Dominant Rio1 kinase/ATPase catalytic mutant induces trapping of late pre-40S biogenesis factors in 80S-like ribosomes

Abstract

During eukaryotic ribosome biogenesis, members of the conserved atypical serine/threonine protein kinase family, the RIO kinases (Rio1, Rio2 and Rio3) function in small ribosomal subunit biogenesis. Structural analysis of Rio2 indicated a role as a conformation-sensing ATPase rather than a kinase to regulate its dynamic association with the pre-40S subunit. However, it remained elusive at which step and by which mechanism the other RIO kinase members act. Here, we have determined the crystal structure of the human Rio1–ATP–Mg2+ complex carrying a phosphoaspartate in the active site indicative of ATPase activity. Structure-based mutations in yeast showed that Rio1's catalytic activity regulates its pre-40S association. Furthermore, we provide evidence that Rio1 associates with a very late pre-40S via its conserved C-terminal domain. Moreover, a rio1 dominant-negative mutant defective in ATP hydrolysis induced trapping of late biogenesis factors in pre-ribosomal particles, which turned out not to be pre-40S but 80S-like ribosomes. Thus, the RIO kinase fold generates a versatile ATPase enzyme, which in the case of Rio1 is activated following the Rio2 step to regulate one of the final 40S maturation events, at which time the 60S subunit is recruited for final quality control check.

Rio1 promotes rDNA stability and downregulates RNA polymerase I to ensure rDNA segregation

“…3d). When plated on 2% galactose medium, the mutant strain died, indicating that the C-terminal region of Rio1 is essential for viability, in agreement with previous observations 12 . Importantly, overexpressing the TEV Protease in wild-type yeast did not affect viability (Fig.…”

“…To examine this possibility, we imaged Rio1, labeled with green fluorescent protein (Rio1-GFP), in exponentially growing S. cerevisiae cells. We identified the protein both in the cytoplasm (consistent with its documented involvement in 20S pre-rRNA maturation 15 and pre-40S ribosome trans-factor recycling 12,17,18 ), and in the nucleus (Fig. 1a).…”

“…Noteworthy, the RIO kinases may act especially as ATPases as they exhibit o0.1% kinase activity in vitro [12][13][14] . Cytoplasmic Rio1 contributes to pre-40S ribosome biogenesis by promoting 20S pre-rRNA maturation and by stimulating the recycling of trans-acting factors at the pre-40S subunit, both in yeast 12,[15][16][17][18] and human cells 19,20 . Roles in the nucleus are unknown for any RIO member, either in yeast or eukaryotes beyond.…”

“…cerevisiae Rio1 belongs to the atypical RIO protein kinase family whose members lack the activation loop and substrate recognition domain present in canonical eukaryotic protein kinases [8][9][10][11] . Noteworthy, the RIO kinases may act especially as ATPases as they exhibit o0.1% kinase activity in vitro [12][13][14] . Cytoplasmic Rio1 contributes to pre-40S ribosome biogenesis by promoting 20S pre-rRNA maturation and by stimulating the recycling of trans-acting factors at the pre-40S subunit, both in yeast 12,[15][16][17][18] and human cells 19,20 .…”

A Puzzle of Life: Crafting Ribosomal Subunits

“…The Rio2 ATPase is strategically positioned between the body and the maturing head in proximity to the decoding center and subsequently may act as a self-releasing checkpoint factor [73,77]. Recruitment of the ATPase Rio1, presumably requiring the prior dissociation of Tsr1 [78], yields late pre-40S ribosomes that are competent to join 60S subunits [79][80][81]. Within these 80S-like ribosomes, Rio1 and the GTPase eIF5B stimulate the Nob1-catalyzed cleavage at site D of the 20S pre-rRNA into mature 18S rRNA [74,80,[82][83][84] (Figures 1 and 2H).…”