Evolution of species-specific promoter-associated mechanisms for protecting chromosome ends by Drosophila Het-A telomeric transposons

Abstract

The non-LTR retrotransposons forming Drosophila telomeres constitute a robust mechanism for telomere maintenance, one which has persisted since before separation of the extant Drosophila species. These elements in D. melanogaster differ from nontelomeric retrotransposons in ways that give insight into general telomere biology. Here, we analyze telomere-specific retrotransposons from D. virilis, separated from D. melanogaster by 40 to 60 million years, to evaluate the evolutionary divergence of their telomeric traits. The telomeric retrotransposon HeT-A from D. melanogaster has an unusual promoter near its 3' terminus that drives not the element in which it resides, but the adjacent downstream element in a head-to-tail array. An obvious benefit of this promoter is that it adds nonessential sequence to the 5' end of each transcript, which is reverse transcribed and added to the chromosome. Because the 5' end of each newly transposed element forms the end of the chromosome until another element transposes onto it, this nonessential sequence can buffer erosion of sequence essential for HeT-A. Surprisingly, we have now found that HeT-A in D. virilis has a promoter typical of non-LTR retrotransposons. This promoter adds no buffering sequence; nevertheless, the complete 5' end of the element persists in telomere arrays, necessitating a more precise processing of the extreme end of the telomere in D. virilis.

Fig. 1.

Diagram of D. melanogaster HeT-A transcription and subsequent transposition. Top diagram shows adjacent elements from the interior of a telomere array. [Dark gray: 5′ and 3′UTR. Light gray: Gag coding regions. White arrowheads: 3′oligo(A). Bent arrows: transcription start sites.] The promoter of the central element (star at transcription start) directs transcription of the element on the right. Steps 1 through 4 show the resulting RNA transcript with a 5′ tag of sequence from the element supplying the promoter. This RNA is reverse transcribed onto a chromosome end, with some or all of the tag remaining on the 5′ end of the newly added element. When this new end is extended by reverse transcription of another RNA, the promoter on the new terminal element (star) directs transcription of an RNA with two tags, sequence from its own 3′ end plus the tag remaining on the element that is transcribed. The chain of tags can grow if this RNA transposes to continue the cycle; however all or part of the 5′-most sequence can also be lost. For simplicity only a minimum of tags are shown here.

Fig. 2.

Sequence at five junctions between head-to-tail D. virilis HeT-A elements, taken from a multiple alignment of tandem HeT-A elements with apparently complete 5′UTRs on the downstream element. This section surrounds the junctions (arrow) between the 3′ ends of upstream elements and the downstream 5′ ends of the neighboring elements. [The 3′ end of each upstream element is indicated by an oligo(A) sequence, the length of which is determined by the site at which reverse transcriptase initiated synthesis on the poly(A) tail of the transcription intermediate and is presumably much shorter than that poly(A) tail.] We have arbitrarily marked the junction on this alignment at the end of the shortest oligo(A) in the set. Nucleotides in lowercase bold immediately after this arrow are the only nucleotides not conserved in all five elements. For their origins, see Discussion. Bent arrows mark the two nucleotides where transcripts started in our experiments (Results). Boxed sequences next to these arrowheads are a match to Inr. Boxed sequences on right are a match to DPE. DPE sequence begins at position +28 nt from the A (+1) at the transcription start site. Sequence 1 was used for the constructs in this study.

Fig. 3.

Diagram of 3′ and 5′UTR sequences separating ORFs of tandem D. virilis HeT-A elements. The line linking the two ORFs summarizes results of a multiple alignment of full length elements, a small section of which is seen in Fig. 2. The 3′ and 5′UTRs make up approximately 3.8 kb of sequence but vary in length slightly from element to element. Thin lines denote regions where elements differ by multiple indels and base changes. The dark gray box on this line marks the region where all five elements are identical or nearly so. Vertical line marks the junction of the two elements (arrow in Fig. 2). As indicated above the line, the region of identity consists of the final 69 bp in the 3′UTR and 1.4 kb in the 5′UTR.

Fig. 4.

Relative promoter activity of sequences from D. virilis HeT-A elements tested in D. virilis cells. Diagram shows junction between two elements showing the 3′UTR of upstream element and the 5′UTR of downstream element. Arrow marks exact junction. Shown below are sequences tested, identified by nucleotide number at either end. Nucleotides are counted from the junction, ignoring the oligo(A). Negative numbers run into the 3′UTR and positive numbers run into the 5′UTR. Activity (±SD) is relative to a very active construct (−49 → +1731) which was set at 100% in each experiment to allow comparison between experiments. CaSpeR, empty expression vector.