Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2001 Nov 1;29(21):4334-40.
doi: 10.1093/nar/29.21.4334.

In vitro 3'-end endonucleolytic processing defect in a human mitochondrial tRNA(Ser(UCN)) precursor with the U7445C substitution, which causes non-syndromic deafness

L Levinger  1 , O Jacobs, M James
Affiliations

In vitro 3'-end endonucleolytic processing defect in a human mitochondrial tRNA(Ser(UCN)) precursor with the U7445C substitution, which causes non-syndromic deafness

L Levinger et al. Nucleic Acids Res. .

Abstract

Eukaryotic tRNAs are transcribed as precursors. A 5'-end leader and 3'-end trailer are endonucleolytically removed by RNase P and 3'-tRNase before 3'-end CCA addition, aminoacylation, nuclear export and translation. 3'-End -CC can be a 3'-tRNase anti-determinant with the ability to prevent mature tRNA from recycling through 3'-tRNase. Twenty-two tRNAs punctuate the two rRNAs and 13 mRNAs in long, bidirectional mitochondrial transcripts. Accurate mitochondrial gene expression thus depends on endonucleolytic excision of tRNAs. Various mitochondrial diseases and syndromes could arise from defective tRNA end processing. The U7445C substitution in the human mitochondrial L-strand transcript (U74C directly following the discriminator base of tRNA(Ser(UCN))) causes non-syndromic deafness. The sequence of the precursor (G/UCU) becomes G/CCU, resembling a 3'-tRNase anti-determinant. We demonstrate that a tRNA(Ser(UCN)) precursor with the U7445C substitution cannot be processed in vitro by 3'-tRNase from human mitochondria. A 3'-end processing defect in this tRNA precursor could thus be responsible for mitochondrial disease.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Eukaryotic tRNA end processing from the 3′-tRNase reaction through aminoacylation. The tRNA has been processed by RNase P to +1. (A) The RNase P product is a substrate for 3′-tRNase (←). N, discriminator base. (B) The 3′-tRNase product is a substrate for CCA addition by tRNA nucleotidyltransferase (tNtase). (C) The tNtase product (mature tRNA) is a substrate for aminoacylation by aminoacyl tRNA synthetase (RS). (D) Charged tRNA. The dashed arrow from (C) to (A, B) with an X through it signifies that mature tRNA with CCA at its 3′-end is a 3′-tRNase anti-determinant (8); it is neither a substrate nor a good inhibitor.
Figure 2
Figure 2
Human mitochondrial tRNASer(UCN) secondary structure and sequence of the double precursor (DP). Horizontal and diagonal arrows signify RNase P and 3′-tRNase cleavage sites, respectively. Thirty-eight nucleotides of 5′-end leader and 20 nt of 3′-end trailer are shown, including the U7445C substitution which changes the sequence GØUCU to GØCCU. /s indicate the positions of nucleotides in canonical tRNA (27,28) which are missing from tRNASer(UCN) and the box encloses an extra base pair (31A, 38A) in the anticodon stem.
Figure 3
Figure 3
A template with a mature 5′-end and fractionation of mitochondrial extract are required to observe 3′-tRNase activity. (A) The 127 nt double precursor (DP) with a 38 nt 5′-end leader and a 20 nt 3′-end trailer was incubated at 37°C with human mitochondrial extract for 0, 10, 20 and 30 min in lanes 1–4, respectively. [–] at left indicates the expected position of the 107 nt intermediate that would be produced by the activity of 3′-tRNase alone. An 89 nt intermediate would be produced by the activity of RNase P alone. Mature tRNA produced by the activity of both RNase P and 3′-tRNase would be 69 nt. (B) The precursor with a mature 5′-end and an 18 nt 3′-end trailer (produced by ligation of -tRNAs) was incubated with extract as in (A). (C) The double precursor [same substrate as in (A)] was incubated with the DEAE–Sepharose 0.1 M KCl fraction. (D) Precursor with a mature 5′-end [same substrate as in (B)] was incubated with the DEAE–Sepharose 0.1 M KCl fraction. ← to the right of (D) indicates the predicted tRNA product of reaction with 3′-tRNase.
Figure 4
Figure 4
tRNA precursor with the U7445C substitution is not a 3′-tRNase substrate. (A and B) 5′-End-labeled wild-type and U7445C precursors were incubated with 3′-tRNase (DEAE–Sepharose 0.1 M KCl fraction) as in Figure 3D and reactions were sampled after 0, 10, 20 and 30 min (lanes 1–4, respectively). An arrow to the left indicates the 3′-tRNase product.
Figure 5
Figure 5
Human mitochondrial 3′-tRNase makes an endonucleolytic cut at the expected processing site. (A and B) As in Figure 4A and B except that tRNAs were labeled at the 3′-end and the gel was run so as to retain the 3′-end trailer fragment [horizontal arrow at left of (A)]. Dot designates a degradation product in the prepared substrate (unincubated lane 1). –s indicate bands that arise from cleavages downstream from the 3′-tRNase site.
Figure 6
Figure 6
Structure probing of 5′-end-labeled wild-type and U7445C tRNASer(UCN) precursors. Precursor tRNASer(UCN) prepared using the double ribozyme method was labeled at its 5′-end and incubated at room temperature for 5 min with increasing concentrations of RNase T1 (0.5, 1 and 2 U/ml) or RNase A (2, 4 and 8 × 10–3 U/ml) as indicated. RNase T1 and RNase A bands are identified by nt number at right. (A) Wild-type; (B) U7445C. (C) Susceptibility of the wild-type and U7445C tRNA precursors to structure probing nucleases. Solid and dashed arrows designate sites susceptible to RNase T1 and A, respectively.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Guerrier-Takada C., Gardiner,K., Marsh,T., Pace,N. and Altman,S. (1983) The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell, 35, 849–857. - PubMed
    1. Lee, J.Y., Rohlman,C.E., Molony,L.A. and Engelke,D.R. (1991) Characterization of RPR1, an essential gene encoding the RNA component of Saccharomyces cerevisiae nuclear RNase P. Mol. Cell. Biol., 11, 721–730. - PMC - PubMed
    1. Solari A. and Deutscher,M.P. (1983) Identification of multiple RNases in Xenopus laevis oocytes and their possible role in tRNA processing. Mol. Cell. Biol., 3, 1711–1717. - PMC - PubMed
    1. Castaño J.G., Tobian,J.A. and Zasloff,M. (1985) Purification and characterization of an endonuclease from Xenopus laevis ovaries which accurately processes the 3′ terminus of human pre-tRNAi–Met (3′Pre-tRNase). J. Biol. Chem., 260, 9002–9008. - PubMed
    1. Frendewey D., Dingermann,T., Cooley,L. and Söll,D. (1985) Processing of precursor tRNAs in Drosophila. Processing of the 3′ end involves an endonucleolytic cleavage and occurs after 5′ end maturation. J. Biol. Chem., 260, 449–454. - PubMed

Publication types

MeSH terms