Silencing of a gene adjacent to the breakpoint of a widespread Drosophila inversion by a transposon-induced antisense RNA

Abstract

Adaptive changes in nature occur by a variety of mechanisms, and Drosophila chromosomal inversions was one of the first studied examples. However, the precise genetic causes of the adaptive value of inversions remain uncertain. Here we investigate the impact of the widespread inversion 2j of Drosophila buzzatii on the expression of the CG13617 gene, whose coding region is located only 12 bp away from the inversion proximal breakpoint. This gene is transcribed into a 2.3-kb mRNA present in all D. buzzatii developmental stages. More importantly, the expression level of CG13617 is reduced 5-fold in embryos of lines homozygous for the 2j inversion compared with lines without the inversion. An antisense RNA that originates in the Foldback-like transposon Kepler inserted at the breakpoint junction in all of the 2j lines and that forms duplexes with the CG13617 mRNA in 2j embryos is most likely responsible for the near silencing of the gene. Few examples of RNA interference caused by transposable elements (TEs) have been previously described, but this mechanism might be prevalent in many organisms and illustrates the potential of TEs as a major source of genetic variation. In addition, because chromosomal rearrangements are usually induced by TEs, position effects might be more common than previously recognized and contribute significantly to the evolutionary success of inversions.

Fig. 1.

Schematic representation of inversion 2j proximal breakpoint sequence in lines st-1 and j-1. Vertical arrows indicate the inversion breakpoint (BP) and separate the inverted and the noninverted segments (to the right and left of the vertical arrows, respectively). Exons of genes flanking the breakpoint are represented as colored boxes, with coding sequences in dark and UTRs in light colors. Transcripts of each gene are shown below the diagram, and 5′and 3′ denote their orientation. Small black arrows indicate PCR primers used in this study. Clones and PCR fragments used for sequencing are represented above each diagram by thin black lines and open bars, respectively. TEs inserted at the breakpoint in line j-1 are depicted as colored boxes with sharp ends. The small duplication (Dup) and insertion (Ins) found at the CG13617 upstream region in line j-1 are also indicated. Primer G11 is absent from line j-1 as the result of an internal rearrangement, but it is represented here for simplicity. Restriction sites: B, BamHI; D, DraI; X, XmnI; S, SalI; H, HindIII; P, PstI; E, EcoRI.

Fig. 2.

Nucleotide sequence of the 3′ end of CG13617 in lines st-1 and j-1. The stop codon, the two putative polyadenylation signals, and the Galileo element inserted in line j-1 are included in shaded boxes. A vertical arrow indicates the 2j inversion proximal breakpoint (BP). Sequences present only in lines with the inversion are shown in italic.

Fig. 3.

Expression analysis of CG13617 in four different developmental stages of two lines with (2j) and without (2st) the inversion. (A) Northern blot analysis of CG13617.(Upper)An ≈2.3-kb transcript was detected in embryos, pupae, and adults. (Lower) rRNA used as a loading control is shown. The RNA amount was higher in embryos, but this fact is not relevant for the comparison between arrangements. (B) Semiquantitative RT-PCR analysis of CG13617. Primers E2/E7 and H1/H2 were used to amplify, respectively, 387 bp of CG13617 mRNA (Upper) and 438 bp of Gapdh mRNA (Lower) as a reference. In 2j embryos, there is an extra band containing intron 3 that corresponds to the CG13617 antisense transcript. E, embryos; L, larvae; P, pupae; A, adults.

Fig. 4.

Relative quantification of CG13617 mRNA and antisense RNA and Pp1α-96A mRNA by real-time RT-PCR. Average expression levels for four 2st and four 2j lines are represented in the graphs. Standard deviation within each arrangement is indicated by error bars. Expression levels are shown in relation to those of lines st-1 (CG13617 and Pp1α-96A mRNA) and j-1 (antisense RNA). A primer spanning the third and fourth exons (E25) and a primer located in the second intron (AS1) were used to ensure that, respectively, only the sense or the antisense transcripts of CG13617 were amplified.

Fig. 5.

Strand-specific RT-PCR of CG13617. Two different cDNAs were synthesized for each sample by using primers specific for the sense (E5) and antisense (E6) transcript and were then amplified with primer pair E2/E7 (RT+). Negative controls for each sample without retrotranscriptase (RT-) are also shown. (A) Amplification of sense and antisense RNAs in embryos from eight different lines. The 387-bp band in the sense mRNA is smaller than that of the DNA and the antisense transcript (470 bp) because of the splicing of the 83-bp intron. (B) Amplification of antisense RNA in four developmental stages of lines st-1 and j-1. E, embryos; L, larvae; P, pupae; A, adults.

Fig. 6.

Detection of CG13617 double-stranded RNA (dsRNA) in st-1 and j-19 embryos. RNA was treated with RNase to degrade single-stranded RNA, denatured, retrotranscribed, and amplified according to the methods of ref. . Amplification was carried out with two successive PCRs using nested primer pairs E11/E12 and E33/E34. A band of 318 bp corresponding to the dsRNA is observed in 2j, but not in 2st, embryos. For each sample, a negative control (NC) without the denaturation step was performed. The first two lanes correspond to genomic DNA of each line.