The architecture of the Schizosaccharomyces pombe CCR4-NOT complex

Abstract

CCR4-NOT is a large protein complex present both in cytoplasm and the nucleus of eukaryotic cells. Although it is involved in a variety of distinct processes related to expression of genetic information such as poly(A) tail shortening, transcription regulation, nuclear export and protein degradation, there is only fragmentary information available on some of its nine subunits. Here we show a comprehensive structural characterization of the native CCR4-NOT complex from Schizosaccharomyces pombe. Our cryo-EM 3D reconstruction of the complex, combined with techniques such as immunomicroscopy, RNA-nanogold labelling, docking of the available high-resolution structures and models of different subunits and domains, allow us to propose its full molecular architecture. We locate all functionally defined domains endowed with deadenylating and ubiquitinating activities, the nucleus-specific RNA-interacting subunit Mmi1, as well as surfaces responsible for protein-protein interactions. This information provides insight into cooperation of the different CCR4-NOT complex functions.

Figure 1. Purification of the active CCR4-NOT complex from fission yeast S. pombe.

(a) SDS-PAGE analysis of the purified CCR4-NOT complex (left) and the CCR4-NOT complex lacking Mmi1 (Mmi1ΔCCR4-NOT complex; right). (b) In vitro deadenylation assay. CCR4-NOT deadenylation was assessed using polyadenylated RNA substrate (34 nucleotides) and the purified CCR4-NOT complex. Samples were collected at 0, 15, 30 and 60 min. The control reaction had no added protein.

Figure 2. Architecture of the CCR4-NOT complex from S. pombe.

(a) Four orthogonal views of the CCR4-NOT 3D reconstruction by negative staining EM (23 Å resolution). (b) The views as in a of the complex by cryo-EM (20 Å resolution). Scale bar, 50 Å.

Figure 3. Location of the Mmi1 subunit in the CCR4-NOT complex.

(a) Three orthogonal views of the 3D reconstruction of the Mmi1ΔCCR4-NOT complex (20 Å resolution). (b) Superimposition of the 3D reconstruction of Mmi1ΔCCR4-NOT (pink, solid volume) on the CCR4-NOT complex (transparent, grey volume). Comparison of the two reconstructions shows an additional mass in the CCR4-NOT complex (black arrow). (c) 2D classes representing the front view of the CCR4-NOT complex. Top, 2D class of the complex used as a reference for particle alignment. Bottom, 2D class of the complex incubated with the nanogold-labelled Mmi1-binding RNA. Black dot, a nanogold particle visualized by EM. Red arrow, position of the Mmi1 subunit. Scale bar, 50 Å.

Figure 4. Immunolocalization of the CCR4-NOT complex subunits.

(a) Front view of the CCR4-NOT 3D reconstruction. (b) 2D class average used as a reference for alignment of the selected particles with bound antibody. (c–h) The same views of the immunocomplexes formed by CCR4-NOT subunits and antibody. The red arrow indicates the additional density in each immunocomplex, which corresponds to the antibody or Fab fragment bound.

Figure 5. 3D reconstruction of the yeast Not2-Not5 heterodimer.

(a) Four orthogonal views of the 3D reconstruction of the Not2-Not5 heterodimer (16 Å resolution). (b) Manual docking of the 3D reconstruction of Not2-Not5 (yellow) within the CCR4-NOT cryo-EM 3D reconstruction (purple). Scale bar, 50 Å.

Figure 6. Functional implications of CCR4-NOT subunit organization.

(a) Pseudo-atomic model generated after model building. (highly disordered regions were removed from the model, but their location for each subunit is suggested by the color codes in b and c (b) Map of the CCR4-NOT complex subunits. Expanded coloured regions of Not2, Not3, Not4 and Mmi1 represent the disordered fragments. (c) Left, binding sites of the known CCR4-NOT-interacting partners Tob/BTG (which binds to Caf1) and Nanos, TTP and DDX6 (which bind to different parts of Not1). The Caf1 deadenylase active site is indicated in yellow and Caf40 W-binding pockets in pink. Centre, proposed mechanism of miRNA substrate degradation. Caf40 recruits RITS complex through GW182 binding; the mRNA 5′ end interacts with DDX6, which enhances its decapping activity, while the 3′ end is deadenylated by Caf1. Right, mechanism for degradation of mRNA recruited by the mRNA-binding proteins Nanos and TTP. These proteins bind to the 3′-UTR of mRNA, which they recruit to the CCR4-NOT complex.