Analysis of the Saccharomyces cerevisiae exosome architecture and of the RNA binding activity of Rrp40p J.S. Luz 1 , J.R. Tavares 1 , F.A. Gonzales 2 , M.C.T. Santos, C.C. Oliveira * Department of Biochemistry, Institute of Chemistry, University of S~ ao Paulo, Av. Prof. Lineu Prestes 748, S~ ao Paulo, SP 05508-900, Brazil Received 4 September 2006; accepted 30 January 2007 Available online 20 February 2007 Abstract The exosome is a complex of eleven subunits in yeast, involved in RNA processing and degradation. Despite the extensive in vivo functional studies of the exosome, little information is yet available on the structure of the complex and on the RNase and RNA binding activities of the individual subunits. The current model for the exosome structure predicts the formation of a heterohexameric RNase PH ring, bound on one side by RNA binding subunits, and on the opposite side by hydrolytic RNase subunits. Here, we report proteineprotein interactions within the exo- some, confirming the predictions of constituents of the RNase PH ring, and show some possible interaction interfaces between the other subunits. We also show evidence that Rrp40p can bind RNA in vitro, as predicted by sequence analysis. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Exosome; RNA binding; RNase; Proteineprotein interaction 1. Introduction The exosome is a multisubunit complex with 3 0 -5 0 exonu- cleolytic activity, which was first identified in yeast [1] and subsequently found in other eukaryotes as well as in archaea [2e6]. The yeast exosome is formed by ten subunits in the cy- toplasm and eleven subunits in the nucleus [7]. Although all subunits show sequence similarity to exoribonucleases, and some of them had their activity demonstrated in vitro [1,8], sin- gle depletion of any subunit leads to similar phenotypes [1,9,10], indicating that only the intact complex is functional in vivo. In addition to the core complex, the exosome associates with other protein cofactors in both cellular compartments. In the nucleus, among other factors, the exosome interacts with Rrp47p, Mtr4p and the TRAMP complex, and in the cytoplasm, with the Ski complex [11e14]. Yeast two-hybrid system and co-purification of complexes with tagged proteins have been used to identify factors interacting with the exo- some, as well as to determine the interactions within the com- plex [1,10,14e18]. Intra-complex interactions have been reported for Rrp43p and Rrp46p [15], Rrp41p and Rrp45p [19,20], and Rrp42p and Mtr3p [19e21], which are the sub- units predicted to form the RNase PH ring of the yeast exosome [22]. The prediction of the RNase PH structure is based on se- quence analysis, electron microscopy data and on the archaeal exosome [4,22e26]. Purification experiments with tagged sub- units have confirmed the presence of all subunits in the com- plex [1,13,27e31]. Purification and analysis of the exosome by mass spectrometry under different conditions revealed in more detail the subunits interactions within the complex, lead- ing to a possible model of the exosome [18]. According to that model, the RNA binding subunits (Rrp4p, Rrp40p and Csl4p) and the hydrolytic RNases (Rrp44p and Rrp6p) bind opposite sides of the RNase PH ring, which is formed by Rrp41p- Rrp45p-Rrp46p-Rrp43p-Mtr3p-Rrp42p [18]. In vitro RNase activities have been reported for the subunits of the S. cerevisiae exosome Rrp4p, Rrp41p, Rrp44p and * Corresponding author. Tel.: þ55 11 3091 3810x208; fax: þ55 11 3815 5579. E-mail address: ccoliv@iq.usp.br (C.C. Oliveira). 1 These authors contributed equally to this work. 2 Present address: Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, CA 90095-1569, USA. 0300-9084/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2007.01.011 Biochimie 89 (2007) 686e691 www.elsevier.com/locate/biochi