Purification, characterization, and cloning of the cDNA of human signal recognition particle RNA 3'-adenylating enzyme.
The 3'-terminal adenylic acid residue in several human small RNAs including signal recognition particle (SRP) RNA, nuclear 7SK RNA, U2 small nuclear RNA, and ribosomal 5S RNA is caused by a post-transcriptional adenylation event (Sinha, K., Gu, J., Chen, Y., and Reddy, R. (1998) J. Biol. Chem. 273, 6853-6859). Using the Alu portion of the SRP RNA as a substrate in an in vitro adenylation assay, we purified an adenylating enzyme that adds adenylic acid residues to SRP/Alu RNA from the HeLa cell nuclear extract. All the peptide sequences obtained by microsequencing of the purified enzyme matched a unique human cDNA corresponding to a new adenylating enzyme having homologies to the well characterized mRNA poly(A) polymerase. The amino terminus region of the human SRP RNA adenylating enzyme showed approximately 75% homology to the amino terminus of the human mRNA poly(A) polymerase that includes the catalytic domain. The carboxyl terminus of the human SRP RNA adenylating enzyme showed less than 25% homology to the carboxyl terminus of poly(A) polymerase, which interacts with other factors and provides specificity. The SRP RNA adenylating enzyme is coded for by a gene located on chromosome 2 in contrast to the poly(A) polymerase gene, which is located on chromosome 14. A recombinant protein for the SRP RNA adenylating enzyme was prepared, and its activity was compared with the purified enzyme from HeLa cells. The data indicate that in addition to the SRP RNA adenylating enzyme, other factors may be required to carry out accurate 3'-end adenylation of SRP RNA.
Perumal
K
K
Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030.
Sinha
K
K
Henning
D
D
Reddy
R
R
D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000201:enzymology; D002874:Chromosome Mapping; D002883:Chromosomes, Human, Pair 14; D002889:Chromosomes, Human, Pair 2; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011062:Polynucleotide Adenylyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D018271:Signal Recognition Particle / Q000378:metabolism
D018076:DNA, Complementary; D018271:Signal Recognition Particle; D009713:Nucleotidyltransferases; C430887:signal recognition particle RNA 3'-adenylating enzyme; D011062:Polynucleotide Adenylyltransferase