Understanding the interactions between phosphoproteome, transcriptome and microRNAome in response to pro-growth signals in pacu (Piaractus mesopotamicus) and gilthead sea bream (Sparus aurata) myotubes.
Whole genome duplication (WGD) events are considered a major feature of the evolution of eukaryotic genomes, providing raw materials in where natural selection can act to promote increasing complexity. Two rapid rounds of WGD (known as 1R and 2R) occurred in the all vertebrates common ancestor around 550 Mya ago. A third round of WGD happened in the base of the teleost lineage (teleost-specific 3rd WGD - Ts3R), estimated to happen 350-320 million years ago (Mya). Around 15-21% of the Ts3R-derived paralogous genes have been retained due to subfunctionalization and/or neofunctionalization mechanisms. Recent studies have demonstrated differences in paralogues retention between the teleost superorders Ostariophysi and Acanthopterygii, many of them key components of the myogenesis, protein synthesis and protein degradation networks. However, despite the progresses done in the identification of these lineage-specific paralogues (LSPs), their physiological roles and regulation remain largely unknown. The pacu (Piaractus mesopotamicus) and the gilthead seabream (Sparus aurata) belong to superorders Ostariophysi and Acanthopterygii, respectively. Both species are economically relevant for the aquaculture and, therefore, objects of an intense research to improve their production.Skeletal muscle is the most abundant tissue in teleost fish, representing up to 60% of the total body mass for some species. Muscle growth is strongly dependent of the balance between protein synthesis and degradation, processes regulated by extrinsic (e.g. temperature, salinity, oxygenation) and intrinsic (e.g. transcriptional factors, hormones, miRNAs) inputs. Among them, amino acids and Igf1 have demonstrated to play an essential role in promoting protein synthesis and muscle growth.The myoblast cell culture is a very useful in vitro model to study the regulation of muscle growth. Cultured myoblasts recapitulate all the main steps during myogenesis such as lineage commitment, proliferation, fusion and myotube formation. Cell cultures can be manipulated under controlled parameters to generate experimental conditions that would allow a better understanding of muscle regulation, growth and development at different levels and under a variety of circumstances. Similarly, cell culture media can be modified to evaluate the role of nutrients or growth factors in the regulation of muscle development.In such context, the generation of high-throughput data, such as transcriptomes, microRNAomes and proteomes, have contributed enormously for the advance in the understanding of the skeletal muscle molecular biology. This approach allows for the obtaining details of the molecular networks involved in muscle physiology under distinct conditions or moments in a detail never achieved before, providing opportunities for the identification of molecular markers and new insights on the signalling pathways that regulate myogenesis.The proposed project intends to obtain the transcriptome, microRNAome and phosphoproteome of pacu and gilthead seabream myoblast cell cultures submitted to treatments with amino acids and Igf1. The integration of transcriptome, microRNAome, and phosphoproteome from these cells will provide new insights into the networks connecting pathways activation and regulation of transcription. In addition, the comparison of these global data between pacu and gilthead seabream will provide new findings on the paralogous genes, miRNAs and pathways that control myogenesis, protein synthesis and protein degradation in skeletal muscle, such as the discovery of lineage-specific markers and the better understanding of their roles in the muscle. (AU)
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