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| Author(s): |
Felipe ten Caten
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Matemática e Estatística (IME/SBI) |
| Defense date: | 2017-02-15 |
| Examining board members: |
Tie Koide;
Angela Kaysel Cruz;
Marilis do Valle Marques;
Helder Takashi Imoto Nakaya;
Daniel Guariz Pinheiro
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| Advisor: | Tie Koide; Ricardo Zorzetto Nicoliello Vencio |
| Abstract | |
The large-scale transcriptome characterization of different organisms revealed a highly complex scenario of gene expression, leading to the identification of numerous transcripts in the genomes of eukaryotes and prokaryotes. This phenomenon has been named pervasive transcription and has been an important source for the search of new RNAs with regulatory functions or involved in the translation of unknown proteins. The abundance of transcriptomic and proteomic data, as well as complete information regarding the genome, allowed the halophilic extremophile Halobacterium salinarum to be an ideal model organism for studies of pervasive transcription. This microorganism belongs to the Archaea group, the last one of the three domains of life to be described, which presents shared characteristics with bacteria and eukaryotes. The use of differential RNA-seq (dRNA-seq) approach, which allows the distinction between primary and processed transcripts, allowed the identification of 179 TSSaRNAs, small RNAs associated with the transcription initiation in H. salinarum. The application of dRNA-seq in RNA samples collected along the growth curve allowed the identification of 4540 transcription start sites (TSS) in H. salinarum NRC-1. Some of these transcription initiation are located upstream to known genes, enabling the identification of TSSs for 1545 genes. 59.2% of these positions are located up to 10 bp away from the translation initiation codon, confirming that most of genes are leaderless. The expression analysis of regions related to antisense TSS under different conditions revealed that most of these regions have a correlated expression profile with genes in the opposite strand, indicating a possible regulatory role. Similarly, analysis of the expression of intergenic TSS allowed the identification of 132 differentially expressed regions that are not related to any other element in H. salinarum NRC-1 genome. Integration with proteomic data reveals that some of these regions may be involved in the production of small proteins. The identification of 1365 TSS located within genes suggests that the production of intragenic RNAs (intraRNAs) is a widely distributed phenomenon in H. salinarum NRC-1 genome. Northern blot experiments confirmed the production of a transcript corresponding to the final portion of VNG_RS05220 gene and Western blot experiments also revealed that the translation of intraRNAs is responsible for producing small proteins corresponding to individual protein domains with important functional role in specific growth conditions. Analysis of TSS upstream to the coding regions of similar protein domains in bacteria and other archaea suggests that the production of coding intraRNAs is a widely distributed phenomenon in prokaryotes and may be responsible for the increased proteome diversity through the generation of protein isoforms from a unique gene. Finally, the RNA-seq data analysis, combined with a search for known signatures for transcription termination in archaea, allowed the identification of the final position of 58 genes. The present work help to give a more complete picture of H. salinarum transcriptional landscape and reveals the presence of new transcripts that can be widely distributed in prokaryotes, with important functional roles. (AU) | |
| FAPESP's process: | 11/14455-4 - RNA-seq: segmentation and modeling of the signal |
| Grantee: | Felipe ten Caten |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |