Eles estão entre os mais abundantes nas células. Ambos têm vários níveis de estrutura e os tRNAs têm diversas espécies e modificações. Todos estes aspetos levam a uma grande dificuldade na deteção de fragmentos de RNAs por métodos de PCR padrão. Esta dissertação trouxe à luz uma nova sequência de PBS sncRNA e um miRNA derivado de rRNA, que poderá ter um impacto num entendimento diferente da infeção por HIV-1 e a relação entre vírus e hospedeiro. Ensaios futuros são necessários para determinar quanto a descoberta de uma nova sequência do PBS sncRNA muda o nosso entendimento da infeção por HIV-1 e qual é a ligação entre o miRNA derivado de rRNA e o HIV-1, para determinar qual é a função exata e como isso afeta o hospedeiro e o HIV-1.
Small non-coding RNAs (sncRNAs) are RNAs that do not encode for proteins and range in size from 18 to 200 nucleotides and are responsible for gene regulation at the transcriptional and post-transcriptional level, RNA processing and modification. In previous studies performed in the laboratory, novel small RNAs (sRNAs) associated with retrotransposons, were identified from sequencing data obtained from RNA of CD4 human naïve T cells non-stimulated and stimulated via T cell receptor (TCR) and non-infected and infected with either HIV-1 or HIV-2. These sRNAs appeared to have new functional categories, distinct from micro RNAs (miRNAs), with characteristics that suggested the potential to represent a new defense mechanism of the host against HIV infection. Distinct from the miRNAs, sncRNAs with about 22 nucleotides in length that are responsible for preventing protein production through binding and then whether by cleavage of messenger RNA (mRNA) or translation repression, these novel sRNAs are shorter and have unknown function or functions. The aim was to test expression alterations in these novel sRNAs, three sRNAs, and characterized their functional impact on the interaction between host cells and HIV virus. For that purpose, biological models were used, a cell line named J-Lat and primary cells, used to build the sequencing libraries. The J-Lat cell line has an integrated, but transcriptionally latent HIV provirus that can be activated. The induction of the NF-κB through the tumour necrosis factor alpha (TNF-α) stimulation leads to production of viral transcripts, making the J-Lat cell line suitable to be a biological model for this study. Given that, non-infected cells and infected primary cells, also suit as a biological model. Our target sRNAs sequences with 15 and 17 nucleotides in length proved difficult for detection by standard PCR methods, because the method needs to be specific while detecting short sequences in length. A reverse transcription with polymerase chain reaction (RT-PCR) method with a specific reverse transcription know as Poly A tailing, proved to be reliable for the detection of short targets in length while also being specific. Appropriate conditions for the reactivation of the latent HIV provirus were established. The J-Lat were cultured and stimulated with TNF-α to lead to the production of viral transcripts. Visualization by florescence microscopy of the coding sequence of the green fluorescence protein (GFP) in the viral genome, plus the detection of viral transcripts by RT-PCR, confirmed the reactivation of the latent virus. Testing of the expression alterations by Poly A tailing and PCR from RNA of TNF-α treated cells and control cells, produced inconsistent results. The targets sRNAs were detected, but there was no differential expression observed along the tested samples. Thus, RNA from the primary cells of the sequencing data was used to verify, if the phenomena could only be observed in primary T cell. Alas, the same results were obtained with target sRNAs detected, but again without differential expression. Bioinformatic analysis was the next step taken, not only to try to check those expression alterations, but also to try to determine the target sRNAs origin. The same sequencing data used for the identification of those novel sRNAs was used for the analysis. The results showed that there was an increase in expression of the sequences in the data of RNA from non-infected cells to infected cells, confirming the earlier assumptions. The fold increase in expression was higher in exact matches for the target sRNAs than in longer reads containing them, which indicates that the target sRNAs are independent from longer reads. In the origin determination, two of the sequences were associated with transfer RNA Lysine 3 (tRNALys3), the primer for reverse transcription of HIV-1 and the other sequence to a ribosomal RNA, 18S rRNA, and two retrotransposons, THE1C and MSTA. ‘Repeat 1’ and ‘Repeat 2’ align with the tRNALys3 with one mismatch and the PBS sncRNA on 12 nucleotides of its total 18. The mismatch position is located on position 58, where there is a modification, A58. On reverse transcription of RNA for sequencing, that position is prone to lead to mismatches. That same location is the one where the nucleotide is different in ‘Repeat 1’ and ‘Repeat 2’. Thus, that lead to believe that those two sequences are one. That sRNA genomic sequence was searched alongside with the PBS sncRNA. The results showed that the amount of PBS sncRNA sequence present in the sequence data is 7 to 32 times lower in non-infected libraries and 32 to 156 times lower in infected libraries, than the sequences for ‘Repeat 1’, ‘Repeat 2’ and sRNA genomic sequence. Therefore, the sRNA genomic sequence is a new PBS sncRNA sequence. Additional research suggests that the sRNA might well be part of a recent class of sRNAs, tRNA-derived fragments (tRFs). They are sncRNAs that are derived from tRNAs and are usually halves of the 5’ end or 3’ end of a tRNA, but there can also exist halves from the middle of the tRNA. Our sequence seems to be part of a new subcategory of these halves. The tRFs have been associated with a lot of processes like RNA silencing, cancer, or, response to viral infections. Regarding the ‘Repeat 3, we determined the relative position of its sequence in the context of the reported secondary structure of the 18S rRNA. The position is located at the base of a stem-loop that is compatible with the canonical miRNA processing pathway. Studies report the existence of rRNA-derived miRNAs in humans and mice. Some of these rRNA-derived miRNAs have been linked to piRNAs and might work as a small guide RNAs. The difficulties in the detection of differential expression of the target sRNAs could be due to the specificities of the tRNAs and rRNAs. They are among the most abundant in the cells. Both have several levels of structure and the tRNAs have several species and modifications. All these aspects lead to a greater difficulty in detecting fragments from these RNAs by standard PCR methods. This dissertation has brought to light a new PBS sncRNA sequence and a rRNA-derived miRNA, which can have an impact in a different understanding of the HIV-1 infection and the relation between virus and host. Future assays are necessary to determine how much the discovery of a new PBS sncRNA sequence changes our understanding of the HIV-1 infection and what is the link between the rRNA-derived miRNA and the HIV-1, to determine what is the exact function and how it affects the host and the HIV-1.
Small non-coding RNAs (sncRNAs) are RNAs that do not encode for proteins and range in size from 18 to 200 nucleotides and are responsible for gene regulation at the transcriptional and post-transcriptional level, RNA processing and modification. In previous studies performed in the laboratory, novel small RNAs (sRNAs) associated with retrotransposons, were identified from sequencing data obtained from RNA of CD4 human naïve T cells non-stimulated and stimulated via T cell receptor (TCR) and non-infected and infected with either HIV-1 or HIV-2. These sRNAs appeared to have new functional categories, distinct from micro RNAs (miRNAs), with characteristics that suggested the potential to represent a new defense mechanism of the host against HIV infection. Distinct from the miRNAs, sncRNAs with about 22 nucleotides in length that are responsible for preventing protein production through binding and then whether by cleavage of messenger RNA (mRNA) or translation repression, these novel sRNAs are shorter and have unknown function or functions. The aim was to test expression alterations in these novel sRNAs, three sRNAs, and characterized their functional impact on the interaction between host cells and HIV virus. For that purpose, biological models were used, a cell line named J-Lat and primary cells, used to build the sequencing libraries. The J-Lat cell line has an integrated, but transcriptionally latent HIV provirus that can be activated. The induction of the NF-κB through the tumour necrosis factor alpha (TNF-α) stimulation leads to production of viral transcripts, making the J-Lat cell line suitable to be a biological model for this study. Given that, non-infected cells and infected primary cells, also suit as a biological model. Our target sRNAs sequences with 15 and 17 nucleotides in length proved difficult for detection by standard PCR methods, because the method needs to be specific while detecting short sequences in length. A reverse transcription with polymerase chain reaction (RT-PCR) method with a specific reverse transcription know as Poly A tailing, proved to be reliable for the detection of short targets in length while also being specific. Appropriate conditions for the reactivation of the latent HIV provirus were established. The J-Lat were cultured and stimulated with TNF-α to lead to the production of viral transcripts. Visualization by florescence microscopy of the coding sequence of the green fluorescence protein (GFP) in the viral genome, plus the detection of viral transcripts by RT-PCR, confirmed the reactivation of the latent virus. Testing of the expression alterations by Poly A tailing and PCR from RNA of TNF-α treated cells and control cells, produced inconsistent results. The targets sRNAs were detected, but there was no differential expression observed along the tested samples. Thus, RNA from the primary cells of the sequencing data was used to verify, if the phenomena could only be observed in primary T cell. Alas, the same results were obtained with target sRNAs detected, but again without differential expression. Bioinformatic analysis was the next step taken, not only to try to check those expression alterations, but also to try to determine the target sRNAs origin. The same sequencing data used for the identification of those novel sRNAs was used for the analysis. The results showed that there was an increase in expression of the sequences in the data of RNA from non-infected cells to infected cells, confirming the earlier assumptions. The fold increase in expression was higher in exact matches for the target sRNAs than in longer reads containing them, which indicates that the target sRNAs are independent from longer reads. In the origin determination, two of the sequences were associated with transfer RNA Lysine 3 (tRNALys3), the primer for reverse transcription of HIV-1 and the other sequence to a ribosomal RNA, 18S rRNA, and two retrotransposons, THE1C and MSTA. ‘Repeat 1’ and ‘Repeat 2’ align with the tRNALys3 with one mismatch and the PBS sncRNA on 12 nucleotides of its total 18. The mismatch position is located on position 58, where there is a modification, A58. On reverse transcription of RNA for sequencing, that position is prone to lead to mismatches. That same location is the one where the nucleotide is different in ‘Repeat 1’ and ‘Repeat 2’. Thus, that lead to believe that those two sequences are one. That sRNA genomic sequence was searched alongside with the PBS sncRNA. The results showed that the amount of PBS sncRNA sequence present in the sequence data is 7 to 32 times lower in non-infected libraries and 32 to 156 times lower in infected libraries, than the sequences for ‘Repeat 1’, ‘Repeat 2’ and sRNA genomic sequence. Therefore, the sRNA genomic sequence is a new PBS sncRNA sequence. Additional research suggests that the sRNA might well be part of a recent class of sRNAs, tRNA-derived fragments (tRFs). They are sncRNAs that are derived from tRNAs and are usually halves of the 5’ end or 3’ end of a tRNA, but there can also exist halves from the middle of the tRNA. Our sequence seems to be part of a new subcategory of these halves. The tRFs have been associated with a lot of processes like RNA silencing, cancer, or, response to viral infections. Regarding the ‘Repeat 3, we determined the relative position of its sequence in the context of the reported secondary structure of the 18S rRNA. The position is located at the base of a stem-loop that is compatible with the canonical miRNA processing pathway. Studies report the existence of rRNA-derived miRNAs in humans and mice. Some of these rRNA-derived miRNAs have been linked to piRNAs and might work as a small guide RNAs. The difficulties in the detection of differential expression of the target sRNAs could be due to the specificities of the tRNAs and rRNAs. They are among the most abundant in the cells. Both have several levels of structure and the tRNAs have several species and modifications. All these aspects lead to a greater difficulty in detecting fragments from these RNAs by standard PCR methods. This dissertation has brought to light a new PBS sncRNA sequence and a rRNA-derived miRNA, which can have an impact in a different understanding of the HIV-1 infection and the relation between virus and host. Future assays are necessary to determine how much the discovery of a new PBS sncRNA sequence changes our understanding of the HIV-1 infection and what is the link between the rRNA-derived miRNA and the HIV-1, to determine what is the exact function and how it affects the host and the HIV-1.
Descrição
Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2019
Palavras-chave
tRNALys3 tRFs miRNA derivados de rRNA PBS sncRNA HIV Teses de mestrado - 2019