Oligoadenylation of 3' decay intermediates promotes cytoplasmic mRNA degradation in Drosophila cells.

Post-transcriptional 3' end addition of nucleotides is important in a variety of RNA decay pathways. We have examined the 3' end addition of nucleotides during the decay of the Hsp70 mRNA and a corresponding reporter RNA in Drosophila S2 cells by conventional sequencing of cDNAs obtained after mRNA circularization and by deep sequencing of dedicated libraries enriched for 3' decay intermediates along the length of the mRNA. Approximately 5%-10% of 3' decay intermediates carried nonencoded oligo(A) tails with a mean length of 2-3 nucleotides. RNAi experiments showed that the oligoadenylated RNA fragments were intermediates of exosomal decay and the noncanonical poly(A) polymerase Trf4-1 was mainly responsible for A addition. A hot spot of A addition corresponded to an intermediate of 3' decay that accumulated upon inhibition of decapping, and knockdown of Trf4-1 increased the abundance of this intermediate, suggesting that oligoadenylation facilitates 3' decay. Oligoadenylated 3' decay intermediates were found in the cytoplasmic fraction in association with ribosomes, and fluorescence microscopy revealed a cytoplasmic localization of Trf4-1. Thus, oligoadenylation enhances exosomal mRNA degradation in the cytoplasm.

Investigation of catalysis by bacterial RNase P via LNA and other modifications at the scissile phosphodiester.

We analyzed cleavage of precursor tRNAs with an LNA, 2'-OCH(3), 2'-H or 2'-F modification at the canonical (c(0)) site by bacterial RNase P. We infer that the major function of the 2'-substituent at nt -1 during substrate ground state binding is to accept an H-bond. Cleavage of the LNA substrate at the c(0) site by Escherichia coli RNase P RNA demonstrated that the transition state for cleavage can in principle be achieved with a locked C3' -endo ribose and without the H-bond donor function of the 2'-substituent. LNA and 2'-OCH(3) suppressed processing at the major aberrant m(-)(1) site; instead, the m(+1) (nt +1/+2) site was utilized. For the LNA variant, parallel pathways leading to cleavage at the c(0) and m(+1) sites had different pH profiles, with a higher Mg(2+) requirement for c(0) versus m(+1) cleavage. The strong catalytic defect for LNA and 2'-OCH(3) supports a model where the extra methylene (LNA) or methyl group (2'-OCH(3)) causes a steric interference with a nearby bound catalytic Mg(2+) during its recoordination on the way to the transition state for cleavage. The presence of the protein cofactor suppressed the ground state binding defects, but not the catalytic defects.