Ro ribonucleoproteins contribute to the resistance of Deinococcus radiodurans to ultraviolet irradiation

Abstract

The genome of the radiation-resistant eubacterium Deinococcus radiodurans contains an ortholog of an RNA-binding protein known as the Ro 60-kD autoantigen. This protein, which was previously identified only in higher eukaryotes, is normally bound to small RNAs known as Y RNAs. We show that the Ro protein ortholog Rsr contributes to the resistance of D. radiodurans to UV irradiation. Rsr binds several small RNAs, encoded upstream of rsr, that accumulate following UV irradiation. One of these RNAs resembles a Y RNA. These results suggest that Ro RNPs could similarly contribute to the recovery of higher cells following UV irradiation.

Figure 1

Comparison of human and worm Ro proteins with D. radiodurans Rsr. Rsr (D.r.) is aligned with the human (H.s.; Deutscher et al. 1988) and C. elegans (C.e.; Van Horn et al. 1995) Ro 60-kD proteins. The alignment was created with MegAlign (Lasergene) using the PAM250 similarity matrix. Similar residues were defined as D = E, H = K = R, A = G, I = L = V, F = W = Y, S = T. The RNA recognition motif (Birney et al. 1993) is boxed. Two conserved sequences within the motif, RNP1 and RNP2, are indicated by overlines. The human and D. radiodurans sequences are 31.8% identical and 45% similar, whereas the C. elegans and D. radiodurans proteins are 29.8% identical and 50% similar.

Figure 2

Rsr contributes to the survival of D. radiodurans following UV irradiation. (A) Genomic DNA was isolated from wild-type cells (lane 1) and cells that were converted to Δrsr as described in Materials and methods (lanes 2,3). Following digestion with StuI and BsrGI, the DNA was subjected to Southern blotting and probed with a random-primed HindIII–NruI fragment containing 226 bp of 3′ coding sequence of rsr and 341 bp of 3′ flanking sequence. In wild-type cells, the probe detects a 2.8-kb StuI–BsrGI fragment that contains most of the rsr coding region and 3′ flanking sequences. In Δrsr cells, this fragment is 4.7 kb, due to replacement of rsr with the cat gene. The increased intensity of the Δrsr band, relative to the wild-type band, is most likely due to gene amplification that occurs during the drug selection step (Agostini et al. 1996). (B) Extracts from wild-type cells (lane 1), Δrsr cells (lane 2), and Δrsr/pMD66(rsr⁺) cells (lane 3) were subjected to Western blot analysis. (C) Wild-type cells (█), Δrsr cells (♦), and Δrsr/pMD66(rsr⁺) cells (○) were subjected to irradiation with the indicated doses of UV light. After irradiation, aliquots were removed, plated on TGY agar, and counted to determine the fraction of surviving cells. Shown are the means for three separate experiments. For each radiation dose, the t-test was used to evaluate the statistical significance of the data. By comparing the ratio of the survival of the wild-type vs. the Δrsr strains, the differences in survival rates were significant at 490 J/m² and higher doses (P < 0.007, using the Bernoulli correction for multiple comparisons). Furthermore, the differences between the survival rates of the wild-type and Δrsr/pMD66(rsr⁺) cells were not significant at any of the depicted doses, although the survival rate of Δrsr/pMD66(rsr⁺) was always less than wild-type cells at 912 J/m² and higher doses. (D) Wild-type cells (lanes 1–3), Δrsr cells (lanes 4–6), and Δrsr/pMD66(rsr⁺) cells were irradiated with 490 J/m² of UV light. Following irradiation, cells were allowed to recover in TGY broth for either 30 min (lanes 2,5,8) or 2 hr (lanes 3,6,9). Cell extracts were prepared and subjected to immunoblotting using anti-Rsr antibodies. Extracts from unirradiated cells were loaded in lanes 1, 4, and 7. Because antibodies to other deinococcal proteins have not been described, the blot was standardized by loading equal amounts of protein as judged by Coomassie blue staining.

Figure 3

Small RNAs encoded upstream of rsr accumulate during recovery from irradiation with UV light. (A) Wild-type (lanes 1–8) and Δrsr (lanes 9–16) cells were either not irradiated (lanes 1,9) or irradiated with 490 J/m² of UV light (lanes 2–8,10–16). At the times indicated, cell aliquots were removed and total RNA extracted and labeled at the 3′ end. (pre-5S) 5S rRNA that is mature at the 3′ end and contains spacer sequences at the 5′ end; (5S*) a form of mature 5S rRNA. Consistent with the presence of a UV cross-link, several attempts to synthesize cDNA complementary to the 5S* RNA were unsuccessful. (B) Map of the rsr locus, showing the positions of small RNAs a–d. The tRNA gene between a and d encodes tRNA^Tyr; tRNA^Asp is encoded 5′ of RNA c. (GenBank accession nos. are AF233654 (a), AF233655 (b), AF233656 (c), and AF233657 (d).

Figure 3

Small RNAs encoded upstream of rsr accumulate during recovery from irradiation with UV light. (A) Wild-type (lanes 1–8) and Δrsr (lanes 9–16) cells were either not irradiated (lanes 1,9) or irradiated with 490 J/m² of UV light (lanes 2–8,10–16). At the times indicated, cell aliquots were removed and total RNA extracted and labeled at the 3′ end. (pre-5S) 5S rRNA that is mature at the 3′ end and contains spacer sequences at the 5′ end; (5S*) a form of mature 5S rRNA. Consistent with the presence of a UV cross-link, several attempts to synthesize cDNA complementary to the 5S* RNA were unsuccessful. (B) Map of the rsr locus, showing the positions of small RNAs a–d. The tRNA gene between a and d encodes tRNA^Tyr; tRNA^Asp is encoded 5′ of RNA c. (GenBank accession nos. are AF233654 (a), AF233655 (b), AF233656 (c), and AF233657 (d).

Figure 4

The major RNA bound by Rsr resembles a Y RNA. (A) Wild-type (lanes 1,2,5,6,9,11) and Δrsr (lanes 3,4,7,8,10,12) cells were either not irradiated (lanes 1–4,9–10) or irradiated with 490 J/m² of UV light (lanes 5–8,11–12). Following recovery, extracts were prepared and subjected to immunoprecipitation with the indicated antibodies. RNAs within immunoprecipitates (lanes 1–8) and total RNA (lanes 9–12) were extracted and labeled at the 3′ end with [³²P]pCp. RNAs a–d in lane 6 were identified by sequencing cDNAs made from the RNAs. The two bands migrating below d were subjected to cDNA synthesis and found to be degradation products of c. (B) Potential secondary structures of human, C. elegans, and D. radiodurans Y RNAs. The human Y3 RNA structure was proposed by O'Brien et al. (1993) and the CeY RNA structure was proposed by Van Horn et al. (1995). The D. radiodurans structure was derived using MFOLD (Mathews et al. 1999).