Determination of protein-RNA interaction sites in the Cbf5-H/ACA guide RNA complex by mass spectrometric protein footprinting.
Pseudouridylation is one of the most common forms of RNA modification. In eukaryotes and archaea, these modifications are carried out by H/ACA ribonucleoprotein (RNP) complexes, composed of an H/ACA guide RNA and four proteins, including the pseudouridine synthase, Cbf5. Remarkable progress has been made toward understanding the structure and function of H/ACA RNPs, both through mapping of RNA-protein and protein-protein interactions and the availability of X-ray structures, including that of the entire RNP. The pseudouridine synthase, Cbf5, is also the protein that specifically recognizes the guide RNAs. In this work, we have investigated the molecular basis of this key interaction. A mass spectrometric protein footprinting approach was employed to determine the amino acids of archaeal Cbf5 involved in interaction with the guide RNA. We found amino acid protections along the same RNA binding track observed in the crystal structure of the fully assembled complex, indicating that this interaction is established in the subcomplex. However, in addition, we observed a set of protections in the D2 subdomain of Cbf5 that appear to represent a unique, additional interaction of the guide RNA with the protein in the subcomplex. On the basis of these results, we present a model for the Cbf5-guide RNA complex that also incorporates other recent findings. Our analysis suggests that the assembly or function of H/ACA RNPs may be accompanied by dynamic changes in RNA-protein interactions.
Baker
Daniel L
DL
Department of Biochemistry and Molecular Biology, and Genetics, University of Georgia, Athens, Georgia 30602, USA.
Seyfried
Nicholas T
NT
Li
Hong
H
Orlando
Ron
R
Terns
Rebecca M
RM
Terns
Michael P
MP
D001483:Base Sequence; D017931:DNA Primers; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016296:Mutagenesis; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D058727:RNA, Small Untranslated
D017931:DNA Primers; D008869:Microtubule-Associated Proteins; D011506:Proteins; D058727:RNA, Small Untranslated