The inhibitory effect of the autoantigen La on in vitro 3′ processing of mammalian precursor tRNAs

Abstract

Mammalian tRNA 3′ processing endoribonuclease (3′ tRNase) can remove a 3′ trailer from various precursor (pre)-tRNAs. We investigated what effect the autoantigen La has on 3′ processing, since the La protein is known to bind to a 3′-terminal uridine tract of pre-tRNAs. We tested sixteen different pre-tRNAArg substrates containing various 3′ trailers with or without a 5′ leader sequence for in vitro processing by pig 3′ tRNase, and for gel-retardation in the presence or absence of human La protein. The R-TUUU series consists of four pre-tRNAs containing 6, 8, 11 and 15 nt 3′ trailers ending with UUU and no 5′ leader, while the R-TAGC series consists of the same four pre-tRNAs as R-TUUU except that the terminal sequence is AGC. The R-6LTUUU and R-6LTAGC series are derived from R-TUUU and R-TAGC, respectively, by adding a 6 nt 5′ leader. La differentially inhibited their processing and bound to the pre-tRNAs; the 50 % inhibitory concentrations for the R-TUUU, R-TAGC, R-6LTUUU, and R-6LTAGC series were 82 to >850, >850, 2 to 292 and 573 to 785 nM, respectively, and the dissociation constants were 10 to 840, >850, 3 to 203 and 155 to 520 nM, respectively. These results indicate that both the terminal sequence UUU and the 5′ leader contribute to more severe inhibition of 3′ processing via tighter interaction with La. With respect to the R-TUUU and R-6LTUUU series, on the whole, the La inhibition was enhanced as the 3′ trailer lengths decreased. Taken together, our results suggest that the La protein sterically hinders 3′ tRNase from binding a pre-tRNA molecule probably near the cleavage site.