Alternative 3? RNA processing of Itgb-1 in the mammary gland

MARTÍN GARCÍA SOLÁ; JULIAN NAIPAUER; MARIA VICTORIA MEDINA; ALBANA GATTELLI; OMAR A. COSO; EDITH C: KORDON

Simposio; SISTAM 2018; 2018

Integrins are heterodimeric cell surface adhesion receptors that play a critical role in the normal development and tumor formation of different tissues. The characterization of the full-length mRNA coding for Subunit B1 (Itgb1) reveals an alternative polyA site (PA1) that produces a previously undescribed Itgb1 mRNA species, 578bp shorter than the one previously reported. Existence of the two different isoforms was confirmed by 3?RACE PCR and SAGE analysis in the mammary gland and other tissues. In order to analyze regulatory events involved in Itgb1 mRNAalternative processing, the short Itgb1 mRNA species lacks two AU-rich elements implicated in regulation of mRNA stability and miRNAs target sequences that might be implicated in translational regulation. In mice, we found that different tissues and differentiation stages during mammary gland development show specific levels of expression for each Itgb1 mRNA variant. We further determined that the AU-binding protein HuR appears to selectively stabilize the longer isoform in the mammary gland. We also hypothesize that Itgb1 protein has different functions depending on whether it was generated by the short or long 3?UTR isoforms. To test this hypothesis, we asked whether green fluorescent protein (GFP), encoded by an mRNA containing the long or the short 3?UTR of Itgb1 would localize differently entering the secretory pathway. We replaced the extracellular domain (ECD) of Itgb1 with GFP, while preserving the Itgb1 signal peptide, transmembrane domains (TMDs) and carboxy terminus. Preliminary results: we observed that GFP-TM encoded by an mRNA containing the long 3?UTR of Itgb1 (St-GFP-Itgb1-Cter-LU) localizes primarily to the cell surface whereas GFP-TM encoded by an mRNA with the short 3?UTR of Itgb1(St-GFP-Itgb1-Cter-SU) localizes predominantly to the endoplasmic reticulum. The localization results were confirmed by fluorescence-activated cell sorting. In summary, the present study identifies a new regulatory instance in the fine-tuning of Itgb1 expression that may have consequences on protein localization and its biological function.