Transposition of a rice Mutator-like element in the yeast Saccharomyces cerevisiae

Abstract

Mutator-like transposable elements (MULEs) are widespread in plants and are well known for their high transposition activity as well as their ability to duplicate and amplify host gene fragments. Despite their abundance and importance, few active MULEs have been identified. In this study, we demonstrated that a rice (Oryza sativa) MULE, Os3378, is capable of excising and reinserting in yeast (Saccharomyces cerevisiae), suggesting that yeast harbors all the host factors for the transposition of MULEs. The transposition activity induced by the wild-type transposase is low but can be altered by modification of the transposase sequence, including deletion, fusion, and substitution. Particularly, fusion of a fluorescent protein to the transposase enhanced the transposition activity, representing another approach to manipulate transposases. Moreover, we identified a critical region in the transposase where the net charge of the amino acids seems to be important for activity. Finally, transposition efficiency is also influenced by the element and its flanking sequences (i.e., small elements are more competent than their large counterparts). Perfect target site duplication is favorable, but not required, for precise excision. In addition to the potential application in functional genomics, this study provides the foundation for further studies of the transposition mechanism of MULEs.

Figure 1.

Schematic Structures of Os3378 and Constructs Used in This Study. (A) Structural comparison between the annotated transposase (LOC_Os05g31510) and Os3378-Z. Black triangles, TIRs; empty boxes, exons; lines linking empty boxes, introns; arrows, transcription start sites; dashed lines, splicing site variations between LOC_Os05g31510 and Os3378-Z. (B) Structural comparison between LOC_Os5g31510 and Os3378-Z at the amino acid level. Shaded regions are in common between the two proteins. HTH, helix-turn-helix. (C) Os3378 element and two artificial nonautonomous Os3378 elements, Os3378NA469 (469 bp in length) and Os3378NA1485 (1485 bp in length), which are formed by combining the TIR and subterminal sequences. (D) The expression vector with Os3378-Z transposase fused downstream of the GAL1 promoter. (E) The reporter vector with Os3378NA inserted in the coding region of the ADE2 gene.

Figure 2.

N-Terminal Deleted Os3378-Z Transposases. (A) Schematic representation of wild-type and N-terminal deleted Os3378-Z transposases. (B) Relative excision frequency induced by wild-type and N-terminal deleted Os3378-Z transposases on 2% galactose. The excision frequency by Os3378-Z-130 was set as 100%. se values were calculated from at least 20 replicates for each form of the Os3378-Z transposase.

Figure 3.

Os3378-Z-105 Transposases with Amino Acid Substitutions and Their Activity. (A) Amino acid substitutions between amino acids 105 and 130 of Os3378-Z-105. (B) Relative excision frequency induced by Os3378-Z-105 and its mutant forms on 2% galactose. The excision frequency by Os3378-Z-130 was set as 100%. se values were calculated from at least 20 replicates for each form of the Os3378-Z transposase.

Figure 4.

Protein Levels of Os3378-Z Transposases and Their Activity at Various Galactose Concentrations. (A) and (B) Protein levels of Os3378-ZCFH (A) and Os3378-Z-130CFH (B) at various galactose levels. Partial images of the total protein are shown at the bottom. (C) Relative excision frequency induced by Os3378-Z and Os3378-Z-130 at various galactose concentrations. The excision frequency by Os3378-Z-130 on 2% galactose was set as 100%.

Figure 5.

The Impact of Os3378NA Size and TSD on Excision Activity Induced by Os3378-Z-130 and Sequences of the Donor Site following Excision of Os3378NA469. (A) Comparison of excision frequency between Os3378NA469 and Os3378NA1485 with and without perfect TSD. (B) Sequences at the donor site on the reporter vector prior to excision and following excisions of Os3378NA469. Os3378NA was integrated into the HpaI restriction site GTTAAC, which is within the coding sequence of the ADE2 gene. Sequences of the Os3378NA integration site in the reporter vectors are shown, where boldface nucleotides are TSD or nonperfect TSD sequences. Six types of the donor site sequence were recovered from yeast ADE2 revertants induced by Os3378NA469noTSD. The numbers in parentheses correspond to numbers of yeast revertant colonies containing that type of sequence at the donor site following excisions. A total of 39 colonies were assayed. Nucleotides in green are extra sequences (compared with original ADE2 sequence) retained after excisions; underlined nucleotides are from the AGC, the mutated portion of the TSD; the boxed nucleotide represents a point mutation.

Figure 6.

Cellular Localization and Activity of N- or C-Terminal EYFP-Tagged Transposases. (A) Relative excision frequency induced by different forms of Os3378-Z on 2% galactose. se values were calculated from at least 20 replicates for each form of the transposase. (B) Cellular localization of EYFP with and without the Os3378-Z amino acids 105 to 130. (C) Cellular localization of various forms of Os3378-Z with EYFP tag at the N termini (left panel) and C termini (right panel). DAPI is a fluorescent stain that binds to DNA.

Figure 7.

Immunoblot Analysis and Activity of Transposases with and without the CFH. (A) Relative excision frequency induced by transposases with and without the CFH on 2% galactose. (B) Protein levels of transposases on 2% galactose. A partial image of the total protein is shown at the bottom.

Figure 8.

Reinsertions of Os3378NA. (A) DNA gel blot analysis of reinsertions of Os3378NA from different reporter vectors. (B) Distribution of reinsertion sites of Os3378NA with regard to genomic sequence features. (C) Ratios of observed distribution of reinsertion sites to that of the fraction of each genomic feature in yeast (S288C). 5′ region, reinsertions upstream of the transcription start site of protein-coding genes on both sides of the reinsertion site (genes are head to head); 3′ region, Os3378NA insertions downstream of the transcription termination site of protein-coding genes on both sides of the reinsertion site (genes are tail to tail); 5′ and 3′ regions, reinsertions located upstream of the transcription start site of one gene and downstream of the transcription termination site of another (genes are head to tail); ARS, autonomously replicating sequence; intergenic, insertions within 1 kb flanking sequence of genes.