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. 2004 Jul 22;32(13):3846-55.
doi: 10.1093/nar/gkh698. Print 2004.

A YY1-binding site is required for accurate human LINE-1 transcription initiation

Jyoti N Athanikar  1 , Richard M Badge, John V Moran
Affiliations

A YY1-binding site is required for accurate human LINE-1 transcription initiation

Jyoti N Athanikar et al. Nucleic Acids Res. .

Abstract

The initial step in Long Interspersed Element-1 (LINE-1) retrotransposition requires transcription from an internal promoter located within its 5'-untranslated region (5'-UTR). Previous studies have identified a YY1 (Yin Yang 1)-binding site as an important sequence in LINE-1 transcription. Here, we demonstrate that mutations in the YY1-binding site have only minor effects on transcription activation of the full-length 5'-UTR and LINE-1 mobility in a single round cultured cell retrotransposition assay. Instead, these mutations disrupt proper initiation of transcription from the +1 site of the 5'-UTR. Thus, we propose that the YY1-binding site functions as a component of the LINE-1 core promoter to direct accurate transcription initiation. Indeed, this sequence may explain the evolutionary success of LINE-1 by enabling full-length retrotransposed copies to undergo autonomous retrotransposition in subsequent generations.

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Figures

Figure 1
Figure 1
An overview of the LINE-1 retrotransposition assay. (A) Rationale of the assay: depicted is a schematic diagram of a full-length retrotransposition-competent L1. ORF1 is indicated by the yellow rectangle. ORF2 is indicated by the blue rectangle. The 5′-UTR and 3′-UTR are indicated by the gray rectangles. A SV40 poly(A) signal present at the 3′ end of the LINE-1 is indicated by the (A)n. The relative position of the YY1-binding site on the antisense strand of the 5′-UTR is indicated, as are the sequences of the YY1-scramble (YY1-s) and YY1-forward (YY1-f) mutants. The mneoI retrotransposition indicator cassette was inserted into the 3′-UTR of wild-type and mutant L1 constructs (15,35). The cassette consists of a backward copy of the neomycin phosphotransferase gene (pink rectangle) and is interrupted by an intron (IVS-2 from the γ-globin gene), which is in the same transcriptional orientation as the L1. SD and SA indicate the splice donor and splice acceptor sites. The cassette also is equipped with its own promoter (P′) and polyadenylation signal (A′). This arrangement ensures that a functional NEO transcript will only be translated following LINE-1 retrotransposition (15). The putative structure of a resultant retrotransposition event that confers G418-resistance (G418R) to HeLa cells is indicated at the bottom of the figure. The horizontal arrows indicate TSDs that are generated upon LINE-1 retrotransposition. (B) The results of the retrotransposition assay: retrotransposition was assayed in HeLa cells using the transient retrotransposition assay (31). Approximately 2 × 105 HeLa cells/well were transfected with JM101/L1.3 (WT), 1–910 EagI, YY1-s or YY1-f. No promoter indicates cells transfected with a LINE-1 construct that lacks a promoter (ΔΔJM101/L1.3) (30).
Figure 2
Figure 2
Effect of the YY1-s mutation on LINE-1 transcriptional initiation. A schematic diagram of the 1–910 EagI wild-type expression construct is indicated at the top of the figure. The YY1-s construct is identical in structure, but harbors the YY1-scramble mutation in the LINE-1 5′-UTR. RPA probes were transcribed from PCR fragments amplified from either the wild-type or YY1-s mutant luciferase constructs; the size of the probe and the predicted size of the expected protected fragment is indicated below the schematic diagram. RPA was performed with total RNAs collected from HeLa, N-Tera 2D1 (N-Tera) and PA-1 cells transfected with either a wild-type (1–910 EagI) or YY1-s (1–910-YY1-s) mutant luciferase-based expression constructs. Experiments in the left-hand panel were conducted with a RPA probe derived from the 1–910 EagI wild-type construct. Experiments in the right-hand panel were conducted with a RPA probe derived from the YY1-s mutant construct. Neg. indicates a RNA sample derived from untransfected HeLa, N-Tera 2D1 or PA-1 cells, respectively; they serve as negative controls in the experiment. The RPA probe also was incubated with yeast RNA in the presence (+) or absence (−) of RNase to visualize the full-length probe and possible probe breakdown products. The center lane (M) indicates a 100 base RNA molecular weight marker (Ambion). The arrow at the left-hand side of the figure indicates the major protected product detected with the wild-type RPA probe. The vertical red bar in the right-hand side of the figure indicates the expected placement of products initiating at or near the first base of the 5′-UTR in the YY1-s experiment. We also detect a less intense band in the Neg. lanes, which probably represents protected RNAs derived from endogenously transcribed LINE-1 elements.
Figure 3
Figure 3
Mapping of the major protected RPA product. RPA was performed as indicated in Figure 2 with total RNAs collected from HeLa cells transfected with either a wild-type luciferase-based expression construct or a mutant luciferase-based expression construct lacking the first five guanosine residues of the LINE-1 5′-UTR (Δ5G). Experiments in the left-hand panel were conducted with a RPA probe derived from the 1–910 EagI wild-type construct. Experiments in the right-hand panel were conducted with a RPA probe derived from the Δ5G mutant. Neg. indicates a RNA sample derived from untransfected HeLa cells and serves as a negative control in the experiment. The center lane (M) indicates a 100 base RNA molecular weight marker (Ambion). The arrow at the left-hand side of the figure indicates the major protected product detected using the wild-type RPA probe. The vertical red bar at the right-hand side of the figure indicates the expected placement of products initiating at or near the first base of the 5′-UTR in the Δ5G mutant construct.
Figure 4
Figure 4
5′-RACE analyses of wild-type and mutant luciferase-based expression constructs. 5′-RACE analysis was performed using poly(A) selected RNA from HeLa cells transfected with either wild-type or mutant luciferase-based expression constructs. The single major product was cloned into the pGEM-T-easy vector (Promega) and was sequenced. The green and blue arrows indicate 5′-RACE products characterized from WT and 1–910 EagI constructs. The gold arrows indicate 5′-RACE products characterized from the Δ5G construct. The blue and light blue arrows indicate 5′-RACE products characterized from the YY1-s and YY1-f constructs, respectively. For the 1–910 EagI and YY1-s constructs, the 5′-RACE analysis was performed twice on two separate RNA samples. The open and filled arrowheads indicate the results from those experiments. The bases outlined in green at positions −22 and −20 are present in the wild-type construct lacking the EagI site (WT).
Figure 5
Figure 5
Analysis of L1 5′-termini distribution and sequence conservation. An annotated non-redundant database of full-length human-specific L1s was used to identify 219 sequences with clearly defined TSDs (32). These elements and their TSDs were aligned and the distance from the base adjacent to the 3′ base of the 5′-TSD to the 3′ end of the YY1 NDC sequence measured. This distribution is plotted against nucleotide position (blue line). For each position of the alignment nucleotide conservation was calculated, and weighted by the number of sequences at that position (red line). Below the plot is aligned the 90% consensus sequence derived from the alignment, and the L1PA1 consensus sequence. The YY1 NDC sequence is underlined.
Figure 6
Figure 6
A model for how the YY1-binding site functions in LINE-1 retrotransposition. The model depicts retrotransposition events arising from a progenitor, full-length LINE-1 element that contains either a wild-type (left panel) or a mutant (right panel) YY1-binding site. In the wild-type scenario, a full-length LINE-1 element containing an intact 5′-UTR predominantly initiates transcription from the +1 site of the 5′-UTR allowing the internal promoter to be regenerated upon retrotransposition. The progenitor as well as the full-length retrotransposed product can then undergo autonomous retrotransposition in subsequent generations. By comparison, LINE-1 elements lacking a functional YY1-binding site cannot initiate transcription at the +1 position of the 5′-UTR and instead may initiate transcription from various positions within the 5′-UTR (represented by the numerous arrows). Thus, the resultant progeny will become progressively shorter, leading to the eventual formation of an element that cannot undergo autonomous retrotransposition. The red question marks (?) in the right-hand panel indicate uncertainties about whether the progeny LINE-1 element will be transcribed or whether it will remain retrotransposition-competent. It is likely that genomic sequences flanking the progenitor and progeny LINE-1 elements will influence their expression and ability to retrotranspose in vivo.
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