Mitochondrial RNA Import in Leishmania tropica: Aptamers Homologous to Multiple tRNA Domains That Interact Cooperatively or Antagonistically at the Inner Membrane

Abstract

A large number of cytoplasmic tRNAs are imported into the kinetoplast-mitochondrion of Leishmania by a receptor-mediated process. To identify the sequences recognized by import receptors, mitochondria were incubated with a combinatorial RNA library. Repeated cycles of amplification of the imported sequences (SELEX) resulted in rapid selection of several import aptamers containing sequence motifs present in the anticodon arm, the D arm, the V-T region, and acceptor stem of known tRNAs, confirming or suggesting the presence of import signals in these domains. As predicted, truncated derivatives of tRNA Ile (UAU) containing the D arm or the V-T region were imported in vitro. Four aptamers were studied in detail. All were imported in vitro as well as in transiently transfected cells, using the same pathway as tRNA, but their individual import efficiencies were different. Two types of aptamers were discernible: the A arm and D arm homologues (type I), which were efficiently transferred across the inner mitochondrial membrane, and the V-T homologues (type II), which were not. Remarkably, subnanomolar concentrations of type I RNAs stimulated the entry of type II RNAs into the matrix, whereas type II RNAs inhibited inner membrane transfer of type I RNAs. Moreover, tRNA Tyr (GUA) and tRNA Ile (UAU) interacted with one another as type I and type II, respectively. Such cooperative and antagonistic interactions may allow the use of a limited number of receptors to recognize a large number of tRNAs of variable affinity and enable the maintenance of a properly balanced tRNA pool for mitochondrial translation.

A bifunctional tRNA import receptor from Leishmania mitochondria

“…This region contains the sequence AGAGY that is also present in the D arms of tRNA Trp and tRNA Arg , as well as in the major sequence class of type I import aptamers obtained by in vitro evolution (10), and could form the core recognition motif for RIC1͞F1␣. In contrast, none of the type II tRNAs tested contain this motif.…”

“…Sequences of tRNA Arg 1 (ACG), tRNA Trp (CCA), tRNA Val (CAC), and tRNA Met-e (CAU) were obtained from the L. major database, and the corresponding genes were amplified from L. tropica genomic DNA using 5Ј and 3Ј primers (Table 2) and cloned downstream of the T7 poly-merase promoter in vector pGEM4Z (Promega). 32 P-labeled tRNAs of high and low specific activity were prepared by run-off transcription as described (10).…”

“…In contrast, kinetoplastids employ a membrane-bound system that recognizes specific import signal motifs on tRNAs, and soluble carriers are not essential (8,9). Moreover, importable RNAs interact cooperatively and antagonistically with one another at the inner membrane of Leishmania mitochondria in vitro and in transiently transfected cells, leading to their classification into type I RNAs, which are directly transported through the inner membrane, and type II RNAs, which require the presence of type I RNAs; conversely, type II RNAs inhibit the inner membrane transfer of type I RNAs (10,11).…”

Selection of RNA aptamers imported into yeast and human mitochondria

“…Thus, for mitochondria of Leishmania tarentolae, the nonspecific import of mini-helices smaller than 17 bases and a structural requirement for longer RNAs was demonstrated; however, the signals for importation of folded RNAs have not been determined (Rubio et al 2000). In Leishmania tropica tRNAs, two import signals were identified: in the variable loop-T region and in the D-arm, the latter consisted of the secondary structure motif and A23 nucleotide identity (Bhattacharyya et al 2002;Goswami et al 2003). In higher plants, mutations abolishing tRNA mitochondrial import have been described in anticodon and D-and T-domains of different tRNAs (Duchene et al 2009), raising the possibility that the 3D structure of tRNA, determined by interaction between D-and T-loops, is essential for import (Salinas et al 2005).…”

Drug Discovery by Aptamers in Protozoan Infectious Diseases

“…Since KMP-11 is a cytoskeleton-associated protein it is believed that its function lies in mobility or is related to the flagellar structure. The SELEX technique has also been employed in gaining cellular knowledge on how the tRNA import into the mitochondrion of L. tropica is achieved (Bhattacharyya et al, 2002). However, approaches towards the development of therapeutic have yet to be carried out due to missing biological activity of the selected aptamers or difficulties in accessing target proteins.…”