Staufen cytoplasmic granules in mRNA targeting and translation.

THOMAS, MG; MARTINEZ TOSAR L J; LOSCHI M;; BOCCACCIO, GL

Iguazú, Misiones, Argentina

Simposio; Symposium “gene expression and RNA Processing”; 2003

Staufen cytoplasmic granules in mRNA targeting and translation. M.G. Thomas, L.J. Martinez Tosar, M. Loschi and Graciela L. Boccaccio, gboccaccio@leloir.org.ar Fundación Instituto Leloir; IIBBA-CONICET; IIB-FCEyN-University of Buenos Aires, Argentina. Cytoplasmic mRNA granules are the functional unit for a variety of post-transcriptional events such as mRNA transport, silencing, activation and degradation. The double-stranded RNA-binding protein Staufen is an RNA granule-forming protein involved in asymmetric distribution of mRNAs in Drosophila’s developing CNS and oocytes. Two highly homologous genes, Staufen 1 and Staufen 2, exist in vertebrates. We isolated several Staufen 1 splicing variants, one of them being a minor transcript and potential target of the Non-sense Mediated Decay surveillance mechanism. Staufen 1 has two putative nuclear localization signals and showed nuclear-cytoplasmic localization. Nuclear export of Staufen 1 was CRM-1 and exportin 5-independent in transiently transfected cells. Specific antibodies against Staufen 1 and 2 were prepared to study these RNA binding proteins in myelinating rodent oligodendrocytes by imaging and biochemical approaches. Sedimentation velocity gradient analysis of oligodendrocyte cytosolic extracts showed that Staufen comigrates mostly with polysomes. Upon treatment of cultured cells with puromycin, Staufen1 granules partially coalesced in larger structures devoid of ER membranes and reminiscent of stress granules, thus indicating the association of Staufen with actively elongating polysomes and suggesting a role in translation. Confocal microscopy analysis of Staufen 1 and Satufen 2 subcellular distribution indicated that these molecules define two distinct populations of 400-800 nm-sized granules at the oligodendrocyte’s processes. We evaluated the colocalization of Staufen proteins with the prototype myelin-targeted MBP mRNA. This messenger was also present in granules with similar size, distribution and abundance. The majority (80 %) of MBP mRNA granules were negative for both Staufen 1 and 2. Altogether these results suggest the existence of specialized RNA complexes involved in post-transcriptional regulation at the myelin compartment. Interference experiments against specific Staufen molecules will be carried out to investigate their potential role in packaging, targeting, anchoring or translation.