Characterization of human RNA polymerase III identifies orthologues for Saccharomyces cerevisiae RNA polymerase III subunits - PubMed (original) (raw)

Characterization of human RNA polymerase III identifies orthologues for Saccharomyces cerevisiae RNA polymerase III subunits

Ping Hu et al. Mol Cell Biol. 2002 Nov.

Abstract

Unlike Saccharomyces cerevisiae RNA polymerase III, human RNA polymerase III has not been entirely characterized. Orthologues of the yeast RNA polymerase III subunits C128 and C37 remain unidentified, and for many of the other subunits, the available information is limited to database sequences with various degrees of similarity to the yeast subunits. We have purified an RNA polymerase III complex and identified its components. We found that two RNA polymerase III subunits, referred to as RPC8 and RPC9, displayed sequence similarity to the RNA polymerase II RPB7 and RPB4 subunits, respectively. RPC8 and RPC9 associated with each other, paralleling the association of the RNA polymerase II subunits, and were thus paralogues of RPB7 and RPB4. Furthermore, the complex contained a prominent 80-kDa polypeptide, which we called RPC5 and which corresponded to the human orthologue of the yeast C37 subunit despite limited sequence similarity. RPC5 associated with RPC53, the human orthologue of S. cerevisiae C53, paralleling the association of the S. cerevisiae C37 and C53 subunits, and was required for transcription from the type 2 VAI and type 3 human U6 promoters. Our results provide a characterization of human RNA polymerase III and show that the RPC5 subunit is essential for transcription.

PubMed Disclaimer

Figures

FIG. 1.

Composition of the tagged RNA polymerase III complex. (A) Purification scheme of the tagged RNA polymerase III complex. (B) Polypeptide composition of the purified tagged RNA polymerase III. Whole-cell extracts (WCE) from either HeLa cells (lanes 1 and 3) or HeLa cells expressing tagged RPC4/HsRPC53 (lanes 2 and 4) were used as starting material for the purification scheme summarized in A. The proteins were separated on an SDS-4 to 20% polyacrylamide gel and stained with Coomassie blue. In lanes 1 and 2, the nickel-agarose beads were washed with a buffer containing 300 mM KCl before elution with 250 mM imidazole. In lanes 3 and 4, the beads were washed with 600 mM KCl. The positions of the molecular size markers are shown on the left (in kilodaltons). The identities of each polypeptide as determined by mass spectrometry analysis are indicated on the right. Solid circles indicate cytoskeletal and other known proteins that were not characterized further. Triangles indicate previously characterized RNA polymerase III subunits. Asterisks indicate polypeptides whose function has not been analyzed before in the context of RNA polymerase III transcription.

FIG. 2.

Structure of HsRPC2. (A) Predicted protein sequence of HsRPC2 (GenBank accession number AY092084). The thick black underlines show the first peptide matches identified by mass spectrometry. These cluster towards the C terminus of the protein because only the C terminus of RPC2 was represented in the database. The blue underlines show the additional matches obtained upon reanalysis of the original mass spectrometry data with the full-length sequence. The homology blocks A to I (52) are shaded in yellow. The invariant E and D residues corresponding to those in close proximity to metal B in the structure of S. cerevisiae RNA polymerase II (11) are boxed. The zinc-binding domain is bracketed in red. (B) The percent identities between H. sapiens (Hs) RPC2 and the second largest subunits of D. melanogaster (Dm), S. pombe (Sc), and S. cerevisiae (Sc) RNA polymerase III (RPC2) and II (RPB2), as well as T. celer (Tc) polypeptide B, are indicated. The sequences were aligned with Blast. (C) The percent identities between HsRPC2 and the indicated second-largest subunits, including the E. coli β subunit (Ec β) in the conserved A to I blocks are shown.

FIG. 3.

HsRPC8 and RPC9 are paralogues of the RNA polymerase II RPB7 and RPB4 subunits, respectively. (A) Clustal W Fast alignment of H. sapiens (Hs) RPC8 and the S. pombe (Sp), S. cerevisiae (Sc), and H. sapiens (Hs) RPB7 subunits performed with default parameters. Stars indicate identical amino acids, colons indicate similar amino acids (A, V, F, P, M, I, L, and W; D and E; R, K, and H; and S, T, Y, H, C, N, G, and Q are considered similar), and periods indicate semiconserved substitutions. (B) Clustal W Fast alignment of H. sapiens (Hs) RPC9 and the S. pombe (Sp), S. cerevisiae (Sc), and H. sapiens (Hs) RPB4 subunits performed with default parameters. The symbols are the same as in A. (C) HsRPC8 and HsRPC9 associate with each other. Untagged and HA-tagged HsRPC8 and HsRPC9 were translated in vitro with [35S]methionine and used for coimmunoprecipitation experiments. Lanes 1, 2, 5, and 6 show 1 μl of each of the in vitro translation reaction mixes loaded directly on the gel (input). In lanes 3, 4, 7, and 8, the proteins indicated above the lanes were used for immunoprecipitation (IP) with anti-HA antibody beads. The immunoprecipitates were washed with a buffer containing 300 mM KCl. The circles and triangles indicate untagged and tagged proteins, respectively.

FIG. 4.

Structure of RPC5. (A) Predicted RPC5 protein sequence (GenBank accession number AY092085). The thick underlines show peptide matches identified by mass spectrometry, the thin underlines indicate synthetic peptides used to generate antibodies (CS1534, amino acids 1 to 9; CS1542, amino acids 207 to 222), and the shaded box shows the glutamic acid-rich domain. (B) Regions of similarity between HsRPC5 and a Mus musculus (Mm) homologue of Drosophila sex-lethal interactor (BAB23761), the Drosophila sex-lethal interactor (Dm SIN, AAF51670 [13]), putative proteins from A. thaliana (At) (NP_199764), C. elegans (Ce) (AAK29861), S. pombe (Sp) (T41323), and S. cerevisiae (Sc) C37 (NP_012950). The percentages show amino acid identities between the colored regions of the various proteins and the regions in HsRPC5 delimited by thin lines of corresponding color as determined by the Blast program. The numbers indicate amino acid numbers. (C) Blast alignment of H. sapiens RPC5 and S. cerevisiae C37.

FIG. 5.

RPC5 copurifies and is associated with RPC1/RPC155 in cells expressing only untagged RNA polymerase III. (A) Conventional purification scheme of an untagged RNA polymerase III complex (49). WCE, whole-cell extract; CV, column volume. (B) Doubly tagged RPC5 expressed in E. coli migrates slightly slower than endogenous RPC5 from HeLa cells. An active P11 phosphocellulose fraction (8 μl, 427 μg/ml) (lanes 1 and 2) or 2 μl of doubly tagged, purified recombinant RPC5 (11.2 μg/ml) (lanes 3 and 4) were fractionated by SDS-PAGE, and the proteins were transferred to nitrocellulose. The membrane was then cut and probed with an anti-RPC5 antibody (CS1542) for lanes 1 and 3 or with the same antibody preincubated with the peptide against which it was raised for lanes 2 and 4. The positions of RPC5 and the molecular size markers (in kilodaltons) are indicated. (C) Endogenous RPC5 is associated with other RNA polymerase subunits in vivo. A HeLa cell nuclear extract was used as the starting material for nondenaturing immunoprecipitations (IP) with the antibodies indicated above the lanes, and the immunoprecipitates were fractionated by SDS-PAGE, transferred to a membrane, and probed by immunoblot with the antibodies indicated at the right of the three panels. preimm., preimmune serum. (D) Copurification of RPC5 and RPC1/RPC155 during Mono Q chromatography. Mono Q fractions 1 to 20 (lanes 1 to 20, respectively) and Mono Q flowthrough (FT, lane 21) were analyzed by immunoblot with the antibodies indicated at the right of the two panels.

FIG. 6.

RPC5 is required for RNA polymerase III transcription. (A) Addition of purified tagged RNA polymerase III (pol III) complex restores transcription in CS1534-treated extracts. HeLa whole-cell extracts were supplemented with 30 ng of recombinant TBP and either 80 ng of recombinant Brf1 (VAI transcription, upper panel) or 30 ng of recombinant TBP, 50 ng of recombinant Brf2, and 400 ng of recombinant SNAPc (third panel, U6 transcription), as indicated on top of the figure. The extracts were then incubated with either preimmune (preimm., lane 1) or anti-RPC5 CS1534 (lane 2 to 7) antibody beads. The treated extracts were complemented with 50 (lane 3) and 100 (lane 4) ng of recombinant RPC5 or with 30 (lane 5), 150 (lane 6), and 300 (lane 7) ng of purified tagged RNA polymerase III complex and tested for VAI or U6 transcription. The bands labeled IC correspond to the internal control signals. (B) Both RPC5 and RPC1/RPC155 are removed from the extracts by CS1534 treatment. The same extracts complemented with TBP and Brf1 or TBP, Brf2 and SNAPc used for VAI (lanes 1 and 2) or U6 (lane 3 and 4) transcription in panel A were analyzed by immunoblotting with the antibodies indicated on the right. (C) RPC5 itself, not only associated proteins, is essential for RNA polymerase III transcription. HeLa whole-cell extracts were complemented with TBP and Brf1 or Brf2 as in A. The extracts were then incubated with either preimmune (lane 1) or anti-RPC5 CS1542 (lanes 2 to 8) antibody beads. The treated extracts were complemented with 10 (lane 3), 50 (lane 4), and 100 (lane 5) ng of recombinant RPC5 or 30 (lane 6), 150 (lane 7), and 300 (lane 8) ng of purified tagged RNA polymerase III complex. (D) RPC1/RPC155 is present in the CS1542-depleted extracts. The same extracts complemented with TBP and Brf1 or TBP and Brf2 and used for VAI (lanes 1 and 3) or U6 (lanes 2 and 4) transcription in panel C were analyzed by immunoblotting with the antibodies indicated on the right.

FIG. 7.

RPC5 associates with HsRPC4/RPC53. (A) RPC5 and RPC4 derivatives used for immunoprecipitations. The regions conserved in the S. cerevisiae counterparts are indicated. The numbers refer to amino acids. (B) Blast alignment of H. sapiens RPC4/RPC53 and S. cerevisiae C53. (C) RPC5 amino acids 1 to 163 are sufficient for association with HsRPC4/RPC53. Full-length and truncated RPC5 proteins as well as HA-tagged full-length HsRPC4/RPC53 were translated in vitro with [35S]methionine and used for coimmunoprecipitation experiments. Lanes 1 to 4 show 1 μl of each of the in vitro translation reaction mixes loaded directly on the gel (input). In lanes 5 to 7, the proteins indicated above the lanes were used for immunoprecipitation (IP) with anti-HA antibody beads. In lanes 8 to 10, the proteins indicated above the lanes were mixed with HA-tagged HsRPC4/RPC53 before immunoprecipitation. The immunoprecipitates were washed with a buffer containing 300 mM KCl. The circles and triangles indicate untagged and tagged proteins, respectively. (D) HsRPC4/RPC53 amino acids 254 to 398 are sufficient for interaction with RPC5 amino acids 1 to 163. The proteins indicated above the lanes were either loaded directly (lanes 1 to 5) or used for coimmunoprecipitation experiments as in C. The circles and triangles are as in C.

FIG. 8.

Subunit-subunit associations. HsRPC4 and HsRPC5 associate with each other, and so do HsRPC8 and HsRPC9, as well as the RNA polymerase II (pol II) subunits RPB7 and RPB4, as symbolized by red arrows. HsRPC8 and HsRPC9 are paralogues of the RNA polymerase II subunits RPB7 and RPB4, as symbolized by blue arrows.

Cited by

16p12.1 Deletion Orthologs are Expressed in Motile Neural Crest Cells and are Important for Regulating Craniofacial Development in Xenopus laevis.
Lasser M, Bolduc J, Murphy L, O'Brien C, Lee S, Girirajan S, Lowery LA. Lasser M, et al. Front Genet. 2022 Mar 24;13:833083. doi: 10.3389/fgene.2022.833083. eCollection 2022. Front Genet. 2022. PMID: 35401697 Free PMC article.
A structural perspective of human RNA polymerase III.
Wang Q, Lei M, Wu J. Wang Q, et al. RNA Biol. 2022;19(1):246-255. doi: 10.1080/15476286.2021.2022293. Epub 2021 Dec 31. RNA Biol. 2022. PMID: 35133940 Free PMC article.
Upregulation of RNA cap methyltransferase RNMT drives ribosome biogenesis during T cell activation.
Galloway A, Kaskar A, Ditsova D, Atrih A, Yoshikawa H, Gomez-Moreira C, Suska O, Warminski M, Grzela R, Lamond AI, Darzynkiewicz E, Jemielity J, Cowling VH. Galloway A, et al. Nucleic Acids Res. 2021 Jul 9;49(12):6722-6738. doi: 10.1093/nar/gkab465. Nucleic Acids Res. 2021. PMID: 34125914 Free PMC article.
Functional assessment of the "two-hit" model for neurodevelopmental defects in Drosophila and X. laevis.
Pizzo L, Lasser M, Yusuff T, Jensen M, Ingraham P, Huber E, Singh MD, Monahan C, Iyer J, Desai I, Karthikeyan S, Gould DJ, Yennawar S, Weiner AT, Pounraja VK, Krishnan A, Rolls MM, Lowery LA, Girirajan S. Pizzo L, et al. PLoS Genet. 2021 Apr 5;17(4):e1009112. doi: 10.1371/journal.pgen.1009112. eCollection 2021 Apr. PLoS Genet. 2021. PMID: 33819264 Free PMC article.
Structural insights into transcriptional regulation of human RNA polymerase III.
Wang Q, Li S, Wan F, Xu Y, Wu Z, Cao M, Lan P, Lei M, Wu J. Wang Q, et al. Nat Struct Mol Biol. 2021 Feb;28(2):220-227. doi: 10.1038/s41594-021-00557-x. Epub 2021 Feb 8. Nat Struct Mol Biol. 2021. PMID: 33558766

References

1. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped Blast and PSI-Blast: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402. - PMC - PubMed
1. Bashaw, G. J., and B. S. Baker. 1997. The regulation of the Drosophila msl-2 gene reveals a function for Sex-lethal in translational control. Cell 89:789-798. - PubMed
1. Bell, L. R., E. M. Maine, P. Schedl, and T. W. Cline. 1988. Sex-lethal, a Drosophila sex determination switch gene, exhibits sex-specific RNA splicing and sequence similarity to RNA binding proteins. Cell 55:1037-1046. - PubMed
1. Bycroft, M., T. J. Hubbard, M. Proctor, S. M. Freund, and A. G. Murzin. 1997. The solution structure of the S1 RNA binding domain: a member of an ancient nucleic acid-binding fold. Cell 88:235-242. - PubMed
1. Chang, C. P., R. V. Pearse, 2nd, S. O'Connell, and M. G. Rosenfeld. 1993. Identification of a seven transmembrane helix receptor for corticotropin-releasing factor and sauvagine in mammalian brain. Neuron 11:1187-1195. - PubMed

Characterization of human RNA polymerase III identifies orthologues for Saccharomyces cerevisiae RNA polymerase III subunits - PubMed (original) (raw)