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. 2005 Jun;11(6):849-52.
doi: 10.1261/rna.2210805. Epub 2005 May 4.

Human Mitochondrial tRNAMet Is Exported to the Cytoplasm and Associates With the Argonaute 2 Protein

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Free PMC article

Human Mitochondrial tRNAMet Is Exported to the Cytoplasm and Associates With the Argonaute 2 Protein

Elisavet Maniataki et al. RNA. .
Free PMC article

Abstract

Argonaute (Ago) proteins are the effector proteins of RNA interference (RNAi) and related silencing mechanisms that are mediated by small RNAs. Ago proteins bind directly to microRNAs (miRNAs) and to short interfering RNAs and are the core protein components of RNA induced silencing complexes (RISCs) and microRNPs (miRNPs). Here we report that an ~70-nt RNA associates specifically with immunopurified Ago2 expressed in human 293 cells. By directional cloning we identified this RNA as the mitochondrial tRNA(Met) (mt tRNA(Met)). Various exported (mt) tRNAs were detected in the cytosol of 293 cells, but Ago2 was found selectively bound to (mt) tRNA(Met). The association in the cytosol of exported (mt) tRNA(Met) with Ago2 complements genetic and microscopic data that link mitochondria with RNAi-related components and events.

Figures

FIGURE 1.
FIGURE 1.
Cloning of the (mt) tRNAMet from immunoprecipitates of myc-tagged Ago2. (A) Immunoprecipitations (IP) with 9E10 (anti-myc) antibody were performed from human 293T cells that had been transfected with constructs expressing the indicated proteins. Immunoprecipitates were analyzed by Western blot with 9E10. myc-Tagged proteins are indicated with arrows. Asterisks indicate antibody chains. (B) RNA was isolated from the indicated immunoprecipitates, 3′-end labeled withT4 RNA ligase, and [5′-32P]-pCp and resolved on 10%UREA-PAGE. The ~70-nt RNA that associated specifically with Ago2-myc (red arrow) was directionally cloned. (C) Results of cloning. (D) Secondary structure of the human (mt) tRNAMet. The post-transcriptionally added CCA triplet is shown in italics.
FIGURE 2.
FIGURE 2.
Specific, cytoplasmic association of FLAG-Ago2 with exported (mt) tRNAMet. (A) A 293 cell line was prepared that expresses FLAG-Ago2 after tetracycline induction. Lysates from non-induced (−) and from tetracycline induced (+) 293 cells were probed on a Western blot with anti-FLAG antibody. (B) Cytosolic lysates were prepared from uninduced and from tetracycline-induced cells. The cells were lysed under conditions that preserve the mitochondrial integrity and the lysates were spun to pellet the mitochondria. The post-mitochondrial supernatants (cytosolic lysates) were subjected to immunoprecipitation (IP) with anti-FLAG antibodies. RNA was isolated, resolved on a 10% UREA-PAGE, blotted on a membrane, and probed with radiolabled oligos against the indicated (mt) tRNAs. (Lane 1) Cytosolic IP from uninduced 293 cells; (lane 2) cytosolic IP from tetracycline-induced 293 cells. The blot also contains total RNA isolated from cytosolic lysates (C, lane 3) and from total lysates (T, lane 4) of tetracycline-induced cells. (C) The cytosolic lysate (C) and the total lysate (T), from tetracycline-induced cells, were analyzed by Western blot with an antibody against the mitochondrial COX4 protein. COX4 is not detected in the cytosolic lysate, confirming the absence of mitochondrial contamination.

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