Transport signals and transcription-dependent nuclear localization of the putative DEAD-box helicase MDDX28.
The human protein MDDX28 is a putative RNA helicase and a nucleocytoplasmic shuttling protein also localized to the mitochondria. Its localization is novel among RNA helicases. We have studied its intracellular targeting signals and show that the first 20 amino acids of MDDX28 are necessary and sufficient for both mitochondrial import and nuclear export of the protein. Mutation of the five leucines in the sequence to alanines abolished the mitochondrial targeting signal as well as greatly reducing the nuclear export signal, indicating that these signal sequences are highly overlapping. Two short stretches of basic amino acids separated by 44 residues were both necessary and sufficient for full nuclear localization. However, they were not absolutely essential, because the protein was present in 7% of the nuclei when both signals were mutated. This indicates that MDDX28 contains another unidentified weak nuclear localization signal(s). Three basic domains in the N-terminal half of the protein and its RNA binding ability were essential for nucleolar localization as well as transcription-inhibition-dependent localization to nuclear subcompartments. Two of these basic domains were the same as those constituting the nuclear localization signal, suggesting that they are responsible for bringing the protein into the nucleus to the sites of RNA binding. Our results indicate that MDDX28 nucleo-cytoplasmic shuttling is dependent on the availability of nascent RNA.
Valgardsdottir
Rut
R
Biotechnology Centre of Oslo, University of Oslo, Gaustadalleen 21, N0349 Oslo, Norway.
Prydz
Hans
H
D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D002467:Cell Nucleus / Q000201:enzymology; D053487:DEAD-box RNA Helicases; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D008928:Mitochondria / Q000201:enzymology; D019913:Nuclear Localization Signals / Q000502:physiology*; D020365:RNA Helicases / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; C434959:DDX28 protein, human; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases