Abstract
A fraction of the signal recognition particle (SRP) RNA from human, rat, Xenopus, and Saccharomyces cerevisiae cells contains a single post-transcriptionally added adenylic acid residue on its 3'-end; in the case of human SRP RNA, over 60% of the SRP RNA molecules contain a nontemplated adenylic acid residue on their 3'-ends (Sinha, K. M., Gu, J., Chen, Y., and Reddy, R. (1998) J. Biol. Chem. 273, 6853-6859). In this study, we investigated the enzyme that is involved in this 3'-end adenylation of SRP RNA. A U1A protein peptide conjugated to albumin completely inhibited the polyadenylation of a SV40 mRNA by HeLa cell nuclear extract in vitro; however, the 3'-end adenylation of human SRP RNA or Alu RNA, which corresponds to 5' and 3'-ends of SRP RNA, was not affected by this U1A peptide conjugate. SRP RNA from mutant strains of S. cerevisiae with a temperature-sensitive mRNA poly(A) polymerase grown at a restrictive temperature of 37 degrees C also contained a post-transcriptionally added adenylic acid residue just like SRP RNA from wild-type cells and mutant cells grown at permissive temperature of 23 degrees C. In addition, binding of SRP 9/14-kDa protein heterodimer was required for adenylation of Alu RNA in vitro. These lines of evidence, along with other data, show that post-transcriptional adenylation of SRP and Alu RNAs is carried out by a novel enzyme that is distinct from the mRNA poly(A) polymerase, CCA-adding enzyme, and nonspecific terminal transferase.
Highlights
Oped an in vitro system in which signal recognition particle (SRP) RNA is accurately processed by the HeLa cell nuclear extract, where three transcriptionally encoded uridylic acid residues are removed on the 3Ј-end and a single adenylic acid residue is added on the 3Ј-end (2)
Several lines of evidence obtained in this study show that mRNA poly(A) polymerase is not involved in the 3Ј adenylation of SRP RNA; a novel adenylating enzyme is responsible for the addition of a single adenylic acid residue to the 3Ј-end of SRP RNA
The 51–70% fraction contained 25% of the total protein and 80% of the SRP adenylating activity. It is known from the published literature that human mRNA poly(A) polymerase fractionates in the 0 – 40% ammonium sulfate fraction (32). These data suggested that SRP/Alu RNA adenylating activity may be distinct from mRNA poly(A) polymerase, since these two activities were fractionating in two different ammonium sulfate fractions
Summary
Chemicals and Isotopes—[␣-32P]ATP and [␣-32P]UTP were purchased from Amersham Pharmacia Biotech. For the polyadenylation of yeast CYC1 pre-mRNA with yeast extract, the plasmid DNA containing the CYC1 gene (a gift from Dr Walter Keller) was linearized with NdeI restriction enzyme, and capped CYC1 pre-mRNA was synthesized in vitro with T7 RNA polymerase including [␣-32P]UTP as the labeled nucleotide. For the polyadenylation of SV40 late pre-mRNA in HeLa cell nuclear extract, the plasmid DNA containing SV40 late pre-mRNA (kindly provided by Dr Susan Berget) was linearized with HpaI, and capped precleaved RNA was transcribed with SP6 RNA polymerase including [␣-32P]UTP as a labeled nucleotide. The 25-l reaction mixture contained 20 mM creatine phosphate, 2 mM ATP, 0.6 mM MgCl2, 0.5% PEG, 0.15 mM dithiothreitol, 40% (v/v) HeLa cell nuclear extract, and labeled substrate RNA (ϳ50,000 cpm). Further quantification was done using the Molecular Dynamics system with the ImageQuant software
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