Biologia 67/5: 845—851, 2012 Section Cellular and Molecular Biology DOI: 10.2478/s11756-012-0085-y Epitope selection for multipeptide proteins: the case of RNA polymerase IV and V Leydi de Rocio Canche Moo 1 , Analy Arroyo Herrera 1 , Luis Rodriguez-Zapata 2 , Victor Suarez 3 & Enrique Castano 1 * 1 Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, A.C. Calle 43 No. 130 Col. Chuburná de Hidalgo, C.P. 97200, Mérida, Yucatán, México; e-mail: Enriquec@cicy.mx 2 Unidad de Biotecnología, Centro de Investigación Científica de Yucatán, A.C. Calle 43 No. 130 Col. Chuburná de Hidalgo, C.P. 97200, Mérida, Yucatán, México 3 Universidad Autónoma de Yucatán, Facultad de Medicina, Unidad Interinstitucional de Investigación Clínica y Epidemi- ológica, Av. Itzáes entre 59 y 59–A, C.P. 97000, Mérida, Yucatán, México Abstract: Large multipeptide protein complexes have provided a challenge for epitope selection, which is required for immunological protocols where native conformations are needed. Immunolocalization requires native conformation of the proteins, which is essential for further understanding of biological activity. RNA polymerase IV and V are multisubunit proteins that interact with other factors in the RNA-directed DNA methylation pathway for control of DNA silencing by small interfering RNA and DNA methylation. DNA silencing is an important process during cell differentiation, nuclear structure and viral control among others. RNA polymerase IV and V are yet to be studied in model monocot systems like Oryza sativa, which may provide further data to define if the genetic silencing mechanism has diverged over time as compared to dicots. Here we show an in silico selection process of exposed sequences and their use for obtaining antibodies against native RNA polymerase IV and V in O. sativa. Key words: antibody; RNA polymerase IV and V; Oryza sativa; somatic embryogenesis; siRNA. Abbreviations: 2,4-D, 2,4-dichlorophenoxy acetic acid; DAPI, 4’,6-diamidino-2-phenylindole; Pol, RNA polymerase; siRNA, small interfering RNA. Introduction Epitope selection for antibody production has long been a difficult task, a myriad of antibodies lack the ability to detect the target proteins in vivo or to carry out co-immunoprecipitations to obtain further complexes. RNA polymerase (Pol) IV and V (also known as pol IV/NRPD1a/NRPD1 and polV/NRPD1b/NRPE1, re- spectively) are only found in plants as a particular mechanism for gene silencing. Due to the multipeptide complexity of the protein and the reduced amounts of expression, the traditional process of purification has not been carried out. Recombinant expression of the protein is nonviable, although for single polypeptide proteins or proteins that are not posttranscriptional modified it is an important part of the antibody pro- teomics technology, as it produces subfragments of pro- teins called protein epitope signature tags (Lindskog et al. 2005). However, for most large complexes this does not work well due to the lack of postrancriptional mod- ifications (if Escherichia coli is used) or improper as- sembly of all the subunits of the recombinant protein. Usually random selections of peptide sequences that are uncommon in a sequence data bank are used for the production of antibodies. Although they may provide a good signal in denaturation assays (e.g., western blot), it often lacks the ability to detect target proteins in vivo. It is possible to utilize the advantage of the struc- tural information from a similar protein Pol II (Han et al. 2004; Armache et al. 2005) together with the in silico prediction to define possible exposed epitopes for two of the largest subunits of Pol IV and V from Oryza sativa. Pol V is a large multipeptide protein that is in- volved in gene silencing. Over the last decade with the emerging view of the RNA-directed DNA methylation pathway (Herr et al. 2005; Matzke et al. 2009; Law et al. 2010), small interfering RNAs (siRNAs) have become an important tool for gene silencing and are required for several processes including viral protection, cell dif- ferentiation and nuclear territory organization (Pontes et al. 2009). The complete mechanism for gene silencing is yet unclear, however it is known that generation and targeting of siRNAs requires Pol IV, which is proposed * Corresponding author c 2012 Institute of Molecular Biology, Slovak Academy of Sciences Unauthenticated Download Date | 7/29/18 4:33 AM