Rice MULEs Transpose in Yeast

Named for their mutational effect, Mutator-like transposable elements (MULEs) in plant genomes transpose frequently and can also acquire pieces of genomic DNA, forming Pack-MULEs; movement and recombination of genomic fragments by Pack-MULEs can alter the regulation of existing genes and may form new genes (Jiang et al., 2004; reviewed in Chénais et al., 2012). MULEs have terminal inverted repeats, transpose via a DNA intermediate, and form small target site duplications on insertion. Most MULEs can’t jump without a little help; rice (Oryza sativa) has an estimated 30,000 MULEs but few autonomous MULEs. Many aspects of the biology of MULEs remain unknown, particularly the host factors required for transposition and the mechanism by which Pack-MULEs acquire genomic DNA fragments.

Recapitulation of transposition in a tractable system would enable rapid analysis of the components of transposition. To this end, Zhao et al. (2015) used the Os3378 MULE from rice to recapitulate transposition in yeast (Saccharomyces cerevisiae). They first isolated a full-length element that encodes a functional transposase, by examining cDNAs and comparing genomic sequences and secondary structures of active MULEs from multiple species. To assay transposition in yeast, they expressed the transposase from a galactose-inducible promoter and examined excision of a nonautonomous element from ADE2 in a reporter construct. Indeed, they observe excision and can also detect the sites of reinsertion of the test element, allowing them to test multiple aspects of MULE function. The founder element of MULEs, MuDR, encodes two proteins; one functions as the transposase, but previous hypotheses suggested that the other protein functions in element reinsertion. Examination of reinsertions of the test element showed that the transposase also catalyzes element reinsertion; therefore, the function of the second protein encoded by MuDR remains a mystery.

The authors also examine the function of the transposase. Consistent with the low copy number of this element in rice, the original transposase gives a low excision rate, 0.47 events/10⁶ cells. However, they found they could enhance excision with transposase variants, including deletions and amino acid substitutions in regions not conserved in other MULEs, and even fusions to enhanced yellow fluorescent protein (EYFP). Moreover, the EYFP fusions showed that the wild-type transposase localizes to the nucleus, consistent with the presence of nuclear localization signals, but some fusions localized to the cytoplasm or formed aggregates (see figure). The authors also changed the levels of transposase by altering the galactose concentration, finding that excision rates increased and then plateaued with increasing galactose. However, immunoblots showed that the excision rate did not strictly increase with increasing levels of transposase.

Activity and subcellular localization of transposase fused to EYFP. N-terminal or C-terminal fusions showed different effects on excision rate of the wild-type Os3378-Z transposase and the truncated Os3378-Z-105 and -130. Examination of the fluorescent signal showed that EYFP-Os3378-Z and EYFP-Os3378-Z-105 localized to the nucleus, while EYFP-Os3378-Z-130 localized all over the cell. Fusion of EYFP at the C terminus induced aggregates in cytoplasm. (Reprinted from Zhao et al. [2015], Figure 6.)

The authors also examined parameters of the element’s DNA sequence. Changing the nonautonomous test element showed that smaller elements excise better than larger ones and that a perfect nine-base target site duplication improves the rate of precise excision. The authors also test a long-standing hypothesis that Pack-MULEs may form by transposition of a single terminus but find that constructs containing only one terminus do not excise.

Thus, the authors used this tractable, rapid system to examine multiple parameters of MULE activity and test long-standing hypotheses. Their findings enable work to advance our understanding of this common genomic repetitive element that shapes gene regulation and genomic evolution. Moreover, this advanced understanding may lead to applications for MULEs in biotechnology and genomics research.