Protein-protein interactions with subunits of human nuclear RNase P

Abstract

A yeast two-hybrid system was used to analyze interactions among the protein subunits of human nuclear RNase P themselves and with other interacting partners encoded in a HeLa cell cDNA library. Subunits hpop1, Rpp21, Rpp29, Rpp30, Rpp38, and Rpp40 are involved in extensive, but weak, protein-protein interactions in the holoenzyme complex. Rpp14, Rpp20, and Rpp30 were found to have strong interactions with proteins encoded in the cDNA library. The small heat shock protein 27, which interacts with Rpp20 in the two-hybrid assay, binds to Rpp20 during affinity chromatography and can be found to be associated with, and enhances the activity of, highly purified RNase P. RNase P activity in HeLa cell nuclei also increases under the stress of heat shock.

Figure 1

Hsp27 in HeLa cell crude extracts binds specifically to Rpp20 on His-binding resin. Histidine-tagged Rpp20 was expressed in E. coli (3) and affinity-purified with the use of His⋅Bind resin as described in Materials and Methods. (A) HeLa cell-free extract was added to resin to which Rpp20 was bound, or to resin alone, and proteins that bound specifically to the column were stripped off as described in Materials and Methods. These proteins were separated in a 15% SDS/PAGE gel and were visualized by silver staining (A) or were electrotransferred to nitrocellulose filters and immunoblotted with monoclonal antibody against Hsp27 (StressGen; see B) and Rpp20 (not shown). Leftmost lane: molecular mass markers, molecular masses (in kDa) are indicated at the left of the figure; lane a: 15 ng Hsp27; lane b: 5 ng Hsp27; lane c: 5 ng Rpp20; lane d: proteins from a nuclear extract of non-heat-treated HeLa cells; lane e: proteins from a nuclear extract of heat-shocked HeLa cells.

Figure 2

Hsp27 copurified with human RNase P activity eluted from the Mono Q column. Purification of human RNase P was performed as described (2). The lane numbers indicate the fractions from a Mono Q column. (A) Aliquots that contained an equal mass of protein from Mono Q fractions were used for RNase P activity assay. (B) Protein concentration of Mono Q fractions (○) and the cleavage of pSupS1 by 0.25 μg of each Mono Q fraction are indicated (■). (C) Aliquots (5 μl) from fractions eluted from Mono Q column were tested by Western blot analysis for the presence of Hsp27 with the use of monoclonal antibody against Hsp27. Lane a: rainbow size marker; lane b: purified Hsp27. RNase P activity assay was performed at 37°C for 5 min as described in Materials and Methods. S, substrate; P, tRNA-containing products; 5′, 5′ leader portion of the products.

Figure 3

Stimulation of RNase P activity by Hsp27. Stimulation by Hsp27 of human nuclear RNase P activity was investigated by mixing 0.115 μg of enzyme purified through a Mono Q column step with different amounts of Hsp27 or 50 ng of BSA. The mixtures were incubated at 37°C (●, Hsp27; ○, BSA) or 43°C (■, Hsp27; □, BSA) for 30 min. After incubation at room temperature for another 30 min, the RNase P activity was assayed at 37°C for 10 min with the use of pSupS1 as substrate (see Materials and Methods).

Figure 4

(A) Heat shock treatment of HeLa cells induced an increase in nuclear RNase P-specific activity. (B) Accumulation of Hsp27 in nuclei. (C) tRNA processing after heat shock. HeLa cell culture and heat shock treatment, preparation of nuclear extracts, and assay of RNase P activity are described in Materials and Methods. (A) Assay of RNase P activity in nuclear extracts performed by mixing 1 μg of nuclear extracts with pSupS1 for 5 min at 37°C. (B) Aliquots of 15 μg of nuclear proteins were subjected to 12% SDS/PAGE and transferred to a nitrocellulose filter. The filter was probed with monoclonal antibody against Hsp27 (as shown in B, lane 1, recombinant Hsp27 from E. coli; lanes 2–4: nuclear extracts of non-heat-treated cells or cells heat treated for 1 and 3 h at 43°C, respectively). (C) Total RNA was extracted from heat-treated HeLa cells (heat shock performed at 43°C for 0.5, 1, 2, 4 h) and non-heat-treated cells (harvested after incubation at 37°C for 2 and 4 h as controls). Ten micrograms of total RNA samples was separated on 8% polyacrylamide/7 M urea gel, transferred to nylon membrane, and blotted with a γ-³²P-labeled oligonucleotide complementary to mature tRNA^Leu (Upper), and the same filter was reprobed with a γ-³²P-labeled oligonucleotide that is complementary to human 5S rRNA (Lower). PT, the primary transcript; tRNA, the mature tRNA.

Figure 5

A model of the results of the two-hybrid analysis of interactions between protein subunits in human RNase P. The scheme is based on the results of the two-hybrid assay (Table 2) that showed a weak interaction between certain pairs of subunits. No information is available regarding the stoichiometry of subunits, and, therefore, each subunit is represented only once. The subunits shown in red are also in contact with H1 RNA, the RNA subunit of human RNase P (see text). The subunits are designated by their apparent molecular masses (–4) (N. Jarrous, D. Wesolowski, C. Guerrier-Takada, and S.A., unpublished observations) rather than by their full designations (e.g., p20 rather than Rpp20) to reduce crowding in the figure.