In an actively growing yeast cell, thousands of ribosomes are synthesized per minute. After rapid assembly, these intricate molecular machines must faithfully translate genetic information into all cellular proteins. Quality control mechanisms during ribosome biogenesis are therefore critical to ensure that immature subunits and functionally defective subunits do not enter the translating pool.
During ribosome biogenesis, ribosomal subunits are pre-assembled in the nucleus and exported to the cytoplasm in an inactive state. Before they can engage in protein synthesis, the immature pre-60S subunits must first undergo cytoplasmic maturation. This pathway involves a series of ATPase and GTPase-driven steps that load critical ribosomal proteins, including Rpl10, and remove accessory factors that block ribosome function. These steps are critical for both the final structure of the ribosome and to ensure ribosomal subunits are competent for translation.
The release of Tif6, an accessory factor that blocks 40S joining, and the release of Nmd3, an export adaptor, are the final known steps in 60S maturation. Two proteins, Sdo1 and Efl1, cooperate to release Tif6 from the pre-60S subunit. Intriguingly, Sdo1 binds in the ribosomal P-site and is a structural mimic of tRNA, while Efl1 is closely related to elongation factor eEF2. We have suggested that this “translation-like” binding of Efl1 and Sdo1 is a functional check of the integrity of the 60S peptidyl-transferase center before its first round of translation. Ribosomes that fail this test do not release Tif6 or Nmd3, and are therefore prevented from engaging in translation.
We have shown that ribosomes carrying mutations in RPL10, including T-cell leukemia associated rpl10-R98S, specifically fail in this quality control step. To understand the consequences of bypassing quality control, extragenic suppressing mutations of RPL10 mutant alleles were isolated. Importantly, the suppressing mutations bypass the biogenesis defect, but allow mutant ribosomes to engage in translation. Through the characterization of extragenic suppressors, we have uncovered several distinct pathways of bypassing 60S quality control.