Structural features and mechanism of translocation of non-LTR retrotransposons in Candida albicans

Abstract

A number of abundant mobile genetic elements called retrotransposons reverse transcribe RNA to generate DNA for insertion into eukaryotic genomes. Non-long-terminal repeat (non-LTR) retrotransposons represent a major class of retrotransposons, and transposons that move by target-primed reverse transcription lack LTRs characteristic of retroviruses and retroviral-like transposons. Yeast model systems in Candida albicans and Saccharomyces cerevisiae have been developed for the study of non-LTR retrotransposons. Non-LTR retrotransposons are divided into LINEs (long interspersed nuclear elements), SINEs (short interspersed nuclear elements), and SVA (SINE, VNTR, and Alu). LINE-1 elements have been described in fungi, and several families called Zorro elements have been detected from C. albicans. They are all members of L1 clades. Through a mechanism named target-primed reverse transcription (TPRT), LINEs translocate the new copy into the target site to initiate DNA synthesis primed by the 3' OH of the broken strand. In this article, we describe some advances in the research on structural features and origin of non-LTR retrotransposons in C. albicans, and discuss mechanisms underlying their reverse transcription and integration of the donor copy into the target site.

Keywords: Candida albicans; LINE; Zorro; non-LTR retrotransposons; target-primed reverse transcription (TPRT).

Figure 1. Structure of non-LTR retrotransposons. (A) Structure of LINEs: LINEs family consists of two open reading frames, ORF1 and ORF2. ORF1 encodes a RNA-binding protein that associates with the LINE transposition intermediate. ORF2 encodes endonuclease (endo), reverse transcriptase (RT), zinc finger domain (zf), and RNase H domains in some cases (not shown). Arrows are TSDs. A represents poly-A tail. (B) Structure of Zorro3 in C. albicans: ORF1 contains two zinc knuckle (zk) motifs called type I ORF1, while human L1s contains a type II ORF1. Zorro3 has no TSDs, with poly-A tract flanking both ends. (C) Structure of Zorro1 in C. albicans. The end of 5′UTR cannot be identified. Unlike another non-LTR retrotransposons, neither a poly-A tract nor a 3′ tandem repeat is apparent at the 3′ end of Zorro1.

Figure 2. TSDs target site sequence flanking by retrotransposons. The direction of TSDs is 5′ to 3′. (A) Four base pairs TSDs target site sequence analyzed by Tca families in Candida albicans. Sample capacity, n = 24. (B) Five base pairs TSDs target site sequence analyzed by Ty families in Saccharomyces cerevisiae. Sample capacity, n = 118.

Figure 3. The neighbor-joining (NJ) phylogenetic tree based on RT amino acid sequences of L1 elements from fungi. The percentage of bootstrap support for major branches is indicated. The clade and families are shown on the right. The distance is the categories distance of the PROTDIST program of PHYLIP.

Figure 4. The neighbor-joining (NJ) phylogenetic tree based on RT sequences of Zorro elements from C. albicans. The percentage of bootstrap support for major branches is indicated. The clade and families are shown on the right. The distance is the categories distance of the PROTDIST program of PHYLIP.

Figure 5. The mechanism of target-primed reverse transcription (TPRT).Transposition begins with the transcription of the LINE element (red) into RNA (blue) which encodes an RNA binding protein and a multifunctional protein with endonuclease and reverse transcriptase activity. These proteins (not shown) associate with the LINE RNA, and the endonuclease nicks the DNA at the target site, which contains a poly T tract, which base-pairs with the poly A sequence in the LINE RNA. The LINE RNA is then copied by the reverse transcriptase into a DNA copy (green), which is covalently attached to the target DNA. A second DNA strand is then synthesized on the template of the DNA copy, and the target DNA at each end is filled in to generate the TSDs that flank these elements.

Figure 6. An assay for Zorro3 retrotransposition. The cloned Zorro3 element has a retrotransposition indicator gene (URA3 promoter, and URA3 ORF, disrupted by an antisense intron) inserted into its 3′ UTR. Reverse transcription and integration of the spliced RNA results in a functional and stably integrated URA3 gene and confers a URA3⁺ phenotype on the host cell.