The DEAD-box Protein Rok1 Orchestrates 40S and 60S Ribosome Assembly by Promoting the Release of Rrp5 from Pre-40S Ribosomes to Allow for 60S Maturation.
DEAD-box proteins are ubiquitous regulators of RNA biology. While commonly dubbed "helicases," their activities also include duplex annealing, adenosine triphosphate (ATP)-dependent RNA binding, and RNA-protein complex remodeling. Rok1, an essential DEAD-box protein, and its cofactor Rrp5 are required for ribosome assembly. Here, we use in vivo and in vitro biochemical analyses to demonstrate that ATP-bound Rok1, but not adenosine diphosphate (ADP)-bound Rok1, stabilizes Rrp5 binding to 40S ribosomes. Interconversion between these two forms by ATP hydrolysis is required for release of Rrp5 from pre-40S ribosomes in vivo, thereby allowing Rrp5 to carry out its role in 60S subunit assembly. Furthermore, our data also strongly suggest that the previously described accumulation of snR30 upon Rok1 inactivation arises because Rrp5 release is blocked and implicate a previously undescribed interaction between Rrp5 and the DEAD-box protein Has1 in mediating snR30 accumulation when Rrp5 release from pre-40S subunits is blocked.
Department of Cancer Biology, The Scripps Research Institute, Jupiter, Florida, United States of America.
Sohail
S
Khoshnevis
Department of Biological Science and the Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida, United States of America.
Isabel
I
Askenasy
Department of Biological Science and the Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida, United States of America.
Matthew C
0000-0002-1477-7801
MC
Johnson
Department of Cancer Biology, The Scripps Research Institute, Jupiter, Florida, United States of America.|The Benjamin School, Palm Beach Gardens, Florida, United States of America.
Maria D
MD
Dattolo
Department of Cancer Biology, The Scripps Research Institute, Jupiter, Florida, United States of America.
Crystal L
CL
Young-Erdos
Department of Biological Science and the Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida, United States of America.
M Elizabeth
ME
Stroupe
Department of Cancer Biology, The Scripps Research Institute, Jupiter, Florida, United States of America.
Katrin
K
Karbstein
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D053487:DEAD-box RNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D008958:Models, Molecular; D008968:Molecular Conformation; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D000072417:Protein Domains; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020537:RNA, Small Nucleolar / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054683:Ribosome Subunits, Large, Eukaryotic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054682:Ribosome Subunits, Small, Eukaryotic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D009687:Nuclear Proteins; D012331:RNA, Fungal; D020537:RNA, Small Nucleolar; D016601:RNA-Binding Proteins; C102025:RRP5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C120858:ROK1 protein, S cerevisiae; D053487:DEAD-box RNA Helicases