Identification of gemin5 as a novel 7-methylguanosine cap-binding protein.
BACKGROUND
A unique attribute of RNA molecules synthesized by RNA polymerase II is the presence of a 7-methylguanosine (m(7)G) cap structure added co-transcriptionally to the 5' end. Through its association with trans-acting effector proteins, the m(7)G cap participates in multiple aspects of RNA metabolism including localization, translation and decay. However, at present relatively few eukaryotic proteins have been identified as factors capable of direct association with m(7)G.


RESULTS
Employing an unbiased proteomic approach, we identified gemin5, a component of the survival of motor neuron (SMN) complex, as a factor capable of direct and specific interaction with the m(7)G cap. Gemin5 was readily purified by cap-affinity chromatography in contrast to other SMN complex proteins. Investigating the underlying basis for this observation, we found that purified gemin5 associates with m(7)G-linked sepharose in the absence of detectable eIF4E, and specifically crosslinks to radiolabeled cap structure after UV irradiation. Deletion analysis revealed that an intact set of WD repeat domains located in the N-terminal half of gemin5 are required for cap-binding. Moreover, using structural modeling and site-directed mutagenesis, we identified two proximal aromatic residues located within the WD repeat region that significantly impact m(7)G association.


CONCLUSIONS
This study rigorously identifies gemin5 as a novel cap-binding protein and describes an unprecedented role for WD repeat domains in m(7)G recognition. The findings presented here will facilitate understanding of gemin5's role in the metabolism of non-coding snRNAs and perhaps other RNA pol II transcripts.
Bradrick
Shelton S
SS
Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, United States of America. shelton.bradrick@duke.edu
Gromeier
Matthias
M
D000595:Amino Acid Sequence; D002460:Cell Line; D006151:Guanosine / Q000031:analogs & derivatives* / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D039381:RNA Cap-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D012315:RNA Caps / Q000737:chemistry; D012319:RNA Polymerase II / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism*; D055532:SMN Complex Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
C449808:GEMIN5 protein, human; D039381:RNA Cap-Binding Proteins; D012315:RNA Caps; D017411:Ribonucleoproteins, Small Nuclear; D055532:SMN Complex Proteins; D006151:Guanosine; C016578:7-methylguanosine; D012319:RNA Polymerase II