A Solitary Stalled 80S Ribosome Prevents mRNA Recruitment to Stress Granules

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Artem G. Fedorovskiy^1,2, Anton V. Burakov¹, Ilya M. Terenin^1,3, Dmitry A. Bykov^1,4,5, Kseniya A. Lashkevich¹, Vladimir I. Popenko⁵, Nadezhda E. Makarova^1#, Ivan I. Sorokin^1&, Anastasia P. Sukhinina^1,6, Vladimir S. Prassolov⁵, Pavel V. Ivanov⁷, and Sergey E. Dmitriev^1,5,6,a*

¹Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119234 Moscow, Russia

²Faculty of Materials Science, Lomonosov Moscow State University, 119991 Moscow, Russia

³Sirius University of Science and Technology, 354340 Sirius, Krasnodar Region, Russia

⁴Department of Biochemistry, Faculty of Biology, Lomonosov Moscow State University, 119234 Moscow, Russia

⁵Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia

⁶Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, 119234 Moscow, Russia

⁷Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, 02115 Boston, MA, USA

^* To whom correspondence should be addressed.

^#Current affiliation: Department of Genetics, Microbiology and Statistics, Faculty of Biology, University of Barcelona (UB), 08028 Barcelona, Catalonia, Spain.

^&Current affiliation: Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9747 AG Groningen, Netherlands.

Received November 23, 2022; Revised August 31, 2023; Accepted September 11, 2023

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In response to stress stimuli, eukaryotic cells typically suppress protein synthesis. This leads to the release of mRNAs from polysomes, their condensation with RNA-binding proteins, and the formation of non-membrane-bound cytoplasmic compartments called stress granules (SGs). SGs contain 40S but generally lack 60S ribosomal subunits. It is known that cycloheximide, emetine, and anisomycin, the ribosome inhibitors that block the progression of 80S ribosomes along mRNA and stabilize polysomes, prevent SG assembly. Conversely, puromycin, which induces premature termination, releases mRNA from polysomes and stimulates the formation of SGs. The same effect is caused by some translation initiation inhibitors, which lead to polysome disassembly and the accumulation of mRNAs in the form of stalled 48S preinitiation complexes. Based on these and other data, it is believed that the trigger for SG formation is the presence of mRNA with extended ribosome-free segments, which tend to form condensates in the cell. In this study, we evaluated the ability of various small-molecule translation inhibitors to block or stimulate the assembly of SGs under conditions of severe oxidative stress induced by sodium arsenite. Contrary to expectations, we found that ribosome-targeting elongation inhibitors of a specific type, which arrest solitary 80S ribosomes at the beginning of the mRNA coding regions but do not interfere with all subsequent ribosomes in completing translation and leaving the transcripts (such as harringtonine, lactimidomycin, or T-2 toxin), completely prevent the formation of arsenite-induced SGs. These observations suggest that the presence of even a single 80S ribosome on mRNA is sufficient to prevent its recruitment into SGs, and the presence of extended ribosome-free regions of mRNA is not sufficient for SG formation. We propose that mRNA entry into SGs may be mediated by specific contacts between RNA-binding proteins and those regions on 40S subunits that remain inaccessible when ribosomes are associated.

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KEY WORDS: stress granules, liquid-liquid phase separation LLPS, small molecule inhibitors of translation, ribosomal inhibitors, elongation, polysomes, harringtonine, lactimidomycin, T-2 toxin, stalled ribosome

DOI: 10.1134/S000629792311010X

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