All four double-stranded RNA binding domains of Staufen2 contribute to efficient mRNA recognition and transcript localization

Proteins of the Staufen (Stau) family are core factors of mRNA localization particles in a number of metazoan organisms and cell types, including Drosophila oocytes and mouse neurons. They consist of three to four double- stranded RNA binding domains (dsRBDs) and a C-terminal dsRBD-like domain. Mouse Staufen2 (mStau2) like Drosophila Stau (dmStau) contains four dsRBDs. Existing data suggest that dsRBDs 3-4 are necessary and sufficient for binding to Stau-target mRNAs. In contrast, dsRBDs 1-2 have been suggested to be non-canonical dsRBDs, fulfilling other functions such as protein-protein interaction. Here, we show that dsRBDs 1 and 2 of mStau2 bind RNA with similar affinities and kinetics as dsRBDs 3 and 4. While RNA binding by these tandem domains is transient and characterized by high on- and off-rates, an mStau2 fragment with all four dsRBDs recognizes its target RNAs with high stability. Rescue experiments with dsRBD1-2 mutant versions of mStau2 in otherwise staufen-deficient Drosophila oocytes confirmed the physiological relevance of these findings. Full-length mStau2 partially rescued Stau-dependent mRNA localization, whereas a rescue with dsRBD1-2 mutant mStau2 failed. In summary, our data show that the dsRBDs 1-2 play essential roles in the mRNA recognition and function of Stau-family proteins of different species. The combinatorial binding of two tandem-dsRBDs allows for the cooperative recognition of more complex secondary structures and thus contributes to our understanding of how Stau proteins achieve selectivity in RNA binding.