Schematic organization of the dAdar gene from the transcriptional start sites to the end of exon 4. (a) Schematic representation of the dAdar gene encompassing the region from the two promoter to the end of exon 4. In the article, to avoid confusion, we refer to the promoter with capital letters (4A and 4B), whereas -4a and -4b are the mutually excluded exons. Exons are shown as boxes and introns as black lines. The black and gray arrowheads mark the position of the embryonic (4A) and adult (4B) transcriptional start sites. The black and gray dashed lines indicate the pattern of alternative splicing that arise from the mutually excluded alternative spliced exons -4a and -4b. The two other alternative spliced exon (-1 and 3a) are indicated by a black and gray box respectively. (b and c) Diagrams of the alternative splicing composition of the main spliced variants that arise from the embryonic and adult promoters.

dAdar expression during the oocyte development. In (a and b) RT–PCR of dAdar in oocytes undergoing maturation to unfertilized mature eggs is shown. The alternative spliced region exon 3/3a and transcripts originating from the 4A promoter were amplified. The gray arrows indicate the relative location of the primers used in the RT–PCR reactions. St.4-8 and St.10-11 are the oocytes stages from which total RNA was prepared and RT–PCR analysis was performed. Black arrowheads indicate the mRNA of the constitutively expressed egalitarian gene (Egl) which was used as an internal control. The stacked column charts below the gels show the relative percentage of the spliced isoforms quantified by optical densitometry. On the left site of each column chart, the color key indicates the different alternative spliced isoforms. The quantification was performed on three independent experiments and the average with SD is shown.

Regulation of the splicing of dAdar exon 3/3a by B52/SRp55. (a) Western blot analysis to determine the level of down regulation of endogenous B52/SRp55 protein after knockdown with dsRNA. NE indicates HeLa nuclear extract. The black arrows indicate the molecular weight of the SR proteins that are identified with the 1H4 monoclonal antibody. (b) RT–PCR analysis of dAdar after dsRNA knockdown of B52/SRp55 in S2 cells. M, is the 1 kb plus DNA marker and the symbol minus, indicates the negative RT–PCR control. (c) Western blot analysis of total protein extracts from female and male gonads during early development (days 1, 3 and 10) probed with 1H4 monoclonal antibody. NE indicates HeLa nuclear extract. The black arrows indicate the SR proteins that are recognized with the 1H4 monoclonal antibody.

RT-PCR splicing pattern and tagmata expression of dAdar exon 3/3a. (a) Schematic representation of the D. melanogaster Adar (CG12‱598) transcription unit in the region of alternative spliced exon 3/3a. Introns are shown as lines and exons as boxes. The alternative spliced exon 3a is represented as a gray box. The dinucleotides of the proximal and distal 5′ss are in bold and some of the intervening sequence of exon 3a is shown. (b) RT–PCR analysis of the alternative splicing pattern of dAdar exon 3/3a from heads, thorax, legs and abdomens of Oregon R males and females adult flies (M, F). The gray arrows show the relative location of the primers used in the RT–PCR reaction. The two black arrows indicate the alternative spliced products generated in the RT–PCR reaction, either including or excluding exon 3a. In the lower panel, a black arrowheads mark the position of rp49 that is used as an internal control. (c) Detection by RT–PCR of the splicing pattern of dAdar exon 3/3a expressed in testis and ovaries (lanes 1 and 3, respectively). Lanes 2 and 4 represent the controls obtained from the male fly bodies without testis and the female bodies without ovaries. Expected band size for the longer transcript that includes exon 3a was 346 bp and for the splice product excluding 3a was 235 bp. Lane M is the 1 Kb plus DNA marker. The stacked column charts below the gels show the relative percentage of amount of exon 3a/3 spliced isoforms quantified by optical densitometry. Gray columns indicate the exon 3a percentile, while white columns indicate the exon 3 percentile. Percentage was measured from three independent experiments; average with SD is shown.

Promoter choice of dAdar and analysis of alternative splice pattern in gonads. (a) In the upper panel, RT–PCR analysis of the adult -4b alternative splicing pattern that is expressed in the testis and ovary (lanes 1 and 2) and the corresponding controls (lanes 3 and 4) is shown. In the lower panel is the RT–PCR of the -4a alternative splicing pattern expressed in testis and ovary (lanes 5 and 6), and the corresponding controls (lanes 7 and 8). A diagram of the alternative spliced isoforms are shown on the left with their exon composition and the black and gray boxes indicate the alternative spliced exons -1 and 3a. Exon -1 is expressed in a tissue-specific manner and is found in the ovary. The splicing pattern of dAdar transcripts in testis and ovary (lanes 5–8) is the inverse to their controls (lanes 7 and 8). The gray arrows indicate the relative location of the primers used in the RT–PCR. The expected band size for the -4b-Ex4 transcripts is 615 bp and 575 bp, respectively. The expected band size for the -4a-Ex4 transcripts is 608 bp for the full length, 568 bp (excluding exon-1) 497bp (excluding exon 3a) and 457bp (excluding exon-1 and exon3a). (b) A time course performed by RT–PCR of the expression pattern of dAdar exon3/3a from male and female gonads. (c) A time course of the alternative splicing pattern of -4a exon from male and female gonads analyzed by RT–PCR. Black arrowheads indicate the position of the constitutively expressed rp49 rRNA used as an internal control in the lower panels of (a), (b) and (c). The stacked column charts below the gels show the relative percentage of the spliced isoforms that was quantified by optical densitometry. On the left of each column chart, a color key indicates the different alternative spliced isoforms. The quantification was performed on three independent experiments and the average with SD is shown.

Exon 3a binds the alternative splicing factor B52/SRp55. (a) Schematic representation of the dAdar region encompassing exon 3/3a showing a partial sequence of exon 3a that includes the B52/SRp55 binding site. (1) The RNA sequence found by SELEX as a high affinity binding site for B52/SRp55. (2) The RNA sequence of dAdar exon 3a used for the RNA affinity technique. This RNAs contains the conserved consensus motif for B52/SRp55 and this is shown in bold. (3) (c) Left panel, RNA-affinity purification of proteins bound to RNA encoding either wild type or mutated exon 3a. Right panel, Western blot analysis of the bound proteins with 1H4 monoclonal antibody. The black arrow indicates phosphorylated B52/SRp55 that is present in S2 nuclear extract. The Beads lane is the negative control. The protein gel in the left panel is stained with Coomassie Blue to visualize the pulled down RNA protein complexes and is a loading control for the experiment.