Identificação de características biológicas associadas a sítios-alvo de proteínas de ligação ao RNA (RBPs)

Author(s)

Felipe Eduardo Ciamponi

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2018-02-16

Examining board members

Katlin Brauer Massirer; Marcelo Alves Mori; Paulo Sérgio Lopes de Oliveira

• Advisor: Katlin Brauer Massirer

Abstract

Part 1: Article ¿ BioFeatureFinder: Flexible, unbiased analysis of biological characteristics associated with genomic regions BioFeatureFinder (BFF) interrogates interesting genomic landmarks (including alternatively spliced exons, DNA/RNA-binding protein binding sites, and gene/transcript functional elements) to identify distinguishing biological features (nucleotide content, conservation, k-mers, secondary structure, protein binding sites and others). BFF uses a flexible underlying model, combining classical statistical tests with big data machine learning strategies, that uses thousands of biological characteristics (features) to interpret category labels in genomic ranges or numerical scales from genome graphs. Our results show that BFF provides a reliable analysis platform for large-scale datasets, capable of recovering several well-known features from the literature for RNA-binding proteins as well as uncovering novel associations for 112 eCLIP-seq datasets. BioFeatureFinder is freely available at https://github.com/kbmlab/BioFeatureFinder/. Part 2: Identification of mRNA-targets and binding site characteristics for CAPRIN-1 RNA-binding protein using eCLIP-seq Stress granules are protein and RNA aggregates found in the cytoplasm of cells, in general are produced in response to a source of stress (ex. Hypoxia, viral infections, nutriente deprivation and heat shock). However, several neurodegenerative diseases, such as amyotrophic lateral sclerosis and Alzheimer¿s disease, have already been associated with pathogenic inclusions of these aggregates with harmful consequences for the cells. Amongst the proteins presente in the stress granule, the complex G3BP1-CAPRIN1-USP10 is essential for the condensation fo the granule and it¿s association with ribosomal subunits, with the ectopic expression of CAPRIN1 being suufficient to induce the formation of these aggregates. Although the molecular mechanisms associated with stress granules are not completely elucidated, the main functions postulated for these structures are: protection of RNAs from harmful situations, degradation of target mRNAs, selection of stress-response mRNAs for translation and reprogamming of overall gene expression. In order to characterize the mRNA-targets of Caprin-1 in stress granules, we used a combination of eCLIP-seq, RIP-seq and RNA-seq techiniques to identify the RNA binding sites for Caprin-1, as well as identifying which functional classes of transcripts are enriched in these samples. Our analysis revealed that Caprin-1 posesses a preference for binding to stemloops RNA secondary structures, additionally these regions were also enriched in GG repeats, which might suggest the formation of secondary structures known as G-quadruplexes, which are more stable than the stemloop model. From the functional standpoint, we found that targets identified by eCLIP-seq are enriched in transcripts coding for other RNA-binding proteins, associated mostly with catabolic processes and cell cycle control. Additionally, we compared the enriched categories with predicted alterations in metabolic pathways obtained from RNA-seq data. Overall, we found 19 pathways which are simultaneously enriched in eCLIP-seq targets and had significant alterations in their activation after CAPRIN1 ectopic expression. Lastly, we identified that Caprin-1 binding are also enriched in target sites for microRNAs and are associated with PUM2, another RNA binding protein. Taken together, our data allowed us to gather important information on the role of Caprin-1 in the stress granules, both for the selection of binding targets as well as the functional alterations resulting from the ectopic expression of this protein. Our finds corroborate, with new approaches, data previously suggested in the literature as well as propose novel mechanisms previously unreported for this model (AU)

• FAPESP's process: 15/25134-5 - Implementation of CLIP-seq computational analyzes for determination of target mRNAs and Caprin-1 protein binding sites
• Grantee: Felipe Eduardo Ciamponi
• Support Opportunities: Scholarships in Brazil - Master