The RNA minihelix, a proposed tRNA precursor, exhibits tRNA-like properties. Sequence-specific RNA minihelices can inhibit cell growth probably due to their binding to the cognate tRNA and naturally occurring non-tRNA substrates for aminoacylation may serve a similar purpose. Thus far, no natural RNA minihelices have been found. in the present study, we found a novel small RNA of 32 nucleotides, which is expressed abundantly in all rat tissues tested. Distinct from all of known endogenous small RNAs, this small RNA (temporarily named as tpl-sRNA) can form an RNA minihelix containing a stem-loop domain followed by ACCA. tpl-sRNA is encoded by the light-strand replication origin (Ori L) of the rat mitochondrial genome, and the 3'-terminal CCA of tpl-sRNA is post-transcriptionally added. Moreover, tpl-sRNA is chargeable in vivo. our study demonstrates for the first time an endogenous small RNA that resembles an ancestral tRNA and exhibits some tRNA-like properties in mammals. (C) 2008 Elsevier Inc. All rights reserved.