Two distinct structural elements of 5S rRNA are needed for its import into human mitochondria - PubMed (original) (raw)
Two distinct structural elements of 5S rRNA are needed for its import into human mitochondria
Alexandre Smirnov et al. RNA. 2008 Apr.
Abstract
RNA import into mitochondria is a widespread phenomenon. Studied in details for yeast, protists, and plants, it still awaits thorough investigation for human cells, in which the nuclear DNA-encoded 5S rRNA is imported. Only the general requirements for this pathway have been described, whereas specific protein factors needed for 5S rRNA delivery into mitochondria and its structural determinants of import remain unknown. In this study, a systematic analysis of the possible role of human 5S rRNA structural elements in import was performed. Our experiments in vitro and in vivo show that two distinct regions of the human 5S rRNA molecule are needed for its mitochondrial targeting. One of them is located in the proximal part of the helix I and contains a conserved uncompensated G:U pair. The second and most important one is associated with the loop E-helix IV region with several noncanonical structural features. Destruction or even destabilization of these sites leads to a significant decrease of the 5S rRNA import efficiency. On the contrary, the beta-domain of the 5S rRNA was proven to be dispensable for import, and thus it can be deleted or substituted without affecting the 5S rRNA importability. This finding was used to demonstrate that the 5S rRNA can function as a vector for delivering heterologous RNA sequences into human mitochondria. 5S rRNA-based vectors containing a substitution of a part of the beta-domain by a foreign RNA sequence were shown to be much more efficiently imported in vivo than the wild-type 5S rRNA.
Figures
FIGURE 1.
Human 5S rRNA secondary structure and mutation map. (A) Human 5S rRNA secondary structure (mfold, corrected manually according to structural studies published). Sites of protein binding are colored (Baudin et al. 1991; Chow et al. 1992; White et al. 1992; Allison et al. 1993; Wimberly et al. 1993; Szymanski et al. 2000; Huber et al. 2001; Lu et al. 2003; Zuker 2003). (B) The overall mutation map of the human 5S rRNA obtained in this work. Colors correspond to different import efficiencies in vitro.
FIGURE 2.
Distinct role of 5S rRNA domains in import into isolated HepG2 mitochondria. (A) 5S rRNA sequences from human and yeast (manual alignment). (B) Import of the human 5S rRNA and a T7 transcript of identical sequence into mitochondria (autoradiograph of imported labeled RNAs). 1 indicates 5% of input RNA; 2, import assay; 3, import assay in absence of mitochondria; 4, import assay in absence of protein fractions directing import in vitro. Here and forth, relative import efficiencies (human wild-type 5S rRNA being 100%) are provided below the import pictures. (C) Import of isolated human 5S rRNA domains: 1 indicates 1% of input RNA; 2, import assay. (D) Import of the yeast 5S rRNA and “hybrid” 5S rRNA molecules: 1 indicates 1% of input RNA; 2, import assay.
FIGURE 3.
Import in vitro of 5S rRNA variants with mutations in the γ-domain. (A) Import of the γ-domain mutant variants: 1 indicates 1% of input RNA; 2, import assay. (B) Secondary and tertiary structures of the loop E-helix IV-loop D region from the Xenopus laevis 5S rRNA (JenaLib, 1un6) (Lu et al. 2003).
FIGURE 4.
Import of 5S rRNA variants with mutations in the 5S rRNA α-domain and with double (α/γ) mutations. (A) 5S rRNA variants mobility in gel. (Upper panel) Denaturing gel. (Lower panel) Native gel. (B) Import of the α-domain mutants: 1 indicates 1% of input RNA; 2, import assay. (C) The secondary structure of the helix I-loop A region of the human 5S rRNA. Nucleotides that were mutated in this work are framed. (D) Import of double (α/γ) mutants: 1 indicates 1% of input RNA; 2, import assay.
FIGURE 5.
Import of 5S rRNA with mutations in the β-domain. (A) Import of 5S rRNA variants with mutations in the β-domain in vitro: 1 indicates 1% of input RNA; 2, import assay. (B) The secondary structure of the β-domain of the 5S rRNA variant with substitution of the helix III and the loop C for a heterologic sequence (bold italic). (C) RT-PCR analysis of presence of the 5S rRNA variant with substitution of the helix III and the loop C in total (T) and mitochondrial (M) RNA from stably transfected human cells. (D) Northern blot analysis of presence of the β-domain substitution 5S rRNA variants in total and mitochondrial RNA preparations two days after transient transfection. Relative import efficiencies are indicated below (the β-domain substitution 5S rRNA variant without any additional mutation being 100%). Probes used for hybridization are indicated in left column.
FIGURE 6.
A scheme of putative pathways of the human 5S rRNA intracellular localization (for details, see text).
References
- Baeyens, K.J., De Bondt, H.L., Holbrook, S.R. Structure of an RNA double helix including uracil-uracil base pairs in an internal loop. Nat. Struct. Biol. 1995;2:56–62. - PubMed
- Baudin, F., Romaniuk, P.J., Romby, P., Brunel, C., Westhof, E., Ehresmann, B., Ehresmann, C. Involvement of “hinge” nucleotides of Xenopus laevis 5 S rRNA in the RNA structural organization and in the binding of transcription factor TFIIIA. J. Mol. Biol. 1991;218:69–81. - PubMed
- Chow, C.S., Hartmann, K.M., Rawlings, S.L., Huber, P.W., Barton, J.K. Delineation of structural domains in eukaryotic 5S rRNA with a rhodium probe. Biochemistry. 1992;31:3534–3542. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources