Naked mole-rat has increased translational fidelity compared with the mouse, as well as a unique 28S ribosomal RNA cleavage

Abstract

The naked mole-rat (Heterocephalus glaber) is a subterranean eusocial rodent with a markedly long lifespan and resistance to tumorigenesis. Multiple data implicate modulation of protein translation in longevity. Here we report that 28S ribosomal RNA (rRNA) of the naked mole-rat is processed into two smaller fragments of unequal size. The two breakpoints are located in the 28S rRNA divergent region 6 and excise a fragment of 263 nt. The excised fragment is unique to the naked mole-rat rRNA and does not show homology to other genomic regions. Because this hidden break site could alter ribosome structure, we investigated whether translation rate and amino acid incorporation fidelity were altered. We report that naked mole-rat fibroblasts have significantly increased translational fidelity despite having comparable translation rates with mouse fibroblasts. Although we cannot directly test whether the unique 28S rRNA structure contributes to the increased fidelity of translation, we speculate that it may change the folding or dynamics of the large ribosomal subunit, altering the rate of GTP hydrolysis and/or interaction of the large subunit with tRNA during accommodation, thus affecting the fidelity of protein synthesis. In summary, our results show that naked mole-rat cells produce fewer aberrant proteins, supporting the hypothesis that the more stable proteome of the naked mole-rat contributes to its longevity.

Keywords: NMR; aging.

Fig. 1.

NMR rRNA shows an unusual fragmented pattern. (A) 28R rRNA from multiple organs of the NMR appears as two fragments. Arrows indicate 28S fragments and an 18S band. (B) The fragmentation of NMR 28S rRNA is not caused by degradation during RNA extraction. Either NMR and mouse RNA were extracted from fibroblasts independently or the cells from both species were mixed together before extraction. RNA was separated on 1% denaturing agarose gel.

Fig. 2.

Structure of the NMR 28S rRNA and localization of break sites. (A) Overall structures of the 28S rRNA in mouse, NMR, and Ctenomys. The mouse 28S rRNA has no cleavage sites. The D6 region of the NMR 28S rRNA contains a unique 118-nt insertion (shaded box). The two cleavage sites then excise a 263-nt fragment. Ctenomys is the only other known mammal with fragmented rRNA. The D6 region of the Ctenomys 28S rRNA contains a unique 106-bp insertion (shaded box) with the cleavage site located within this unique sequence. Thick black lines represent the 28S rRNA, the white box represents the D6 region, the arrows denote the cleavage sites, and the size of the D6 region and the distance between the two cleavage sites are indicated. (B) The alignment of the NMR and mouse 28S rDNA sequences. The box highlights the D6 region, the arrows indicate the cleavage sites, and hyphens indicate the gap.

Fig. 3.

NMR and mouse have similar translation rates. (A) [35S]-cystein/methionine incorporation assay performed on growing fibroblasts showing NMR translation occurs at roughly equal speed to that of mouse fibroblasts. The assays were performed at 32 °C and 37 °C for both mouse and NMR cells. NMR SF are primary NMR skin fibroblast and MSF are primary mouse skin fibroblast. (B) Western blot against RPS6, a protein component of the mature ribosome (Upper) and β-actin as loading control (Lower).

Fig. 4.

NMR has higher fidelity of translation than the mouse. Translation fidelity of NMR and mouse skin fibroblasts was quantified as a frequency of amino acid misincorporation into a set of firefly luciferase reporters with a mutated critical lysine 529 (Fig. S3). Misincorporation events can rescue luciferase activity. Luminescence of mutated firefly luciferase was measured and normalized to the luminescence of Renilla luciferase, both under control of the CMV promoter. The positive control (wild-type luciferase) ratio was several orders of magnitude higher and is cropped for visibility. The mutant reporters E - K529E; I - K529I; N - K529N measured misincorporation into the first, second, and third codon positions, respectively; the STOP reporter contained a premature stop codon mutation at amino acid 81; FS+ contained a positive frameshift mutation by nucleotide insertion at position 81; and FS contained a negative frameshift mutation by nucleotide deletion at position 81. Results for the assay are shown for NMR and mouse cells grown at 32 °C (A) and 37 °C (B). Error bars show standard deviation. Asterisks indicate values significantly different between NMR and mouse (P < 0.005) Student t test.