Binding of ATP to UAP56 is necessary for mRNA export.
The major-histocompatibility-complex protein UAP56 (BAT1) is a DEAD-box helicase that is deposited on mRNA during splicing. UAP56 is retained on spliced mRNA in an exon junction complex (EJC) or, alternatively, with the TREX complex at the 5' end, where it might facilitate the export of the spliced mRNA to the cytoplasm. Using confocal microscopy, UAP56 was found to be concentrated in RNA-splicing speckled domains of nuclei but was also enriched in adjacent nuclear regions, sites at which most mRNA transcription and splicing occur. At speckled domains, UAP56 was in complexes with the RNA-splicing and -export protein SRm160, and, as measured by FRAP, was in a dynamic binding equilibrium. The application of an in vitro FRAP assay, in which fluorescent nuclear proteins are photobleached in digitonin-extracted cells, revealed that the equilibrium binding of UAP56 in complexes at speckled domains was directly regulated by ATP binding. This was confirmed using a point mutant of UAP56 that did not bind ATP. Point mutation of UAP56 to eliminate ATP binding did not affect RNA splicing, but strongly inhibited the export of mRNA to the cytoplasm.
Kota
Krishna P
KP
Department of Cell Biology S7-214, University of Massachusetts Medical School, Worcester, MA 01655, USA.
Wagner
Stefan R
SR
Huerta
Elvira
E
Underwood
Jean M
JM
Nickerson
Jeffrey A
JA
D000255:Adenosine Triphosphate / Q000378:metabolism*; D034961:Antigens, Nuclear / Q000378:metabolism; D001216:Asparagine / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism / Q000648:ultrastructure; D053487:DEAD-box RNA Helicases / Q000378:metabolism* / Q000648:ultrastructure; D036681:Fluorescence Recovery After Photobleaching; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000235:genetics; D008938:Mitosis; D050505:Mutant Proteins / Q000378:metabolism; D034521:Nuclear Matrix-Associated Proteins / Q000378:metabolism; D017354:Point Mutation / Q000235:genetics; D011485:Protein Binding; D012326:RNA Splicing; D034443:RNA Transport; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism
D034961:Antigens, Nuclear; D050505:Mutant Proteins; D034521:Nuclear Matrix-Associated Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C111710:SRRM1 protein, human; D001216:Asparagine; D000255:Adenosine Triphosphate; C487886:DDX39B protein, human; D053487:DEAD-box RNA Helicases; D008239:Lysine