RESUMO
Ribosome biogenesis begins in the nucleolus with the formation of 90S pre-ribosomes, from which pre-40S and pre-60S particles arise that subsequently follow separate maturation pathways. Here, we show how structurally related assembly factors, the KH domain proteins Krr1 and Dim2, participate in ribosome assembly. Initially, Dim2 (Pno1) orchestrates an early step in small subunit biogenesis through its binding to a distinct region of the 90S pre-ribosome. This involves Utp1 of the UTP-B module, and Utp14, an activator of the DEAH-box helicase Dhr1 that catalyzes the removal of U3 snoRNP from the 90S. Following this dismantling reaction, the pre-40S subunit emerges, but Dim2 relocates to the pre-40S platform domain, previously occupied in the 90S by the other KH factor Krr1 through its interaction with Rps14 and the UTP-C module. Our findings show how the structurally related Krr1 and Dim2 can control stepwise ribosome assembly during the 90S-to-pre-40S subunit transition.
Assuntos
Biogênese de Organelas , Proteínas de Ligação a RNA/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Nucléolo Celular , RNA Helicases DEAD-box/metabolismo , Escherichia coli , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiaeRESUMO
We describe a method to genetically manipulate Chaetomium thermophilum, a eukaryotic thermophile, along with various biochemical applications. The transformation method depends on a thermostable endogenous selection marker operating at high temperatures combined with chromosomal integration of target genes. Our technique allows exploiting eukaryotic thermophiles as source for purifying thermostable native macromolecular complexes with an emphasis on the nuclear pore complex, holding great potential for applications in basic science and biotechnology.
