Olivier Ferrigno ¹, Thierry Virolle ¹, Zied Djabari ¹, Jean-Paul Ortonne ¹, Robert J. White ², and Daniel Aberdam ¹

¹ U385 INSERM, Faculté de Médecine, Nice, France.
² Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK.

Correspondence should be addressed to D Aberdam.
e-mail: aberdam@unice.fr

Short interspersed elements (SINEs) are highly abundant components of mammalian genomes that are propagated by retrotransposition. SINEs are recognized as a causal agent of human disease and must also have had a profound influence in shaping eukaryotic genomes [1]. The B2 SINE family constitutes approximately 0.7% of total mouse genomic DNA (ref. 2) and is also found at low abundance in humans [3]. It resembles the Alu family in several respects, such as its mechanism of propagation. B2 SINEs are derived from tRNA and are transcribed by RNA polymerase (pol) III to generate short transcripts that are not translated [4, 5]. We find here, however, that one B2 SINE also carries an active pol II promoter located outside the tRNA region. Indeed, a B2 element is responsible for the production of a mouse Lama3 transcript. The B2 pol II promoters can be bound and stimulated by the transcription factor USF (for upstream stimulatory factor), as shown by transient transfection experiments. Moreover, this pol II activity does not preclude the pol III transcription necessary for retrotransposition [6]. Dispersal of B2 SINEs by retrotransposition may therefore have provided numerous opportunities for creating regulated pol II transcription at novel genomic sites. This mechanism may have allowed the evolution of new transcription units and new genes.

One B2 member is found within an intron of the mouse gene Lama3, which encodes isoforms of the laminin a3-chain [7, 8]. The laminin a3C transcript is derived from the strand opposite to the tRNA-like transcript synthesized by RNA pol III (Fig. 1a; refs. 7, 8). This antisense transcription is carried out exclusively by pol II, as shown by its sensitivity to low doses of a-amanitin (Fig. 1b, lanes 1–3). Moreover, transcription from the same site can be carried out efficiently using a reconstituted pol II system (Fig. 1b, lanes 4–7). Although the B2 family evolved from a tRNA gene, a unique downstream region is also propagated as part of this SINE (refs. 3, 4). The size of the RNA produced by in vitro transcription (Fig. 1b) shows that the transcript initiates in the unique region of the B2 sequence. This assignment was verified in vivo by RT–PCR on poly(A)⁺ RNA extracted from NIH-3T3 cells and mouse lung using an alternative set of primers (Fig. 1c), but differs from our previous conclusion based on primer extension [7]. The discrepancy might be explained if the 5' end of the B2 RNA adopts secondary structure that is resistant to primer extension; use of alternative primers and the more sensitive RT–PCR approach allowed positive identification of the start site used in vivo.

Figure 1: Identification of a pol II promoter in a B2 SINE.

Figure 1: Identification of a pol II promoter in a B2 SINE.

a, The B2 SINE within mouse Lama3 is flanked by 20-bp direct repeats (hatched boxes) and contains pol III promoter elements (A- and B-blocks). The region of B2 sequence with homology to tRNA is indicated, as are the start site (+1), the TATA box of the pol II promoter and the putative ATG translational start site of the Lama3 transcript [7]. The asterisk represents the location of the 5' end of the a3C cDNA previously misassigned by primer extension [7]. Also shown are the orientations of the pol II and pol III transcripts and the restriction sites for AflIII (A), PvuII (P), DraI (D), BstY (BstY), BlpI (B) and EagI (E).

b, In vitro transcription assays using HeLa whole cell extract (lanes 1–3) or a reconstituted system containing purified pol II, TFIIA and TFIIH along with recombinant TBP, TFIIB and TFIIE (lanes 4–7). Templates were 50 ng (lane 1) or 100 ng (lanes 2, 3) of PCR-amplified fragment containing DraI-BlpI (DB), or 50 ng of linearized pGL2 plasmid containing the AflII-EagI (AE, lane 4), DraI-EagI (DE, lane 5), AflII-BlpI (AB, lane 6) or DraI-BlpI (DB, lane 7) restriction fragments. Reaction 3 contained a-amanitin. Run-off transcripts are shown. The precise start site was mapped by alignment with sequencing reactions.

c, RT–PCR assays using poly(A)⁺ RNA extracted from NIH-3T3 cells and mouse lung were performed with alternative nested primers to demonstrate that a Lama3 transcript initiates within the B2 element in vivo. The position and orientation of the primers are indicated by arrows under the AE sequence. Each primer (bold) is tested by PCR using AE construct as template. Amplification products are obtained using primers L3–L5, but not with primers L1 or L2; this places the in vivo start site between primers L2 and L3. No amplification is detected when the reverse transcriptase was omitted (not shown). The transcription start site mapped in (b) is boxed (+1).

d, The Lama3 B2 SINE supports expression of the luciferase gene in vivo. NIH-3T3 cells were transfected with luciferase reporter pGL2-basic driven by the different Lama3 B2 fragments, TK or VEGF gene minimal promoters. Values shown are given relative to the TK construct, which is assigned a value of 100.

Our evidence indicates that B2 SINEs have the potential to distribute a functional pol II promoter throughout the genome. During the course of evolution, this is likely to have resulted in many novel mRNA molecules, which would provide raw material for natural selection. A clear example of this phenomenon is provided by the Lama3 transcript [7]. Further examples may be provided by ESTs in the database, which begin at the pol II start site we have mapped in B2 genes (data not shown). In addition, several well-characterized pol II promoters show sequence homology with B2 genes over their core promoter regions (Fig. 5) and are therefore likely to be relics of a SINE insertion event. Although retrotransposition is often thought to be random, chromosomal hybridization has shown that SINEs tend to cluster around the R bands, where active genes are concentrated [16, 17]. This feature would have maximized the impact of their insertion. It is known that SINEs can influence expression of nearby genes [18-20], but we have presented the first evidence that these repeat elements can supply a pol II promoter. We propose that the B2 family has contributed in a novel way to evolution, by serving to distribute functional and regulatable pol II promoters.

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1. Allen TA, Von Kaenel S, Goodrich JA, and Kugel JF, "The SINE-encoded mouse B2 RNA represses mRNA transcription in response to heat shock", Nature Structural and Molecular Biology, Published online: 08 August 2004; | doi:10.1038/nsmb813

2. Espinoza CA, Allen TA, Hieb AR, Kugel JF, and Goodrich JA, "B2 RNA binds directly to RNA polymerase II to repress transcript synthesis",  Nature Structural and Molecular Biology, Published online: 08 August 2004; | doi:10.1038/nsmb812

3. Sorek R, Lev-Maor G, Reznick M, Dagan T, Belinky F, Graur D, and Ast G, "Minimal conditions for exonization of intronic sequences: 5' splice site formation in Alu exons", Mol. Cell vol. 14, no. 2, pp. 221-231 (April, 2004).

4. Otieno AC, Carter AB, Hedges DJ, Walker JA, Ray DA, Garber RK, Anders BA, Stoilova N, Laborde ME, Fowlkes JD, Huang CH, Perodeau B, and Batzer MA, "Analysis of the Human Alu Ya-lineage", J. Mol. Biol., vol. 342, no. 1: pp. 109-18, (September 3, 2004).

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