A novel helicase-type protein in the nucleolus: protein NOH61.
We report the identification, cDNA cloning, and molecular characterization of a novel, constitutive nucleolar protein. The cDNA-deduced amino acid sequence of the human protein defines a polypeptide of a calculated mass of 61.5 kDa and an isoelectric point of 9.9. Inspection of the primary sequence disclosed that the protein is a member of the family of "DEAD-box" proteins, representing a subgroup of putative ATP-dependent RNA helicases. ATPase activity of the recombinant protein is evident and stimulated by a variety of polynucleotides tested. Immunolocalization studies revealed that protein NOH61 (nucleolar helicase of 61 kDa) is highly conserved during evolution and shows a strong accumulation in nucleoli. Biochemical experiments have shown that protein NOH61 synthesized in vitro sediments with approximately 11.5 S, i.e., apparently as homo-oligomeric structures. By contrast, sucrose gradient centrifugation analysis of cellular extracts obtained with buffers of elevated ionic strength (600 mM NaCl) revealed that the solubilized native protein sediments with approximately 4 S, suggestive of the monomeric form. Interestingly, protein NOH61 has also been identified as a specific constituent of free nucleoplasmic 65S preribosomal particles but is absent from cytoplasmic ribosomes. Treatment of cultured cells with 1) the transcription inhibitor actinomycin D and 2) RNase A results in a complete dissociation of NOH61 from nucleolar structures. The specific intracellular localization and its striking sequence homology to other known RNA helicases lead to the hypothesis that protein NOH61 might be involved in ribosome synthesis, most likely during the assembly process of the large (60S) ribosomal subunit.
Zirwes
R F
RF
Division of Cell Biology, German Cancer Research Center, D-69120 Heidelberg, Germany.
Eilbracht
J
J
Kneissel
S
S
Schmidt-Zachmann
M S
MS
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002466:Cell Nucleolus / Q000737:chemistry* / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D053487:DEAD-box RNA Helicases; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009865:Oocytes; D020365:RNA Helicases / Q000737:chemistry* / Q000302:isolation & purification; D012270:Ribosomes / Q000737:chemistry; D014162:Transfection; D014982:Xenopus laevis
D000251:Adenosine Triphosphatases; C410330:DDX56 protein, human; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases