Abstract
The R2TP is a recently identified Hsp90 co-chaperone, composed of four proteins as follows: Pih1D1, RPAP3, and the AAA(+)-ATPases RUVBL1 and RUVBL2. In mammals, the R2TP is involved in the biogenesis of cellular machineries such as RNA polymerases, small nucleolar ribonucleoparticles and phosphatidylinositol 3-kinase-related kinases. Here, we characterize the spaghetti (spag) gene of Drosophila, the homolog of human RPAP3. This gene plays an essential function during Drosophila development. We show that Spag protein binds Drosophila orthologs of R2TP components and Hsp90, like its yeast counterpart. Unexpectedly, Spag also interacts and stimulates the chaperone activity of Hsp70. Using null mutants and flies with inducible RNAi, we show that spaghetti is necessary for the stabilization of snoRNP core proteins and target of rapamycin activity and likely the assembly of RNA polymerase II. This work highlights the strong conservation of both the HSP90/R2TP system and its clients and further shows that Spag, unlike Saccharomyces cerevisiae Tah1, performs essential functions in metazoans. Interaction of Spag with both Hsp70 and Hsp90 suggests a model whereby R2TP would accompany clients from Hsp70 to Hsp90 to facilitate their assembly into macromolecular complexes.
Highlights
Mammalian RNA polymerase II-associated protein 3 (RPAP3) recruits heat shock protein 90 (Hsp90) to assemble cellular machineries such as RNA polymerases
We show that Spag protein binds Drosophila orthologs of R2TP components and Hsp90, like its yeast counterpart
Using null mutants and flies with inducible RNAi, we show that spaghetti is necessary for the stabilization of snoRNP core proteins and target of rapamycin activity and likely the assembly of RNA polymerase II
Summary
This family of structurally related RNPs includes the snoRNPs, necessary for ribosomal RNA maturation, the spliceosomal snRNA U4, and mRNP encoding selenoproteins This function is conserved from yeast to mammals and requires a dedicated adaptor, called Rsa in S. cerevisiae and Nufip in mammals [3, 5]. This function in PIKK stabilization is dependent on an adaptor called Tel2 [7] In all these processes, R2TP appears to stabilize newly synthesized proteins by recruiting Hsp and to assemble them into macromolecular complexes by yet poorly understood mechanisms [8]. R2TP appears to stabilize newly synthesized proteins by recruiting Hsp and to assemble them into macromolecular complexes by yet poorly understood mechanisms [8] These studies reveal that mammalian R2TP plays a role in the formation of cellular machineries that are necessary for cell growth and proliferation [8]. It is of particular interest to determine whether the function of Spag could be similar to that of the mammalian RPAP3 and, if so, whether Spag would be part of a multimeric Hsp co-chaperone R2TP complex
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