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

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.

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