Discovery of transcription start, transcription termination and transcript processing sites in Halobacterium salinarum NRC-1 using dRNA-seq
Autor(a) principal: | |
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Data de Publicação: | 2023 |
Tipo de documento: | Tese |
Idioma: | eng |
Título da fonte: | Biblioteca Digital de Teses e Dissertações da USP |
Texto Completo: | https://www.teses.usp.br/teses/disponiveis/95/95131/tde-17082023-215744/ |
Resumo: | Differential RNA sequencing (dRNA-seq) has been proven to be a valuable tool for studying bacterial and archaeal genomes by identifying transcription start sites (TSS). With the help of statistical analysis, researchers can analyze dRNA-seq reads from treated and untreated libraries based on the activity of the 5-monophosphate dependent terminator RNA exonuclease (TEX). Our study focuses on Halobacterium salinarum NRC-1, a type of extremophilic archaeon that is commonly found in highly saline environments and is a model organism for studying molecular biology and genetics in extreme environments. The objective of our study is to identify and map Alternative Transcription Start Sites (alTSS), Transcript Process Sites (TPS), and Transcription Termination Sites (TTS) using the dRNA-seq data in H. salinarum NRC-1. We modified the TSSAR tool to detect TSSs from dRNA-seq data, assuming that sequencing reads start at the exact position during transcription and follow a Gamma-Poisson distribution. We annotated alTSSs into four types based on the number of dRNA-seq library reads and differences between two main TSS locations. Alternative TSSs have lower RNA reads than primary ones and can have upstream open reading frames, leading to changes in gene regulation output, 5UTR isoform, and gene transcription pausing. Mapping alTSSs allowed us to explain changes in cell response to growth conditions and gene expression across different growth stages. Our findings revealed a significant number of falsely annotated internal transcription start sites (iTSSs) that were redefined as alternative TSSs in previous genome annotations. These alternative TSSs produced different protein isoforms depending on the length of the amino acid chain and the open reading frame. Additionally, alternative TSSs were identified not only in H. salinarum but also in other organisms, suggesting a crucial role in regulating gene expression across various species. Furthermore, we conducted a re-analysis of dRNA-seq data, focusing on non-primary transcripts (monophosphorylated RNAs) instead of the traditional method of enriching for primary transcripts (triphosphorylated RNAs) to identify genome-wide transcript processing sites (TPS) in H. salinarum NRC-1. We also applied this approach to Haloferax volcanii for comparative analysis. Lastly, we used dRNA-seq data to identify hairpin structures and mapped them onto the genome, providing insights into the potential role of transcription termination sites (TTS). |
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Discovery of transcription start, transcription termination and transcript processing sites in Halobacterium salinarum NRC-1 using dRNA-seqDescoberta de locais de início de transcrição, término de transcrição e processamento de transcritos em Halobacterium salinarum NRC-1 usando dRNA-seqAlternative TSSCaloi-seqCaloi-seqdRNA-seqdRNA-seqExpressão gênicaGene expressionHalobacterium salinarum NRC-1Halobacterium salinarum NRC-1Post-transcriptional regulationRegulação pós-transcricionalRNA processing sitesSítios de processamento de RNASítios de processamento de transcriçãoTranscription processing sitesTSS alternativoTTSTTSUTRsUTRsDifferential RNA sequencing (dRNA-seq) has been proven to be a valuable tool for studying bacterial and archaeal genomes by identifying transcription start sites (TSS). With the help of statistical analysis, researchers can analyze dRNA-seq reads from treated and untreated libraries based on the activity of the 5-monophosphate dependent terminator RNA exonuclease (TEX). Our study focuses on Halobacterium salinarum NRC-1, a type of extremophilic archaeon that is commonly found in highly saline environments and is a model organism for studying molecular biology and genetics in extreme environments. The objective of our study is to identify and map Alternative Transcription Start Sites (alTSS), Transcript Process Sites (TPS), and Transcription Termination Sites (TTS) using the dRNA-seq data in H. salinarum NRC-1. We modified the TSSAR tool to detect TSSs from dRNA-seq data, assuming that sequencing reads start at the exact position during transcription and follow a Gamma-Poisson distribution. We annotated alTSSs into four types based on the number of dRNA-seq library reads and differences between two main TSS locations. Alternative TSSs have lower RNA reads than primary ones and can have upstream open reading frames, leading to changes in gene regulation output, 5UTR isoform, and gene transcription pausing. Mapping alTSSs allowed us to explain changes in cell response to growth conditions and gene expression across different growth stages. Our findings revealed a significant number of falsely annotated internal transcription start sites (iTSSs) that were redefined as alternative TSSs in previous genome annotations. These alternative TSSs produced different protein isoforms depending on the length of the amino acid chain and the open reading frame. Additionally, alternative TSSs were identified not only in H. salinarum but also in other organisms, suggesting a crucial role in regulating gene expression across various species. Furthermore, we conducted a re-analysis of dRNA-seq data, focusing on non-primary transcripts (monophosphorylated RNAs) instead of the traditional method of enriching for primary transcripts (triphosphorylated RNAs) to identify genome-wide transcript processing sites (TPS) in H. salinarum NRC-1. We also applied this approach to Haloferax volcanii for comparative analysis. Lastly, we used dRNA-seq data to identify hairpin structures and mapped them onto the genome, providing insights into the potential role of transcription termination sites (TTS).O sequenciamento diferencial de RNA (dRNA-seq) tem se mostrado uma ferramenta valiosa para o estudo de genomas bacterianos e arqueanos por meio da identificação de sítios de início de transcrição (TSS, do inglês Transcription Start Sites). Com o auxílio da análise estatística, os pesquisadores podem analisar as leituras de dRNA-seq de bibliotecas tratadas e não tratadas com base na atividade da exonucleasse de RNA terminador dependente de 5\'-monofosfato (TEX). Nosso estudo se concentra em Halobacterium salinarum NRC-1, um tipo de arqueia extremófila comumente encontrada em ambientes altamente salinos e é um organismo modelo para estudar a biologia molecular e genética em ambientes extremos. O objetivo de nosso estudo é identificar e mapear sítios alternativos de início de transcrição (alTSS, do inglês Alternative Transcription Start Sites), sítios de processamento de transcrição (TPS, do inglês Transcript Proces Sites) e sítios de terminação de transcrição (TTS, do inglês Transcription Termination Sites) usando os dados dRNA-seq em H. salinarum NRC-1. Modificamos a ferramenta TSSAR para detectar TSSs a partir de dados dRNA-seq, assumindo que as leituras de sequenciamento começam na posição exata durante a transcrição e seguem uma distribuição Gamma-Poisson. Anotamos os alTSSs em quatro tipos com base no número de leituras da biblioteca dRNA-seq e diferenças entre duas localizações principais de TSS. Os TSSs alternativos possuem leituras de RNA mais baixas do que as primárias e podem ter fase de leitura aberta upstream, levando a mudanças na produção de regulação gênica, isoformas 5\'UTR e pausas na transcrição gênica. O mapeamento de alTSSs nos permitiu explicar as mudanças na resposta celular às condições de crescimento e expressão gênica em diferentes estágios de crescimento. Nossas descobertas revelaram um número significativo de sítios de início de transcrição internos (iTSSs, do inglês Internal Transcription Start Sites) erroneamente anotados que foram redefinidos como TSSs alternativos nas anotações genômicas anteriores. Esses TSSs alternativos produzem diferentes isoformas de proteína dependendo do comprimento da cadeia de aminoácidos e do quadro de leitura aberto. Além disso, foram identificados TSSs alternativos não apenas em H. salinarum, mas também em outros organismos, sugerindo um papel crucial na regulação da expressão gênica em várias espécies. Ademais, executamos uma reanálise dos dados de dRNA-seq, com ênfase em transcritos não primários (RNAs monofosforilados) ao invés do método tradicional de enriquecimento em transcritos primários (RNAs trifosforilados) para identificar sítios de processamento de transcrição (TPS, do inglês Transcript Proces Sites em todo o genoma em H. salinarum NRC-1. Também aplicamos essa abordagem ao Haloferax volcanii para análise comparativa. Por fim, utilizamos dados de dRNA-seq para identificar estruturas em hairpin e mapeá-las no genoma, fornecendo informações sobre o potencial papel dos locais de terminação da transcrição (TTS).Biblioteca Digitais de Teses e Dissertações da USPVencio, Ricardo Zorzetto NicolielloIbrahim, Amr Galal Abd El-Raheem2023-06-28info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisapplication/pdfhttps://www.teses.usp.br/teses/disponiveis/95/95131/tde-17082023-215744/reponame:Biblioteca Digital de Teses e Dissertações da USPinstname:Universidade de São Paulo (USP)instacron:USPLiberar o conteúdo para acesso público.info:eu-repo/semantics/openAccesseng2023-09-28T11:00:06Zoai:teses.usp.br:tde-17082023-215744Biblioteca Digital de Teses e Dissertaçõeshttp://www.teses.usp.br/PUBhttp://www.teses.usp.br/cgi-bin/mtd2br.plvirginia@if.usp.br|| atendimento@aguia.usp.br||virginia@if.usp.bropendoar:27212023-09-28T11:00:06Biblioteca Digital de Teses e Dissertações da USP - Universidade de São Paulo (USP)false |
dc.title.none.fl_str_mv |
Discovery of transcription start, transcription termination and transcript processing sites in Halobacterium salinarum NRC-1 using dRNA-seq Descoberta de locais de início de transcrição, término de transcrição e processamento de transcritos em Halobacterium salinarum NRC-1 usando dRNA-seq |
title |
Discovery of transcription start, transcription termination and transcript processing sites in Halobacterium salinarum NRC-1 using dRNA-seq |
spellingShingle |
Discovery of transcription start, transcription termination and transcript processing sites in Halobacterium salinarum NRC-1 using dRNA-seq Ibrahim, Amr Galal Abd El-Raheem Alternative TSS Caloi-seq Caloi-seq dRNA-seq dRNA-seq Expressão gênica Gene expression Halobacterium salinarum NRC-1 Halobacterium salinarum NRC-1 Post-transcriptional regulation Regulação pós-transcricional RNA processing sites Sítios de processamento de RNA Sítios de processamento de transcrição Transcription processing sites TSS alternativo TTS TTS UTRs UTRs |
title_short |
Discovery of transcription start, transcription termination and transcript processing sites in Halobacterium salinarum NRC-1 using dRNA-seq |
title_full |
Discovery of transcription start, transcription termination and transcript processing sites in Halobacterium salinarum NRC-1 using dRNA-seq |
title_fullStr |
Discovery of transcription start, transcription termination and transcript processing sites in Halobacterium salinarum NRC-1 using dRNA-seq |
title_full_unstemmed |
Discovery of transcription start, transcription termination and transcript processing sites in Halobacterium salinarum NRC-1 using dRNA-seq |
title_sort |
Discovery of transcription start, transcription termination and transcript processing sites in Halobacterium salinarum NRC-1 using dRNA-seq |
author |
Ibrahim, Amr Galal Abd El-Raheem |
author_facet |
Ibrahim, Amr Galal Abd El-Raheem |
author_role |
author |
dc.contributor.none.fl_str_mv |
Vencio, Ricardo Zorzetto Nicoliello |
dc.contributor.author.fl_str_mv |
Ibrahim, Amr Galal Abd El-Raheem |
dc.subject.por.fl_str_mv |
Alternative TSS Caloi-seq Caloi-seq dRNA-seq dRNA-seq Expressão gênica Gene expression Halobacterium salinarum NRC-1 Halobacterium salinarum NRC-1 Post-transcriptional regulation Regulação pós-transcricional RNA processing sites Sítios de processamento de RNA Sítios de processamento de transcrição Transcription processing sites TSS alternativo TTS TTS UTRs UTRs |
topic |
Alternative TSS Caloi-seq Caloi-seq dRNA-seq dRNA-seq Expressão gênica Gene expression Halobacterium salinarum NRC-1 Halobacterium salinarum NRC-1 Post-transcriptional regulation Regulação pós-transcricional RNA processing sites Sítios de processamento de RNA Sítios de processamento de transcrição Transcription processing sites TSS alternativo TTS TTS UTRs UTRs |
description |
Differential RNA sequencing (dRNA-seq) has been proven to be a valuable tool for studying bacterial and archaeal genomes by identifying transcription start sites (TSS). With the help of statistical analysis, researchers can analyze dRNA-seq reads from treated and untreated libraries based on the activity of the 5-monophosphate dependent terminator RNA exonuclease (TEX). Our study focuses on Halobacterium salinarum NRC-1, a type of extremophilic archaeon that is commonly found in highly saline environments and is a model organism for studying molecular biology and genetics in extreme environments. The objective of our study is to identify and map Alternative Transcription Start Sites (alTSS), Transcript Process Sites (TPS), and Transcription Termination Sites (TTS) using the dRNA-seq data in H. salinarum NRC-1. We modified the TSSAR tool to detect TSSs from dRNA-seq data, assuming that sequencing reads start at the exact position during transcription and follow a Gamma-Poisson distribution. We annotated alTSSs into four types based on the number of dRNA-seq library reads and differences between two main TSS locations. Alternative TSSs have lower RNA reads than primary ones and can have upstream open reading frames, leading to changes in gene regulation output, 5UTR isoform, and gene transcription pausing. Mapping alTSSs allowed us to explain changes in cell response to growth conditions and gene expression across different growth stages. Our findings revealed a significant number of falsely annotated internal transcription start sites (iTSSs) that were redefined as alternative TSSs in previous genome annotations. These alternative TSSs produced different protein isoforms depending on the length of the amino acid chain and the open reading frame. Additionally, alternative TSSs were identified not only in H. salinarum but also in other organisms, suggesting a crucial role in regulating gene expression across various species. Furthermore, we conducted a re-analysis of dRNA-seq data, focusing on non-primary transcripts (monophosphorylated RNAs) instead of the traditional method of enriching for primary transcripts (triphosphorylated RNAs) to identify genome-wide transcript processing sites (TPS) in H. salinarum NRC-1. We also applied this approach to Haloferax volcanii for comparative analysis. Lastly, we used dRNA-seq data to identify hairpin structures and mapped them onto the genome, providing insights into the potential role of transcription termination sites (TTS). |
publishDate |
2023 |
dc.date.none.fl_str_mv |
2023-06-28 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
format |
doctoralThesis |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
https://www.teses.usp.br/teses/disponiveis/95/95131/tde-17082023-215744/ |
url |
https://www.teses.usp.br/teses/disponiveis/95/95131/tde-17082023-215744/ |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
|
dc.rights.driver.fl_str_mv |
Liberar o conteúdo para acesso público. info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
Liberar o conteúdo para acesso público. |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.coverage.none.fl_str_mv |
|
dc.publisher.none.fl_str_mv |
Biblioteca Digitais de Teses e Dissertações da USP |
publisher.none.fl_str_mv |
Biblioteca Digitais de Teses e Dissertações da USP |
dc.source.none.fl_str_mv |
reponame:Biblioteca Digital de Teses e Dissertações da USP instname:Universidade de São Paulo (USP) instacron:USP |
instname_str |
Universidade de São Paulo (USP) |
instacron_str |
USP |
institution |
USP |
reponame_str |
Biblioteca Digital de Teses e Dissertações da USP |
collection |
Biblioteca Digital de Teses e Dissertações da USP |
repository.name.fl_str_mv |
Biblioteca Digital de Teses e Dissertações da USP - Universidade de São Paulo (USP) |
repository.mail.fl_str_mv |
virginia@if.usp.br|| atendimento@aguia.usp.br||virginia@if.usp.br |
_version_ |
1809090808206327808 |