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Distinct Regions of RPB11 Are Required for Heterodimerization with RPB3 in Human and Yeast RNA Polymerase II Wagane J

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Distinct Regions of RPB11 Are Required for Heterodimerization with RPB3 in Human and Yeast RNA Polymerase II Wagane J Distinct regions of RPB11 are required for heterodimerization with RPB3 in human and yeast RNA polymerase II Wagane J. Benga, Sylvie Grandemange, George V. Shpakovski, Elena K. Shematorova, Claude Kedinger, Marc Vigneron To cite this version: Wagane J. Benga, Sylvie Grandemange, George V. Shpakovski, Elena K. Shematorova, Claude Kedinger, et al.. Distinct regions of RPB11 are required for heterodimerization with RPB3 in hu- man and yeast RNA polymerase II. Nucleic Acids Research, Oxford University Press, 2005, 33 (11), pp.3582-3590. 10.1093/nar/gki672. hal-02981340 HAL Id: hal-02981340 https://hal.archives-ouvertes.fr/hal-02981340 Submitted on 27 Oct 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. 3582–3590 Nucleic Acids Research, 2005, Vol. 33, No. 11 doi:10.1093/nar/gki672 Distinct regions of RPB11 are required for heterodimerization with RPB3 in human and yeast RNA polymerase II Wagane J. Benga, Sylvie Grandemange1, George V. Shpakovski2, Elena K. Shematorova2, Claude Kedinger and Marc Vigneron* Unite´ Mixte de Recherche 7100 CNRS–Universite´ Louis Pasteur, Ecole Supe´rieure de Biotechnologie de Strasbourg, Boulevard Se´bastien Brandt, BP 10413, 67412 Illkirch Cedex, France, 1Ge´ne´tique des Maladies Inflammatoires, Institut de Ge´ne´tique Humaine UPR 1142, CNRS. 141, rue de la Cardonille, 34396 Montpellier Cedex 5, France and 2Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, GSP-7, 117997 Moscow, Russia Received April 20, 2005; Revised and Accepted June 7, 2005 ABSTRACT in the eubacterial model that its initial step is the formation of a homodimer of the alpha subunit (1). The eukaryotic counter- In Saccharomyces cerevisiae, RNA polymerase II part of this homodimer appears to be a heterodimer that com- assembly is probably initiated by the formation of prises, in the case of Saccharomyces cerevisiae, the subunits the RPB3–RPB11 heterodimer. RPB3 is encoded by a encoded by the RPB3 and RPB11 genes, henceforth referred to single copy gene in the yeast, mouse and human gen- as Sc3 and Sc11 subunits, respectively (2,3). omes. The RPB11 gene is also unique in yeast and In the yeast genome all RNAP II subunits, including Sc3 mouse, but in humans a gene family has been identi- and Sc11, are encoded by unique genes. While the mouse fied that potentially encodes several RPB11 proteins genome also contains single RPB3 and RPB11 genes, the differing mainly in their C-terminal regions. We com- situation is different in the human genome: in contrast to pared the abilities of both yeast and human proteins to the RPB3 gene (encoding subunit Hs3) which is unique, a heterodimerize. We show that the yeast RPB3/RPB11 family of RPB11 genes was identified [(4), Table 1]. In the present study we shall consider only two members of this gene heterodimer critically depends on the presence of the family, RPB11a and RPB11b. The RPB11a gene is the pre- C-terminal region of RPB11. In contrast, the human dominantly transcribed gene in all human cells and tissues heterodimer tolerates significant changes in RPB11 tested so far. Its unique transcript encodes the Hs11a protein C-terminus, allowing two human RPB11 variants to that is found in RNAP II from all cells analysed to date. The heterodimerize with the same efficiency with RPB3. In RPB11b gene yields several mRNAs resulting from alternative keepingwiththisobservation,theinteractionsbetween splicing processes, which can be detected ubiquitously in the conserved N-terminal ‘a-motifs’ is much more human cells as well. Whether these mRNAs are translated important for heterodimerization of the human sub- at all in vivo remains to be established (4). In this study, units than for those in yeast. These data indicate that we shall consider only the Hs11ba isoform, although other the heterodimerization interfaces have been modified isoforms have been described (see Table 1 and Discussion). during the course of evolution to allow a recent diver- The structure of the yeast RNAP II has been solved at atomic resolution (2,5), and is widely considered as a repres- sification of the human RPB11 subunits that remains entative model for all eukaryotic RNAP IIs, including the compatible with heterodimerization with RPB3. human RNAP II. This assumption is supported not only by sequence homologies but also by the observation that a num- ber of human RNAP II subunits are indeed able to functionally INTRODUCTION replace their yeast counterparts (6,7). Nevertheless, the alpha- The assembly of the RNA polymerase II (RNAP II) multipro- like subunits Hs3 and Hs11a constitute a notable exception, tein complex is still poorly understood. It has been established since they cannot substitute for their yeast homologs (4). *To whom correspondence should be addressed. Tel: +33 3 90 24 47 82; Fax: +33 3 90 24 47 70; Email: [email protected] The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors Ó The Author 2005. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact [email protected] Nucleic Acids Research, 2005, Vol. 33, No. 11 3583 Table 1. The human POLR2J gene family Gene Genbank accession No Chromosomal cDNA Genbank accession No location hRPB11a NC_000007 [101 679 870–101 674 430] 7q22 hRPB11a AA937330, X98433, X82385 hRPB11b NC_000007 [101 872 683–101 841 531] 7q22 hRPB11ba CR596358, CR614811, CR606303, AX405711, AJ277739 hRPB11bb AJ277740, R85011 hRPB11c NC_000007 [101 773 636–101 742 353] 7q22 hRPB11ca AJ277741 hRPB11cb AA306683 hRPB11cg AF468111 hRPB11d NC_000007 [43 799 592–43 768 503] 7p13 hRPB11dg BC017250, AL526460, AL554541 In an attempt to explain this lack of complementation, we The pGEN vector, a 2 mm origin-containing plasmid exhib- performed a detailed genetic analysis of the RPB3/RPB11 iting an expression cassette driven by a PGK promoter (6), was heterodimerization process both in human and yeast systems. used to insert the coding sequence of interest into a unique Among the various methods available, we chose the two- NheI restriction site. hybrid assay in S.cerevisiae, which should be optimal for The pCM vector, which exhibits a centromeric replication assaying Sc3 and Sc11 yeast proteins. The main limitation origin and an ADH1 promoter-driven expression cassette, was of this method is that fusion proteins have to be used instead used to insert the sequences of interest between unique XhoI of the endogenous subunits. It may be stressed, however, that and BamHI restriction sites [pCM185, (9)]. the yeast fusion proteins, LexA-Sc11 and VP16-Sc3, were Each wild-type RNAP II subunit coding sequence was found to be functional in vivo since they were able to restore modified by Taq polymerase-mediated amplification using viability to yeast deleted for these essential genes (strains appropriate primers, so as to be cloned either into the NheI YGVS-072 and D138-1d, respectively, see Supplementary site of pVP or pGEN, or between the XhoI and BamHI sites of Table 2), after chase of the balancer plasmid (data not pLex or pCM (see Supplementary Table 2). shown). Hence, we inferred that these proteins must be able to Deletion mutants were similarly obtained by Taq interact in a way closely mimicking the endogenous subunits. polymerase-mediated amplification, using the appropriate We show here that this ability to interact extends to homo- primers (see Supplementary Table 2). logous human subunits. Our analysis, however, is based on the Site directed mutagenesis of the various coding sequences comparison of effects of similar mutations affecting the pro- was performed by the Pfu-mediated amplification of plasmids, ducts of orthologous genes which are valid only if using the using mispaired oligonucleotide primers (Stratagen). same interaction assay. When analysing the results, one should Random mutagenesiswasperformed bytreatingpVPSc3and keep in mind that the levels of induced b-galactosidase activity pVP Hs3 plasmids with 40 mM hydroxylamin at 37C over- represent an indirect measurement of the efficiency of the night, before transforming Escherichia coli (DH5a strain). The heterodimer formation in vivo, which cannot be formally resulting libraries consisted of 100 000 independent clones. correlated with an affinity constant. Individual clones were obtained after screening in yeast, and Our results provide the first direct evidence that this initial their sequences were determined after recovery by electropora- step in the RNAP II assembly process has significantly tion of the yeast extracts into the DH5a strain of E.coli. diverged, allowing the human RPB11 C-terminal domain to be diversified without affecting the stability of the complex, Two hybrids assay while the integrity of its yeast counterpart appears to be essen- tial. The new alleles of RPB11 genes present in the human The L40 yeast strain [ade2, trp1-901, leu2-3,112, his3D200, genome, therefore, encode proteins that potentially exhibit LYS2::(LexAop)4HIS3, URA3::(LexAop)8lacZ, (8)] was trans- human-specific functions within the RNAP II complex.
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