Functional consequence of SF1 inhibition and overexpression in hematopoietic cell line

Author(s)

Illy Enne Gomes Pereira

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2019-06-07

Examining board members

Leticia Fröhlich Archangelo; Maria Helena de Souza Goldman; Katlin Brauer Massirer

• Advisor: Leticia Fröhlich Archangelo

Abstract

SF1 protein recognizes the 3 \'regions of the introns during the early steps of spliceosome formation, but is not required for the constitutive splicing. Thus, SF1 acts as an alternative splicing factor on a sub-set of cellular pre-mRNAs. Recent studies demonstrated the occurrence of SF1mutations in hematological diseases, but it is not yet clear whether these are gain or loss of function mutations and no study has demonstrated the functional consequence of differential expression of SF1 in the hematopoietic system. Therefore, the objective of this research was to evaluate the functional consequence of inhibition and overexpression of the SF1 splicing factor in hematopoietic cell line. Namalwa cells were transduced with lentiviruses and retroviruses for inhibition and overexpression of SF1, respectively. Efficiently transduced cells were puromycin selected (inhibition) or sorted by FACS (overexpression) and used for functional assays such as apoptosis (Annexin V), proliferation (Ki67, MTT) and colony forming unit to investigate the possible consequences of altered SF1 expression in this cell lineage. Our results demonstrate that SF1 depleted or overexpressing cells (inhibition n = 1 and n = 2 overexpression) formed fewer colonies in relation to the control. Similarly, SF1 overexpressing cells formed colonies 2 times larger than those formed by the control cells. The MTT viability assay demonstrated less viability of both inhibited and SF1 overexpressing cells compared to control. Both apoptosis and proliferation was not affected in the cell with altered SF1 expression. Thus, our preliminary data indicates that altered SF1 expression does not play role in proliferation and apoptosis of hematopoietic cells; but results in reduced metabolic activity, changing the phenotype of the colony forming units which presents a fewer number as consequence of reduction of viability and, on the other hand, increased size does not has cause known yet (AU)

• FAPESP's process: 16/19658-4 - Functional consequence of the splicing factor SF1 inhibition in leukemic cell line
• Grantee: Illy Enne Gomes Pereira
• Support Opportunities: Scholarships in Brazil - Master