Two retrotransposons maintain telomeres in Drosophila

Abstract

Telomeres across the genus Drosophila are maintained, not by telomerase, but by two non-LTR retrotransposons, HeT-A and TART, that transpose specifically to chromosome ends. Successive transpositions result in long head-to-tail arrays of these elements. Thus Drosophila telomeres, like those produced by telomerase, consist of repeated sequences reverse transcribed from RNA templates. The Drosophila repeats, complete and 5'-truncated copies of HeT-A and TART, are more complex than telomerase repeats; nevertheless, these evolutionary variants have functional similarities to the more common telomeres. Like other telomeres, the Drosophila arrays are dynamic, fluctuating around an average length that can be changed by changes in the genetic background. Several proteins that interact with telomeres in other species have been found to have homologues that interact with Drosophila telomeres. Although they have hallmarks of non-LTR retrotransposons, HeT-A and TART appear to have a special relationship to Drosophila. Their Gag proteins are efficiently transported into diploid nuclei where HeT-A Gag recruits TART Gag to chromosome ends. Gags of other non-LTR elements remain predominantly in the cytoplasm. These studies provide intriguing evolutionary links between telomeres and retrotransposable elements.

Figure 1

Diagram of the two D. melanogaster telomere retrotransposons, drawn as their putative RNA transposition intermediates. Coding regions and 3′ UTRs are labeled. (A)_n indicates the poly(A) tail on the RNAs. It is the source of the (dA/T)_n that joins each DNA copy to the chromosome when the element transposes. HeT-A elements are ~ 6 kb. The 5′ end of TART has not yet been completely defined but subfamilies appear to be 10–12 kb.

Figure 2

Model for extension of chromosome ends by telomeric retrotransposons. Retrotransposons yield sense-strand transcripts that serve as both mRNAs and transposition intermediates. This diagram shows our current model for the path of these RNAs from transcription until they are reverse transcribed to add another repeat onto the telomere array. Gray arrows represent HeT-A (dark) and TART (light) elements attached to the end of the chromosome. A poly(A) sense-strand RNA is transcribed from a member of the array (step 1). For the telomeric retrotransposons, there is evidence suggesting that this RNA must be translated (step 2) before serving as a template (step 3) for telomere additions. This suggestion is now supported by the finding that translation products (Gags) of these RNAs appear capable of delivering the transposition template specifically to its target at the telomere. Gray circles in diagram represent Gags of either HeT-A or TART. Analogy with retroviruses suggests that reverse transcriptase is also included in the Gag-RNA complex; however, there is no evidence on this point. Reproduced from The Journal of Cell Biology, 2002, 158:398 by copyright permission of the Rockefeller University Press.

Figure 3

Intracellular localization of GFP (Green Fluorescent Protein)-tagged Gags in transiently transfected cultured Drosophila cells of the SL2 line. Fluorescence micrographs show each cell in two panels. DNA in all cells is stained with DAPI (false-colored red). Left panels show merged GFP and DAPI superimposed on the DIC image. Right panels show GFP alone. Transfectants shown: a. HeT-A Gag – a cell with large Het-dots in the nucleus and Het-body in the cytoplasm; b. TART Gag--small clusters in the nucleus, more diffuse than Het-dots. c. Doc (a non-telomeric retrotransposon) Gag – irregular clusters in cytoplasm, more concentrated near the nucleus.

Figure 4

Fluoresence micrographs of cells from transgenic Drosophila flies expressing HeT-A Gag-GFP protein under the control of Gal4 driven by the daughterless promoter. Gag protein does not enter the nuclei of polyploid cells (a, b). In polytene salivary gland cells (a), a large part of Gag-GFP is in an irregular aggregation (possibly the Golgi apparatus) adjacent to the nucleus. In polyploid tubule cells (b) the protein is entirely cytoplasmic and forms small clusters of approximately equal size. In diploid cells (c) the protein makes Het-dots in the nucleus with a small possible Het-body in the cytoplasm (but notice the magnification compared to a and b). Gag-GFP protein is green. DNA is stained with DAPI (red). These data stacks were deconvolved using Deltavision software.

Figure 5

Intracellular localization of TART Pol-GFP in transiently transfected cultured Drosophila cells. (a) Left panel shows merged GFP and DAPI (red) superimposed on the DIC image. Right panel shows GFP alone. Pol-GFP is not detected in the nucleus. b. Left panel shows parts of four untransfected cells surrounding a cell co-transfected with TART Pol-YFP (Yellow Fluorescent Protein) and HeT-A Gag-CFP (Cyan Fluorescent Protein). Panels on right show YFP (shown as yellow-green) and CFP (shown as blue) only. HeT-A Gag forms nuclear Het-dots but does not recruit TART Pol into the nucleus; however the two proteins apparently have some interaction, as evidenced by the recruitment of TART Pol into the cytoplasmic Het-body.

Figure 6

HeT-A Gag recruits TART Gag, but not Doc Gag, to nuclear Het-dots. (a) Cell co-transfected with HeT-A Gag-CFP and TART Gag-YFP. Left panel shows merged CFP, YFP and DAPI (red) superimposed on the DIC image. Right panels show CFP and YFP alone. HeT-A Gag recruits TART Gag to both nuclear Het-dots and cytoplasmic Het-body (although this cell has no Het-body). (b) Cell co-transfected with HeT-A Gag-CFP and Doc Gag-YFP. Bottom panel shows merged CFP, YFP and DAPI (red) superimposed on the DIC image. Top panels show CFP and YFP alone. HeT-A Gag does not carry Doc Gag into the nucleus but is associated with it in the cytoplasmic Het-body.