Accurate 3' end processing and adenylation of human signal recognition particle RNA and Alu RNA in vitro

Abstract

Human signal recognition particle (SRP) RNA is transcribed by RNA polymerase III and terminates with -GUCUCUUUU(OH) on its 3' end. Our previous studies showed that the three terminal uridylic acid residues of human SRP RNA are post-transcriptionally removed and a single adenylic acid residue is added, resulting in a 3' end sequence of-GUCUCUA(OH) (Sinha, K. M., Gu, J., Chen, Y., and Reddy, R. (1998) J. Biol. Chem. 273, 6853-6859). In this study we show that the Alu RNA, corresponding to the 5' and 3' ends of SRP RNA, is also accurately processed and adenylated in vitro. Alu RNAs containing 7 or 11 additional nucleotides on the 3' end were accurately processed and then adenylated. Deletion analysis showed that an 87-nucleotide-long motif comprising of the 5' and 3' ends, including stem IV of the Alu RNA, is sufficient and necessary for the 3' end processing and adenylation. A 73- nucleotide-long construct with deletion of stem IV, required for the binding of SRP 9/14-kDa proteins, was neither processed nor adenylated. The adenylated Alu RNA as well as adenylated SRP RNA were bound to the SRP 9/14- kDa heterodimer and were immunoprecipitated by specific antibodies. A significant fraction of SRP RNA in the nucleoli was found to be processed and adenylated. These data are consistent with nascent SRP and/or Alu RNAs first binding to SRP 9/14-kDa protein heterodimer, followed by the removal of extra sequence on the 3' end and then the addition of one adenylic acid residue in the nucleus, before transport into the cytoplasm.