An Rpb4/Rpb7-like complex in yeast RNA polymerase III contains the orthologue of mammalian CGRP-RCP

Abstract

The essential C17 subunit of yeast RNA polymerase (Pol) III interacts with Brf1, a component of TFIIIB, suggesting a role for C17 in the initiation step of transcription. The protein sequence of C17 (encoded by RPC17) is conserved from yeasts to humans. However, mammalian homologues of C17 (named CGRP-RCP) are known to be involved in a signal transduction pathway related to G protein-coupled receptors, not in transcription. In the present work, we first establish that human CGRP-RCP is the genuine orthologue of C17. CGRP-RCP was found to functionally replace C17 in Deltarpc17 yeast cells; the purified mutant Pol III contained CGRP-RCP and had a decreased specific activity but initiated faithfully. Furthermore, CGRP-RCP was identified by mass spectrometry in a highly purified human Pol III preparation. These results suggest that CGRP-RCP has a dual function in mammals. Next, we demonstrate by genetic and biochemical approaches that C17 forms with C25 (encoded by RPC25) a heterodimer akin to Rpb4/Rpb7 in Pol II. C17 and C25 were found to interact genetically in suppression screens and physically in coimmunopurification and two-hybrid experiments. Sequence analysis and molecular modeling indicated that the C17/C25 heterodimer likely adopts a structure similar to that of the archaeal RpoE/RpoF counterpart of the Rpb4/Rpb7 complex. These RNA polymerase subunits appear to have evolved to meet the distinct requirements of the multiple forms of RNA polymerases.

FIG. 1.

In vivo visualization of C17 cellular localization. Phase contrast (A), DAPI staining (B), and green fluorescence (C) images of the same C160-GFP-expressing cells. Phase contrast (D), DAPI staining (E), and green fluorescence (F) images of the same C17-GFP-expressing cells. Phase contrast (G), DAPI staining (H), and green fluorescence (I) images of the same control (YPH500) cells.

FIG. 2.

Heterocomplementation of RPC17 by its H. sapiens orthologue. Haploid strains containing the Δrpc17 null allele and multicopy plasmids expressing C17 (strain YMS1), HsC17 (YMS2), HA-C17 (YMS3), or HA-HsC17 (YMS4) were obtained by plasmid shuffling. Cell growth was tested by spotting 5 μl of liquid cell cultures on YPD plates, and growth was observed after incubation of the plates at 16, 24, 30, 34, or 37°C for 7, 5, 3, 3, or 4 days, respectively.

FIG. 3.

Subunit composition and transcriptional activity of mutant RNA Pol III. (A) Subunit composition. RNA Pol III fractions (1 μg), purified from strains YMS1, YMS2, YMS3, and YMS4 containing C17, HsC17, HA-C17, or HA-HsC17 as indicated, were analyzed by SDS-PAGE on a 4 to 15% polyacrylamide gel and revealed by silver staining. The positions of Pol III subunits are indicated on the right. Asterisks point to the bands corresponding to the untagged or HA-tagged versions of C17 or HsC17. (B) Western analysis. An RNA Pol III fraction (1 μg), purified from strain YMS4 expressing HA-HsC17, was analyzed by SDS-PAGE on a 4 to 13% polyacrylamide gel, and subunits were revealed by Western blotting with a mixture of anti-HA and anti-Pol III antibodies. Fourteen subunits, indicated on the left, were revealed in this experiment. The asterisk points to the band revealed by anti-HA antibodies corresponding to HA-HsC17. (C) Specific transcription of the SUP4 tRNA^Tyr gene. The SUP4 tRNA^Tyr gene was transcribed in vitro for 45 min at 25°C in the presence of TFIIIC, TFIIIB, and 50 ng of RNA Pol III purified from strains YMS1 toYMS4 containing C17, HsC17, HA-C17, or HA-HsC17, as indicated. Transcripts were separated in urea gel and revealed by autoradiography. (D) Comparison of in vivo start site selection pattern. YMS1 or YMS2 cells expressing C17 or HsC17 were grown at 37°C for 1, 10, 24, or 33 h, as indicated. RNAs were extracted, and primer extension analysis on the tRNA^Ile gene was performed as described in Materials and Methods with an oligonucleotide hybridizing within the tRNA^Ile gene intron. The lower band corresponds to the 5′-end-processed transcript, and the upper band (6 pb longer) corresponds to the primary transcript.

FIG. 4.

Dosage-dependent suppression of rpc17 or rpc25 mutants. The growth of the YMS4 (expressing HA-HsC17) or DS3-6b (rpc25-S100P) mutant strain transformed by various multicopy plasmids was tested by spotting 5 μl of liquid cell cultures on YPD plates. Plates were incubated for 3 days at 24, 30, 34, or 37°C as indicated. (A) High-copy-number suppression of the YMS4 mutant by RPC25 or RPC160. The growth of strain YMS4 transformed by the pFL44L void vector was compared to the growth of the same strain transformed by two plasmids isolated from a yeast genomic library (pMS1 and pMS17) or pFL44L harboring RPC17, RPC25, or RPC160, as indicated. (B) High-copy-number suppression of the rpc25-S100P mutation by RPC17. The growth of strain DS3-6b transformed by the pFL44L void vector was compared to the growth of the same strain transformed by two plasmids (pCZ1021 and pCZ4036) isolated from a genomic library or by pFL44L harboring RPC25 or RPC17, as indicated.

FIG. 5.

Physical interaction between C17 and C25. (A) Two-hybrid interaction between C25 and C17. The RPC25 ORF was fused in frame with the activating domain (G_AD), and the RPC17 ORF was fused in frame with the DNA-binding domain (G_BD) of GAL4. Transcriptional activation of the lacZ reporter gene was assayed by growing yeast cells transformed by a combination of plasmids as indicated on selective medium and overlaying the cells with X-Gal agar. White or blue coloration of cell patches on X-Gal plates is shown. (B) Coimmunopurification of C17 with C25. Bacterial crude extracts (240 μg) prepared from cells expressing T7-C17 or C25-HA or coexpressing both recombinant proteins were incubated with magnetic beads coated with anti-HA antibodies (HA beads) or uncoated (Control beads), as indicated. The beads were washed, and bound proteins were eluted by boiling the beads in loading buffer. The input, unbound, and eluted fractions were analyzed by SDS-PAGE and Western blotting with anti-T7 or anti-HA antibodies, as indicated. Asterisks indicate the positions of immunoglobulin heavy or light chains. The positions and molecular masses (in kilodaltons) of marker bands are indicated on the left. The positions of C25-HA and T7-C17 are indicated on the right.

FIG. 6.

Sequence alignments of the C17/Rpb4/RpoF and C25/Rpb7/RpoE families. Protein sequences (the first and last residues shown in the alignments are indicated) of C17, Rpb4, and RpoF (A) or C25, Rpb7, and RpoE (B) from several species were aligned with PSI-BLAST and HCA (22). The lengths of the loops not included in the alignments are indicated within brackets. Identical amino acid residues (at least five at the same position) are highlighted with a black background. Conserved residues are highlighted with a gray background. White letters indicate hydrophobic amino acids or amino acids that can substitute for them in some circumstances (A, C, T, and S). The secondary structures experimentally determined by Todone et al. (67) for the MjRpoF/MjRpoE complex are indicated above the sequences. The highly conserved hydrophobic amino acids mainly involved in the maintenance of the fold are marked with stars. The vertical arrow shows the strictly conserved tyrosine of the C17/Rpb4/RpoF family, which makes contact with the other subunit. GenBank identifiers are as follows: MjRpoF, 17943305; MjRpoE, 17943304; SsRpoF, 15897651; SsRpoE, 13813566; HsC17, 7656977; HsC25, 5304853; HsRpb4, 4758574; HsRpb7, 929921; SpC17, 13624765; SpC25, 7490488; SpRpb4, 9297039; SpRpb7, 2529241; ScC17, 6322449; ScC25, 516561; ScRpb4, 6322321; ScRpb7, 464672. Mj, Methanococcus jannaschii; Ss, Sulfolobus solfataricus; Hs, H. sapiens; Sp, S. pombe; Sc, S. cerevisiae

FIG. 7.

Molecular modeling of the C17/C25 complex. Ribbon representation of the model of the three-dimensional structure of the C17/C25 complex (left panel) that was constructed on the basis of the experimental structure of the RpoE/RpoF complex (right panel) (67). The C17/C25 complex was modeled on the basis of the alignments shown in Fig. 6. The large insertions in the C17 (pink) and C25 (blue) sequences, which cannot be modeled accurately, are indicated by broken lines (with the number of residues in the loop). Secondary structures are labeled according to the results of Todone et al. (67). Major differences between the left and right panels are located in the loops linking H1 to H2 and H2 to H3 (C17) as well as in the loop linking B4 to B5 (C25). Two of them (H1-H2 and B4-B5) are located in close proximity, suggesting that these two loops could interact with each other.