The rat mitochondrial Ori L encodes a novel small RNA resembling an ancestral tRNA Chun-Hong Yu 1 , Jian-You Liao 1 , Hui Zhou, Liang-Hu Qu * Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory for Biocontrol, Zhongshan University, Guangzhou 510275, PR China article info Article history: Received 13 May 2008 Available online 2 June 2008 Keywords: RNA minihelix Small RNA tRNA Mitochondria Rat abstract 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 0 -terminal CCA of tpl-sRNA is post-transcriptionally added. More- over, 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. Ó 2008 Elsevier Inc. All rights reserved. Mammalian cells harbor diverse small RNAs involved in a wide range of developmental and physiological pathways. Many small RNAs can be grouped into specific RNA classes on the basis of size, structure, sequence motifs, protein partners, or subcellular location [1]. tRNAs that serve as essential components of the protein-syn- thesizing machinery have a 3 0 -terminal CCA and form a cloverleaf secondary structure. MicroRNAs of about 21–23 nt are processed from hairpin precursor structures and regulate gene expression by binding to mRNAs via antisense elements [2]. Piwi-interacting RNAs (piRNAs) of 26–31 nt in length have been found to be associ- ated with Piwi-subclade members of the Argonaute protein family and play a role in gametogenesis [3–7]. The increasing number of small RNAs reveals the small RNA world to be much greater and more complicated than previously anticipated. The majority of known small RNAs are encoded by the nuclear genome. In addition to the nucleus, the mitochondrion is another DNA-containing cell organelle. Mitochondria have their own genetic system, a vestigial genome originating from an endosymbi- otic proteobacterial ancestor [8]. The mammalian mitochondrial genome encodes 13 proteins, 22 tRNAs and 2 rRNAs. Furthermore, mammalian mitochondrial DNA (mtDNA) has one small and one large non-coding region [9]. The larger region is called the D-loop region and contains the main regulatory sequences for transcrip- tion and replication initiation. The smaller region is an 30 nucle- otide-long segment which contains the origin of light-strand replication (Ori L) and is located inside a tRNA gene cluster. Previ- ous studies showed that D-loop region of mammalian mitochon- drial genomes can encode non-coding RNAs [10,11]. Sbisà et al. detected some transcripts surrounding the L-strand replication origin of rat mitochondrial DNA, such as precursors of the tRNAs clustered in the Ori L region [12]. Here, whilst mining for novel small RNAs from the rat, we discovered a novel small RNA of 32 nt encoded by Ori L. Notably, the novel small RNA can fold into a stable stem-loop structure and has a 3 0 -terminal CCA that is not encoded by the mitochondrial genome. Similar to tRNAs, this small RNA can be charged in vivo. These features are distinct from those of known small RNAs isolated from a broad spectrum of organisms to date and strongly resemble that of the RNA minihelix, a proposed primitive form of tRNAs. Materials and methods Construction and screening of cDNA libraries. Total RNAs were isolated from several different tissues of adult rat (Rattus norvegi- cus) according to the procedure described previously [13]. The seven different tissues are testis, heart, kidney, lung, liver, spleen and brain. About 20–27 nt-long mixed small RNAs from seven different tissues of adult rat were recovered from a 12% denaturing polyacrylamide gel and next used to construct a cDNA library according to the described procedure [14] with modifications in the sequences of 5 0 -adapter and miRT-primer used for RT, miPri- mer1 and miPrimer2 for PCR. Likewise, RNAs of 20–36 nt from adult brains were gel-purified and used to construct the second 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.05.092 * Corresponding author. Present address: Biotechnology Research Center, Zhong- shan University, Guangzhou 510275, PR China. Fax: +86 20 84036551. E-mail address: lssqlh@mail.sysu.edu.cn (L.-H. Qu). 1 These authors contributed equally to this work. Biochemical and Biophysical Research Communications 372 (2008) 634–638 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc