Contributions of the individual domains in human La protein to its RNA 3'-end binding activity.
The autoantigen La regulates the maturation of RNA polymerase III transcripts by binding to their poly(U) termination signal. The modular protein harbors a N-terminal RNA recognition motif (RRM), RRM1, and in the C-terminal domain, a second, atypical RRM2, in addition to a phosphorylation site, and a putative nucleotide binding site. This study presents a detailed investigation into the RNA 3'-end binding properties of La by using binding titration and competition assays with subsequent gel mobility shift analysis. Two truncation mutants containing one (La-RRM1) or both (La-RRM1-RRM2) RNA-binding domains were constructed, overexpressed, and purified. A K(d) value of 25 +/- 10 nm for La binding to a nonameric RNA ligand with the oligouridylate recognition sequence was obtained, discriminating with a specificity ratio of approximately 100 for this probe over a RNA ligand with a 3'-poly(A) stretch. The N-terminal La-RRM1 region was identified as the major contributor of these properties to La, manifested in a 5-fold lower K(d) of 5 +/- 3 nm and a slightly increased specificity ratio of 120 for the RNA ligand. The atypical RRM2 in the C-terminal domain of La has an unprecedented negative effect on 3'-end RNA recognition, as indicated by a higher K(d) value of 90 +/- 10 nm for the La-RRM1-RRM2 mutant but comparable specificity. Thus the C-terminal regions beyond RRM2 positively modulate the RNA binding affinity of La. Negative regulation, however, occurs through Ser(366) phosphorylation decreasing the binding affinity by 2-fold. ATP had no influence on RNA complex formation. The functional implications of these findings for the mechanism of action of La are discussed.
Ohndorf
U M
UM
Max-Planck-Institut für Biochemie, Abteilung Strukturforschung, Am Klopferspitz 18a, D-82152 Planegg-Martinsried, Germany. ohndorf@biochem.mpg.de
Steegborn
C
C
Knijff
R
R
Sondermann
P
P
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001324:Autoantigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D017931:DNA Primers; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D000097585:SS-B Antigen
D000255:Adenosine Triphosphate; D001324:Autoantigens; D017931:DNA Primers; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D000097585:SS-B Antigen