Mutagenesis of ARS2 Domains To Assess Possible Roles in Cell Cycle Progression and MicroRNA and Replication-Dependent Histone mRNA Biogenesis

Abstract

ARS2 is a regulator of RNA polymerase II transcript processing through its role in the maturation of distinct nuclear cap-binding complex (CBC)-controlled RNA families. In this study, we examined ARS2 domain function in transcript processing. Structural modeling based on the plant ARS2 orthologue, SERRATE, revealed 2 previously uncharacterized domains in mammalian ARS2: an N-terminal domain of unknown function (DUF3546), which is also present in SERRATE, and an RNA recognition motif (RRM) that is present in metazoan ARS2 but not in plants. Both the DUF3546 and zinc finger domain (ZnF) were required for association with microRNA and replication-dependent histone mRNA. Mutations in the ZnF disrupted interaction with FLASH, a key component in histone pre-mRNA processing. Mutations targeting the Mid domain implicated it in DROSHA interaction and microRNA biogenesis. The unstructured C terminus was required for interaction with the CBC protein CBP20, while the RRM was required for cell cycle progression and for binding to FLASH. Together, our results support a bridging model in which ARS2 plays a central role in RNA recognition and processing through multiple protein and RNA interactions.

ACKNOWLEDGMENTS

This work was supported by an NSERC Discovery Grant and a Foundation Fighting Blindness operating grant awarded to P.L.H. and an NSERC Discovery Grant to R.L.C.

We thank Chris Nelson for critically reviewing the manuscript and for antibodies and reagents and help with experiments. We also thank Stephen Evans and Omid Haji-Ghassemi for providing assistance and advice on homology modeling. The let-7c DsRed sensor was a gift from Gregory Hannon, and pCK-Drosha-FLAG was a gift from V. Narry Kim. Anti-ARS2 (XL12.2 and LX186.3) was generously provided by the Ludwig Institute for Cancer Research.