mRNA Location and Translation Rate Determine Protein Targeting to Dual Destinations

Numerous proteins are targeted to two or multiple subcellular destinations where they exert distinct functional consequences. The balance between such differential targeting is thought to be determined post-translationally, relying on protein sorting mechanisms. Here, we show that protein targeting can additionally be determined by mRNA location and translation rate. Our model, the NET1 protein, distributes between the nucleus and cytosol and functions in cell motility. N-terminal NLSs and an internal PH domain competitively determine NET1 distribution. Peripheral localization of the NET1 mRNA and fast translation lead to higher cytosolic retention through the PH domain. By contrast, perinuclear mRNA location and/or slower translation rate favor nuclear targeting through importin β1. NET1 mRNA location is modulated by physiological stimuli and profoundly impacts NET1 function in cell motility. Indeed, perinuclear NET1 mRNA localization causes reduced RhoA activity, impedes focal adhesion maturation and results in slower cell migration to an extent similar as that observed upon knockdown of NET1 expression. Overall, these results reveal that the location of protein synthesis and the rate of translation elongation act in coordination to influence the ability of competing domains within a polypeptide to determine protein distribution and function. Ribosome and disome profiling were performed to identify sites of ribosome stalling and collisions on the NET1 mRNA.