Using the Yeast Three-Hybrid System to Identify Proteins that Interact with a Phloem-Mobile mRNA

Abstract

Heterografting and RNA transport experiments have demonstrated the long-distance mobility of StBEL5 RNA, its role in controlling tuber formation, and the function of the 503-nt 3' untranslated region (UTR) of the RNA in mediating transport. Because the 3' UTR of StBEL5 is a key element in regulating several aspects of RNA metabolism, a potato leaf cDNA library was screened using the 3' UTR of StBEL5 as bait in the yeast three-hybrid (Y3H) system to identify putative partner RNA-binding proteins (RBPs). From this screen, 116 positive cDNA clones were isolated based on nutrient selection, HIS3 activation, and lacZ induction and were sequenced and classified. Thirty-five proteins that were predicted to function in either RNA- or DNA-binding were selected from this pool. Seven were monitored for their expression profiles and further evaluated for their capacity to bind to the 3' UTR of StBEL5 using β-galactosidase assays in the Y3H system and RNA gel-shift assays. Among the final selections were two RBPs, a zinc finger protein, and one protein, StLSH10, from a family involved in light signaling. In this study, the Y3H system is presented as a valuable tool to screen and verify interactions between target RNAs and putative RBPs. These results can shed light on the dynamics and composition of plant RNA-protein complexes that function to regulate RNA metabolism.

Keywords: BEL1 family; Solanum tuberosum; StBEL5; mobile RNA; potato; yeast three-hybrid.

Figure 1

Schematic diagram of the Y3H system for screening interacting partner proteins from a leaf cDNA library using the 503-nt 3′ UTR of StBEL5 as bait.

Figure 2

List of seven B5RBPs (A) and a quantitative analyses of their interactions with the 503-nt 3 ′ UTR of StBEL5 using a β-galactosidase assay (B). The 3′ UTR of StBEL5 with StPTB6-pAD was used as a positive control (B), and an empty pAD (B) or the pIIIA/MS2-1 bait vectors (C) were used as negative controls. The numbers above each bar (B) represent the means for β-galactosidase activity in triplicate, and standard errors are shown for each mean (B,C). The RNA produced by the pIIIA/MS2-1 vector in yeast, including the 60-nt MS2 sites, is approximately 272 nt in length (Bernstein et al., 2002).

Figure 3

Protein structure and prediction of conserved domains of the seven B5RBPs. The deduced amino acid sequences of the B5RBPs were analyzed using SMART (http://smart.embl-heidelberg.de/) and NCBI’s Conserved Domain Search. RRM, RNA recognition motif; DUF, domain of unknown function; GRM, glycine-rich motif; KOW, from Kyrpides et al. (1996); CC, coiled-coil motif; ZF-CCCH, zinc finger, CCCH-type.

Figure 4

Phylogenetic analysis (A) and alignment of the aa sequence (B) of the family of LSH proteins of potato (St). Included in the dendrogram (A) are the 10 LSH proteins of Arabidopsis (At). Arrows indicate the two StLSHs, LSH3 and -10, identified from the current yeast three-hybrid screening. The nuclear localization signal (NLS) is boxed (B). Asterisks below the aligned amino acids (B) indicate conserved residue identity.

Figure 5

Mobility shift assays for in the vitro interaction of the 3′ UTR of StBEL5 with select B5RBPs. (A) Full-length 3′ UTR of StBEL5 with B5RBP2, -3, and -5. The iron response element (IRE) is included as a negative control (B). Approximately 5 fmole of biotin-labeled bait RNA and protein concentrations ranging from 30 to 250 nM were used in each reaction.

Figure 6

Analysis of β-galactosidase activity for the interaction of select StBEL5 RNA-binding proteins (B5RBPs) with sequences within the 3′ UTR of StBEL5. The values in each graph are normalized to activity relative to the full-length UTR. 3′ UTR, full-length UTR; D1; a 178-nt sequence within the UTR starting from the stop codon; T2, a UAGU-rich region within the UTR; UA, UA-rich region toward the 3′ end of the UTR; MS2-1, RNA sequence from the bait vector serving as a negative control. See Figure 7 for details on these truncated StBEL5 bait sequences.

Figure 7

Schematic diagram of the bait RNA sequences from the 3′ UTR of StBEL5 used in Figure 6. Included are three truncated sequences within the full-length 503-nt 3′ UTR: the 178-nt D1 sequence, the 106-nt T2 region, and the 123-nt UA-rich region (UA). Both UA-and CU-rich motifs are underlined. The UAGU motifs of the T2 sequence are boxed.

Figure A1

Coomassie-stained gels of protein purification of three B5RBPs. Each protein was induced in E. coli, the soluble fraction was extracted, and the protein purified once or twice (B5RBP3) using the HisPur Cobalt purification kit (Pierce). The farthest right-hand lanes (arrows) represent from 15 to 100 ng of protein loaded and these samples were used for the RNA EMSAs. (+) indicates the addition of IPTG; (−) no IPTG; S, soluble fraction; IS, insoluble fraction.