RESUMEN
Eukaryotic ribosome biogenesis is a highly orchestrated process involving numerous assembly factors including ATP-dependent RNA helicases. The DEAH helicase DHX37 (Dhr1 in yeast) is activated by the ribosome biogenesis factor UTP14A to facilitate maturation of the small ribosomal subunit. We report the crystal structure of DHX37 in complex with single-stranded RNA, revealing a canonical DEAH ATPase/helicase architecture complemented by a structurally unique carboxy-terminal domain (CTD). Structural comparisons of the nucleotide-free DHX37-RNA complex with DEAH helicases bound to RNA and ATP analogs reveal conformational changes resulting in a register shift in the bound RNA, suggesting a mechanism for ATP-dependent 3'-5' RNA translocation. We further show that a conserved sequence motif in UTP14A interacts with and activates DHX37 by stimulating its ATPase activity and enhancing RNA binding. In turn, the CTD of DHX37 is required, but not sufficient, for interaction with UTP14A in vitro and is essential for ribosome biogenesis in vivo. Together, these results shed light on the mechanism of DHX37 and the function of UTP14A in controlling its recruitment and activity during ribosome biogenesis.
Asunto(s)
Adenosina Trifosfatasas/química , Adenosina Trifosfato/análogos & derivados , ARN Helicasas DEAD-box/química , Biogénesis de Organelos , ARN Helicasas/química , ARN/química , Ribonucleoproteínas Nucleolares Pequeñas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Cinética , Ratones , Modelos Moleculares , Unión Proteica , Biosíntesis de Proteínas , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , ARN/metabolismo , ARN Helicasas/genética , ARN Helicasas/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas/genética , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Especificidad por SustratoRESUMEN
CRISPR-Cas induced homology-directed repair (HDR) enables the installation of a broad range of precise genomic modifications from an exogenous donor template. However, applications of HDR in human cells are often hampered by poor efficiency, stemming from a preference for error-prone end joining pathways that yield short insertions and deletions. Here, we describe Recursive Editing, an HDR improvement strategy that selectively retargets undesired indel outcomes to create additional opportunities to produce the desired HDR allele. We introduce a software tool, named REtarget, that enables the rational design of Recursive Editing experiments. Using REtarget-designed guide RNAs in single editing reactions, Recursive Editing can simultaneously boost HDR efficiencies and reduce undesired indels. We also harness REtarget to generate databases for particularly effective Recursive Editing sites across the genome, to endogenously tag proteins, and to target pathogenic mutations. Recursive Editing constitutes an easy-to-use approach without potentially deleterious cell manipulations and little added experimental burden.