Transcription of endogenous and exogenous R2 elements in the rRNA gene locus of Drosophila melanogaster

Abstract

R2 retrotransposons insert into the rRNA-encoding units (rDNA units) that form the nucleoli of insects. We have utilized an R2 integration system in Drosophila melanogaster to study transcription of foreign sequences integrated into the R2 target site of the 28S rRNA genes. The exogenous sequences were cotranscribed at dramatically different levels which closely paralleled the level of transcription of the endogenous R1 and R2 elements. Transcription levels were inversely correlated with the number of uninserted rDNA units, variation in this number having been brought about by the R2 integration system itself. Females with as few as 20 uninserted rDNA units per X chromosome had expression levels of endogenous and exogenous insertion sequences that were 2 orders of magnitude higher than lines that contained over 80 uninserted rDNA units per chromosome. R2 insertions only 167 bp in length exhibited this range of transcriptional regulation. Analysis of transcript levels in males suggested R2 insertions on the Y chromosome are not down-regulated to the same extent as insertions on the X chromosome. These results suggest that transcription of the rDNA units can be tightly regulated, but this regulation gradually breaks down as the cell approaches the minimum number of uninserted genes needed for survival.

FIG. 1.

D. melanogaster lines containing B. mori sequences within their rDNA arrays. (A) Diagram of the D. melanogaster rDNA unit, with the rRNA genes represented by diagonally hatched boxes. Gene sequences upstream of the R2 site have diagonal hatching in the opposite orientation from sequences downstream of the R2 site. Also shown is an enlarged area spanning the R1Dm and R2Dm insertion sites. Distances in base pairs are relative to the R2 insertion site. (B) Diagrams of eight insertion lines generated by injection of RNA containing the R2Bm 3′ UTR and additional upstream sequences. Dark gray boxes correspond to various lengths of either the R2Bm 5′ UTR, R2Bm ORF, or GFP ORF (see Materials and Methods). If the insertion gave rise to more than a 5-bp deletion of 28S sequences at the 5′ junction, then the length of the deletion is indicated above the junction. A 150-bp R1Dm insertion (black box) is located downstream of the insertion in SWIFF 4. SWIFF 1 and SWIFF 2 were previously referred to as HR4-1 and 10R21, respectively (12). (C) Diagrams of the inserted 28S units in six lines generated by injecting RNA containing the 250-nt R2Bm 3′ UTR and 170 bp of upstream B. mori 28S sequences. The 170-bp B. mori 28S sequences were inserted in most cases resulting in tandem duplications (gray hatched boxes). An asterisk indicates that the entire upstream 170 bp was not duplicated: 23 bp of 28S sequences were duplicated in SWIFF 13, and no duplication occurred in SWIFF 14. A 500-bp R1Dm element is downstream of the SWIFF 11 insertion. Many of the insertions generated with this RNA had deletions of the R2Bm 3′ UTR sequences or downstream 28S sequences at the 3′ junction of the insertion. (D) Diagram of the inserted 28S unit generated by injecting an R2Bm transcript containing 170 bp of upstream and 350 bp of downstream D. melanogaster 28S gene sequences. Integration of this insert involved recombination events that regenerated the region of the 28S gene containing the R1 and R2 sites (see text). The chromosomal location of each insertion on either the X or Y chromosome is shown to the right of each diagram.

FIG. 2.

Transcription levels of the R2Bm insertions in each SWIFF line. (A) Total RNA from adult females or adult males (as indicated) was subjected to RNase protection. A labeled, antisense probe corresponding to the 250-bp 3′ UTR of R2Bm was hybridized to the RNA, the mixture was digested with RNase T1 and RNase A, and the resistant RNA was run on a 5% polyacrylamide denaturing gel. Numbers above the lanes indicate the SWIFF line used. Due to deletions at the 3′ end of the insertion, the protected fragment for SWIFF 11 is 240 nt and that for SWIFF 12 is 134 nt. The latter is indicated by dots in the lower region of the gel. Locations of single-stranded DNA size standards are indicated to the right. (B) Comparison of transcript levels in males and females for three R2Bm insertions on the X chromosome (SWIFF 6, 11, and 9) and one insertion on the Y chromosome (SWIFF 7).

FIG. 3.

Transcription from endogenous R2Dm elements in the SWIFF lines. (A) Schematic representation of the R2Dm element with the 5′ UTR, ORF, and 3′ UTR indicated. Vertical lines represent the extent to which various R2Dm elements in the w1118 line are 5′ truncated. Horizontal lines represent the 32 full-length and 20 5′-truncated R2 copies in w1118. The positions within the element of the R2Dm antisense RNA probes used in the RNase protection assays are shown at the bottom of the diagram. (B) RNase-protected fragment using either antisense probe 1 (left panel) or antisense probe 2 (right panel). RNA was isolated from adult males (SWIFF 7), adult females (SWIFF 1 and 11), and 0- to 6-h embryos (w1118). The panel on the left was exposed 10 times longer than that on the right. (C) RNase-protected fragments from adult females and males of each line, determined by using antisense probe 3. The upper band represents the level of transcripts from all insertions greater than 240 bp. The lower band represents the level of transcripts arising from the smallest truncated element, a 167-bp insertion on the X chromosome. (D) The RNase-protected fragments from adult females generated using antisense probe 4. The upper band corresponds to the level of transcripts derived from all insertions greater than 600 bp in length. The lower band corresponds to the level of transcripts derived from the second-shortest R2Dm element, a 516-bp insertion on the X chromosome. Numbers above the lanes indicate the SWIFF line used, and w indicates the w1118#12 line. Size standards are indicated to the right of each gel.

FIG. 4.

Transcription from endogenous R1Dm elements in the SWIFF lines. Total RNA from adult females of each line was subjected to RNase protection by using a probe from the extreme end of the 3′ UTR of the R1Dm element (see Materials and Methods). All R1Dm elements in these lines are greater than 240 bp in length; thus, the protected bands represent the level of transcripts derived from all R1 insertions. Size standards are indicated to the right of the gel.

FIG. 5.

Stability of endogenous transcript levels over many generations. (A) The relative level of transcripts corresponding to the 167-bp truncated element was examined in both males and females for five stocks of the w1118 line separately maintained as small populations for 18 months. RNase protection assays were carried out with probe 2 (Fig. 3A). (B) The relative level of the 167-bp transcript level was monitored in two of the SWIFF lines over an 18-month period. Total RNA was isolated from adult females at the indicated generation, and RNase protection assays were performed as for panel A.

FIG. 6.

Relationship in females between the transcript levels of various insertions and the number of uninserted units in the X rDNA locus. (A) Schematic diagrams of the number of each type of rDNA unit present in w1118#12 and the various SWIFF lines. The length of each box represents the number of units, based on the scale shown. Unshaded boxes represent uninserted units, hatched boxes represent R1 inserted units, gray boxes represent R2 inserted units, and hatched gray boxes represent doubly inserted units. (B) Plot of the number of uninserted rDNA units per X chromosome (shown in panel A) versus relative levels of transcripts from various R1Dm and R2Dm insertions (Table 2). The transcript level in w1118#12 was defined as 1.0 for each assay. Symbols used to indicate transcript levels for the 167-bp R2Dm insertion, the 516-bp R2Dm insertion, and the 3′ UTR of all R1Dm elements are indicated on the figure.

FIG. 7.

Transcript levels in heterozygous females. (A) Males from SWIFF 15 and w1118#12 were mated to females from SWIFF 6. Total RNA from the adult female progeny from these crosses as well as from the parental females were subjected to RNase protection assays using probe 2 (Fig. 3A) to monitor transcription of the 167-bp R2Dm insertion. Lane a, SWIFF 6/SWIFF 6; lane b, SWIFF 6/SWIFF 15; lane c, SWIFF 15/SWIFF 15; lane d, w1118#12/w1118#12; lane e, SWIFF 6/w1118#12; lane f, SWIFF 6/SWIFF 6. (B) Schematic diagrams of the number of uninserted units in the parental and heterozygous females for the two crosses assayed in panel A. Lanes a to f refer to the lanes in panel A, and the numbers at right correspond to the relative level of transcripts in each lane, with the transcript level in w1118 (lane d) defined as 1.0.