Abstract
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.
Highlights
Most eukaryotic RNA molecules are synthesized as precursor molecules and are subsequently processed
The carboxyl terminus of the human signal recognition particle (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 3Ј-end analysis of labeled SRP RNA and Alu RNA showed that a single adenylic acid residue is added to the 3Ј end of these RNAs, which is identical to the single A addition on the 3Ј end of SRP RNA in vivo [8, 16]
Summary
Chemicals and Isotopes—[␣-32P]ATP was purchased from ICN Biochemicals, Inc. Prepacked columns and chromatography media were purchased from Amersham Pharmacia Biotech. The hydroxyapatite media was from Bio-Rad. The reverse transcription-PCR kit was from Qiagen. Restriction enzymes were from Life Technologies, Inc. The T7 RNA polymerase was from New England Biolabs. Preparation of Substrate RNAs—Plasmid DNA corresponding to the Alu portion of canine SRP RNA (p7Alu) under the T7 promoter [18] was a gift from Dr Katharina Strub. This paper is available on line at http://www.jbc.org cDNA of the Human SRP RNA 3Ј-Adenylating Enzyme. 1 unit of specific activity is defined as 2 fmol of ATP transferred to the 3Ј end of Alu RNA by 1 mg of protein in 1 hour
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