Structure and expression of mobile ETnII retroelements and their coding-competent MusD relatives in the mouse

Abstract

ETnII elements are mobile members of the repetitive early transposon family of mouse long terminal repeat (LTR) retroelements and have caused a number of mutations by inserting into genes. ETnII sequences lack retroviral genes, but the recent discovery of related MusD retroviral elements with regions similar to gag, pro, and pol suggests that MusD provides the proteins necessary for ETnII transposition in trans. For this study, we analyzed all ETnII elements in the draft sequence of the C57BL/6J genome and classified them into three subtypes (alpha, beta, and gamma) based on structural differences. We then used database searches and quantitative real-time PCR to determine the copy number and expression of ETnII and MusD elements in various mouse strains. In 7.5-day-old embryos of a mouse strain in which two mutations due to ETnII-beta insertions have been identified (SELH/Bc), we detected a three- to sixfold higher level of ETnII-beta and MusD transcripts than in control strains (C57BL/6J and LM/Bc). The increased ETnII transcription level can in part be attributed to a higher number of ETnII-beta elements, but 70% of the MusD transcripts appear to have been derived from one or a few MusD elements that are not detectable in C57BL/6J mice. This element belongs to a young MusD subgroup with intact open reading frames and identical LTRs, suggesting that the overexpressed element(s) in SELH/Bc mice might provide the proteins for the retrotransposition of ETnII and MusD elements. We also show that ETnII is expressed up to 30-fold more than MusD, which could explain why only ETnII, but not MusD, elements have been positively identified as new insertions.

FIG. 1.

Similarity of ETn and MusD LTRs. (a) Alignment of LTR sequences. 5′-LTR sequences of representative elements of ETnII (E2_AC074208, E2_Y17106, E2_9_76405783, and E2_AC099629), MusD (M_BK001485, M_AF246633, and M_17_92416751), and ETnI (E1_M16478, E1_9_85784030, and E1_2_22373000) are shown. ETnII and MusD LTRs are designated type 2 sequences, and ETnI LTRs are designated type 1 sequences. AC074208 is an ETnII element that has integrated into the wiz gene of B6 mice (1) and is located on chromosome 17 at position 31588150. E2_Y17106 was found as a new insertion in SELH mice (9). M_BK001485 is located on chromosome 2 of B6 mice and contains full-length open reading frames for gag, pro, and pol. This element is a member of the young MusD subfamily discussed in the text. M_AF246633 corresponds to the MusD2 sequence identified by Mager and Freeman (24). E1_M16478 represents the first ETnI element, which was found by Brûlet et al. (2) in BALB/c mice. A structural comparison of the full-length elements is shown in Fig. 2 for all four ETnII elements, M_BK001485, E1_M16478, and E1_9_85784030. Points indicate identity with the E2_AC074208 sequence, whereas dashes indicate gaps in the sequence. The inverted repeats (IR) and the locations of the purine-pyrimidine stretch (pu-py), putative TATA box, and poly(A) signal are marked as suggested by Brûlet et al. (2). Note that a MatInspector search (32) identified nt 178 to 195 of E2_AC074208 as a mammalian C-type LTR TATA box with a core similarity of 1.0 and a matrix similarity of 0.921. The locations of a 13- or 14-bp insertion, putative transcription factor binding sites, and the borders of U3, R, and U5 are marked. (b) Comparison of 5′ and 3′ LTR sequences. The identity of 5′ and 3′ LTRs of 37 MusD, 30 ETnII, and 26 ETnI elements was determined with the Diblast program on the National Center for Biotechnology Information server. The y axis shows the accumulated number of elements.

FIG. 2.

Schematic representation of ETnII elements. Thirty-five ETnII elements (for reference, see Table 3) were sorted into three different groups (α, β, and γ) according to structural similarities. Representatives of each group are shown, with their lengths (in base pairs) given on the right. Black bars, LTRs; gray bars, retroviral genes (gag, pro, pol); white bars, nonretroviral sequences of unknown origin; hatched bars, sequences specific to ETnI. To better illustrate the position of homologous regions, length differences are indicated by dotted lines. Some nucleotide positions are shown, with the numbers referring to the respective element. The identities and the overall number of elements in the B6 draft sequence showing the same structure are indicated on the left of the respective exemplary element. ETnII group α encompasses 9 elements, group β comprises 21 elements, and group γ comprises 5 elements. Four of the 17 group β elements which share an identical structure were isolated from non-B6 mice (indicated by “13x + 4”), and all other elements were from B6. In element 33, the 5′ LTR was replaced by an unknown sequence, and element 34 contains a MERVL sLTR. The locations of group α-, β-, and γ-specific primer pairs used for expression analysis are indicated by arrows.

FIG. 3.

Analysis of retroviral copy number by quantitative real-time PCR. The relative copy number of elements in the strains C57BL/6J (B6), LM/Bc, SELH/Bc, A/J, and CD-1 was determined by using primers specific for ETnII-α, ETnII-β, MusD, and IAP elements (see Materials and Methods). Note that due to different primer efficiencies, graphs can only be compared within a group.

FIG. 4.

Expression of retroviral elements in embryos and decidua. (a) Relative RNA levels of different elements in 7.5- and 9.5-day-old embryos and in surrounding tissue (decidua) are shown. RNA quantification was performed by real-time PCR. For this representative experiment, expression was determined in LM/Bc mice (E7.5, three samples; E9.5, two samples; decidua, one sample). Similar results were obtained for SELH/Bc mice (at least two samples each for E7.5, E9.5, and decidua; data not shown). Note that values are only comparable within a boxed group. (b) Comparison of ETnII and MusD expression in 7.5-day-old embryos of LM and SELH mice. The relative numbers of ETnII-α, ETnII-β, and MusD transcripts were determined by real-time PCR. Serial dilutions of plasmids containing the retroviral amplicons were used to construct standard curves (see Materials and Methods). n, number of mice tested.

FIG. 5.

Comparison of retroviral expression between 7.5-day-old embryos of different mouse strains. Relative quantification of ETnI, ETnII, MusD, and IAP expression in LM, SELH, B6, and CD-1 mice by real-time PCR is shown. n, number of mice tested; n.d., not determined. Note that values are only comparable within a boxed group.

FIG. 6.

Dendrogram of transcribed MusD sequences and elements from the B6 genome. Phylogeny for 1.2-kb MusD fragments (nt 680 to 1880) was deduced from SELH and LM transcripts (10 each) and from 30 randomly chosen MusD DNA sequences of B6 mice. Six young MusD elements found in the B6 genome were submitted to GenBank as distinct entries, and their names and accession numbers are printed in bold. Names of the remaining MusD sequences refer to their chromosomal location or to the GenBank accession number from which each element was identified. The branch in the young subgroup containing the seven SELH transcripts with a G-to-A transition at position 1103 is indicated. Distances were calculated using the Kimura two-parameter model with a transition/transversion ratio of 2. The length of the bar corresponds to a nucleotide sequence divergence of 0.2%.