Atomic modeling of the ITS2 ribosome assembly subcomplex from cryo-EM together with mass spectrometry-identified protein-protein crosslinks

Abstract

The assembly of ribosomal subunits starts in the nucleus, initiated by co-transcriptional folding of nascent ribosomal RNA (rRNA) transcripts and binding of ribosomal proteins and assembly factors. The internal transcribed spacer 2 (ITS2) is a precursor sequence to be processed from the intermediate 27S rRNA in the nucleoplasm; its removal is required for nuclear export of pre-60S particles. The proper processing of the ITS2 depends on multiple associated assembly factors and RNases. However, none of the structures of the known ITS2-binding factors is available. Here, we describe the modeling of the ITS2 subcomplex, including five assembly factors Cic1, Nop7, Nop15, Nop53, and Rlp7, using a combination of cryo-electron microscopy and cross-linking of proteins coupled with mass spectrometry approaches. The resulting atomic models provide structural insights into their function in ribosome assembly, and establish a framework for further dissection of their molecular roles in ITS2 processing.

Keywords: CXMS; Cic1; ITS2; Nop15; Nop53; Nop7; Rlp7; cross-linking; cryo-EM; ribosome assembly.

Figure 1

Structural overview of the pre‐60S particle and its “foot‐like” region. (A): Cryo‐EM map of the pre‐60S particle with unassigned density colored dark gray and density corresponding to the mature 60S subunit colored light gray. (B): Previously known assembly factors are highlighted in different colors. (C): The segmented map of the “foot‐like” region of the pre‐60S particle. (D): Two views of the “foot‐like” region are shown, with density of the ITS2 rRNA colored yellow.

Figure 2

Cross‐linking data related to the “foot‐like” region. (A): Three assembly factors (Cic1, Nop15, and Nop53) are cross‐linked to ribosomal proteins around the “foot‐like” region. Red dots represent cross‐linking sites in ribosomal proteins. For clarification, only structures of the ribosomal proteins (gray) from the crystal structure of the yeast ribosome (PDB id: 3u5e)32 are shown in ribbon representation, with those involved in cross‐linking to assembly factors highlighted in wheat. L25(K6), L8(K21), and L22(A2) are absent in the structure, so their closest residues in sequence are shown instead. (B): Eleven protein–protein cross‐links are shown involving five assembly factors and six ribosomal proteins.

Figure 3

Modeling of Cic1 and Nop15. (A): The predicted model of the RNA recognition motif (residues 91–169) of Nop15 is fitted into the map as a rigid body. (B): Final atomic model of Nop15 (residues 88–220) superimposed with the map. (C): The predicted models of two α/β subdomains of Cic1 are fitted into the density map as two rigid‐bodies. (D): Final atomic model of Cic1 superimposed with the map. (E): The C‐terminal helix of Nop15 interacts with Cic1. (F): The spatial relationship between Nop15 and Cic1 in the ITS2 region. Density of the ITS2 RNA is colored yellow.

Figure 4

Modeling of Rlp7. (A): A long helix in the N‐terminal region of Rlp7 is identified in the map according to its cross‐linking sites with Nop15. The secondary structure prediction of Rlp7 is shown in the lower right panel. (B): The predicted ferredoxin‐like fold domain of Rlp7 is fitted into the density map as a rigid body. (C): Final atomic model of Rlp7 superimposed with the map. (D): Rlp7 directly interacts with the linkage region of the 5.8S, ITS2, and 25S rRNAs (marked by asterisk). The ITS2 rRNA is colored yellow.

Figure 5

Modeling of Nop53. Two helices identified in the density map according to the cross‐linking of Nop53 to ribosomal proteins L35 and L27, respectively. The sites of cross‐linking and distances are labeled.

Figure 6

Modeling of Nop7. (A): Cryo‐EM density of a representative region of Nop7, superimposed with the atomic model. The predicted secondary structure of the N‐terminal region of Nop7 is shown on the right panel. (B): The poly‐alanine skeleton of the N‐terminal half of Nop7. (C): The predicted BRCT domain of Nop7 is fitted into the density map as a rigid body. (D): Final model for the BRCT domain of Nop7. (E): The final atomic model of Nop7 after chain extension.

Figure 7

The N‐terminal helical region of Nop7 interacts with multiple rRNA helices. M1 represents the first amino acid (methionine) of Nop7. Different rRNA helices that interact with Nop7 are highlighted and labeled in separate colors.

Figure 8

Interaction of Nop53 with the rRNA and ribosomal proteins. (A): Nop53 interacts with two assembly factors (Nop7 and Rlp7). (B): Nop53 interacts with three ribosomal proteins (L35, L25, and L27). L25‐N denotes the N terminus of L25. (A) and (B) are rotated 180° about the vertical axis. (C): Both the N‐ and C‐termini of Nop53 interact with multiple rRNA helices. These rRNA helices involved in interactions with Nop53 are highlighted and labeled in separate colors.