Human RioK3 is a novel component of cytoplasmic pre-40S pre-ribosomal particles.

Centre National de la Recherche Scientifique
RNA biology (Impact Factor: 5.38). 02/2012; 9(2):162-74. DOI: 10.4161/rna.18810
Source: PubMed

ABSTRACT Maturation of the 40S ribosomal subunit precursors in mammals mobilizes several non-ribosomal proteins, including the atypical protein kinase RioK2. Here, we have investigated the involvement of another member of the RIO kinase family, RioK3, in human ribosome biogenesis. RioK3 is a cytoplasmic protein that does not seem to shuttle between nucleus and cytoplasm via a Crm1-dependent mechanism as does RioK2 and which sediments with cytoplasmic 40S ribosomal particles in a sucrose gradient. When the small ribosomal subunit biogenesis is impaired by depletion of either rpS15, rpS19 or RioK2, a concomitant decrease in the amount of RioK3 is observed. Surprisingly, we observed a dramatic and specific increase in the levels of RioK3 when the biogenesis of the large ribosomal subunit is impaired. A fraction of RioK3 is associated with the non ribosomal pre-40S particle components hLtv1 and hEnp1 as well as with the 18S-E pre-rRNA indicating that it belongs to a bona fide cytoplasmic pre-40S particle. Finally, RioK3 depletion leads to an increase in the levels of the 21S rRNA precursor in the 18S rRNA production pathway. Altogether, our results strongly suggest that RioK3 is a novel cytoplasmic component of pre-40S pre-ribosomal particle(s) in human cells, required for normal processing of the 21S pre-rRNA.

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    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-Mg(2+) 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.
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    ABSTRACT: Biogenesis of 40S pre-ribosomal subunits requires many trans-acting factors, among them several protein kinases. In this study we show that the human CK1 isoforms δ and ε are required for cytoplasmic maturation steps of 40S subunit precursors. We show that both CK1 δ and ε isoforms are components of pre-40S subunits, where they phosphorylate the ribosome biogenesis factors ENP1/BYSL and LTV1. CK1 inhibition or co-depletion of CK1δ and ε result in failure to recycle a series of trans-acting factors including ENP1/BYSL, LTV1, RRP12, DIM2/PNO1, RIO2 and NOB1 from pre-40S particles after nuclear export. Further, CK1δ/ε co-depletion leads to defects in 18S-E pre-rRNA processing. Together, these data demonstrate that CK1δ and ε play a decisive role in triggering late steps of pre-40S maturation that are required for acquisition of functionality of 40S ribosomal subunits in protein translation.
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