Long 5' leaders inhibit removal of a 3' trailer from a precursor tRNA by mammalian tRNA 3' processing endoribonuclease

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Abstract
Mammalian tRNA 3′ processing endoribonuclease (3′ tRNase) can remove a 3′ trailer from various pre-tRNAs without 5′ leader nucleotides. To examine how 5′ leader sequences affect 3′ processing efficiency, we performed in vitro 3′ processing reactions with purified pig 3′ tRNase and pre-tRNA Arg s containing a 13-nt 3′ trailer and a 5′ leader of various lengths. The 3′ processing was slightly stimulated by 5′ leaders containing up to 7 nt, whereas leaders of 9 nt or longer severely inhibited the reaction. Structure probing indicated that the 5′ leader sequences had little effect on pre-tRNA folding. Similar results were obtained using pre-tRNA Val s containing a 5′ leader of various lengths. We also investigated whether 3′ tRNase can remove 3′ trailers that are stably base-paired with 5′ leaders to form an extended acceptor stem. Even such small 5′ leaders as 3 and 6 nt, when base-paired with a 3′ trailer, severely hindered removal of the 3′ trailer by 3′ tRNase.