Mapping of the RNA-binding and endoribonuclease domains of NIPP1, a nuclear targeting subunit of protein phosphatase 1.
NIPP1 (351 residues) is a major regulatory and RNA-anchoring subunit of protein phosphatase 1 in the nucleus. Using recombinant and synthetic fragments of NIPP1, the RNA-binding domain was mapped to the C-terminal residues 330-351. A synthetic peptide encompassing this sequence equalled intact NIPP1 in RNA-binding affinity and could be used to dissociate NIPP1 from the nuclear particulate fraction. An NIPP1 fragment consisting of residues 225-351 (Ard1/NIPP1gamma), that may be encoded by an alternatively spliced transcript in transformed B-lymphocytes, displayed a single-strand Mg(2+)-dependent endoribonuclease activity. However, full-length NIPP1 and NIPP1(143-351) were not able to cleave RNA, indicating that the endoribonuclease activity of NIPP1 is restrained by its central domain. The endoribonuclease activity was also recovered in the RNA-binding domain, NIPP1(330-351), but with a 30-fold lower specific activity. Thus, the endoribonuclease catalytic site and the RNA-binding site both reside in the C-terminal 22 residues of NIPP1. The latter domain does not conform to any known nucleic-acid binding motif.
Jin
Q
Q
Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium.
Beullens
M
M
Jagiello
I
I
Van Eynde
A
A
Vulsteke
V
V
Stalmans
W
W
Bollen
M
M
D017398:Alternative Splicing; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D020134:Catalytic Domain; D002417:Cattle; D002467:Cell Nucleus / Q000201:enzymology* / Q000378:metabolism; D004722:Endoribonucleases / Q000378:metabolism*; D006868:Hydrolysis / Q000187:drug effects; D008274:Magnesium / Q000494:pharmacology; D009843:Oligoribonucleotides / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011072:Poly U / Q000235:genetics / Q000378:metabolism; D011233:Precipitin Tests; D054645:Protein Phosphatase 1; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D015003:Yeasts / Q000235:genetics
D009843:Oligoribonucleotides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011072:Poly U; D012313:RNA; D004722:Endoribonucleases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D008274:Magnesium