Visualization of the joining of ribosomal subunits reveals the presence of 80S ribosomes in the nucleus

• Published in: RNA (Cambridge) 2013-12, Vol.19 (12), p.1669-1683
• Main Authors: Al-Jubran, Khalid, Wen, Jikai, Abdullahi, Akilu, Roy Chaudhury, Subhendu, Li, Min, Ramanathan, Preethi, Matina, Annunziata, De, Sandip, Piechocki, Kim, Rugjee, Kushal Nivriti, Brogna, Saverio
• Format: Article
• Language: English
• Subjects: Animals; Bacterial Proteins - biosynthesis; Cell Line; Cell Nucleolus - metabolism; Cell Nucleus - metabolism; Drosophila; Drosophila melanogaster - cytology; Drosophila melanogaster - genetics; Drosophila melanogaster - metabolism; Drosophila Proteins - metabolism; Luminescent Proteins - biosynthesis; Microscopy, Fluorescence; Peptidyl Transferases - metabolism; Polytene Chromosomes - metabolism; Protein Binding; Recombinant Fusion Proteins - biosynthesis; Ribosomal Proteins - metabolism; Ribosomes - metabolism; RNA, Messenger - genetics; RNA, Messenger - metabolism; Transcription, Genetic
• ISSN: 1355-8382; 1469-9001
• DOI: 10.1261/rna.038356.113

In eukaryotes the 40S and 60S ribosomal subunits are assembled in the nucleolus, but there appear to be mechanisms preventing mRNA binding, 80S formation, and initiation of translation in the nucleus. To visualize association between ribosomal subunits, we tagged pairs of Drosophila ribosomal proteins (RPs) located in different subunits with mutually complementing halves of fluorescent proteins. Pairs of tagged RPs expected to interact, or be adjacent in the 80S structure, showed strong fluorescence, while pairs that were not in close proximity did not. Moreover, the complementation signal is found in ribosomal fractions and it was enhanced by translation elongation inhibitors and reduced by initiation inhibitors. Our technique achieved 80S visualization both in cultured cells and in fly tissues in vivo. Notably, while the main 80S signal was in the cytoplasm, clear signals were also seen in the nucleolus and at other nuclear sites. Furthermore, we detected rapid puromycin incorporation in the nucleolus and at transcription sites, providing an independent indication of functional 80S in the nucleolus and 80S association with nascent transcripts.