RNAi Related Mechanisms Affect Both Transcriptional and Posttranscriptional Transgene Silencing in Drosophila  Manika Pal-Bhadra, Utpal Bhadra, James A Birchler  Molecular Cell  Volume 9, Issue 2, Pages 315-327 (February 2002) DOI: 10.1016/S1097-2765(02)00440-9

Figure 1 Silencing of Full-Length Adh Transgenes (A) Structure of the Adh transgene. The rosy sequence acts as a transformation marker. (B) Expected products from RNase protection assay using the Adh DP probe are shown. The transcript of Adh is shown at the top. The length and restriction limits of the DP probe are presented below. The normal transcript of the full-length Adh gene protects two fragments of 142 and 160 bp. The transcript of the Adhfn6 endogenous allele protects a 355 bp fragment. (C) RNase protection by RNA produced by the one to ten dosage series of adult females. Expected sizes of the protected fragments based on the splicing pattern are noted on the right, and the protected RNAs are noted on the left. A 70 bp fragment of β-tubulin RNA acts as a loading control. The copy number of transgenes for each lane is noted below. (D) Histogram of Northern analyses of one to ten copies of Adh transgenes using different insertion sites to achieve the same dosage. The values presented are the Adh/β-tubulin ratios from triplicate gels. The composition of each dose was derived by combining insertions on the X chromosome or on the 2L, 2R, 3L, and 3R chromosome arms as noted. The results demonstrate that stocks with the same copy number derived from different insertions have similar expression levels. Molecular Cell 2002 9, 315-327DOI: (10.1016/S1097-2765(02)00440-9)

Figure 2 Transcriptional Analysis (A) Nuclear run-on of Adh expression in adult female flies carrying one to ten copies of Adh transgenes. Each column is an individual hybridization blot. The labeled RNAs were hybridized to three different denatured DNAs from the Adh, β-tubulin (as an internal control), and the lac-Z (negative control) genes as indicated on the left. The copy number of the Adh genes is noted at the top. (B) Comparison of mRNA synthesis and steady-state levels. Quantification of Adh transcription is from the average of triplicate experiments. The mean ratios of run-on values (Adh/β-tubulin ± SD) in each genotype were plotted after deducing the amount of transcription of two Adh endogenous copies present in each case (dotted line). A similar graph was plotted using the Adh/β-tubulin (± SE) ratios which were derived from triplicate Northern gels using the same genotypes (from Pal-Bhadra et al., 1997). (C) Nuclear run-on assays using flies with zero to six copies of w-Adh. The copy number of the w-Adh transgene is noted at the top, and the sex from which the sample was derived is labeled at the bottom. Each row shows the hybridization signal from the genes indicated on the left. (D) A comparison of synthesis and steady-state Adh RNA determined from triplicate run-on and Northern analyses using flies with zero to six copies of w-Adh. Data were analyzed as described for (B). (E) Nuclear run-on analysis of Adh-w silencing by w-Adh transgenes. All flies carry a deficiency for the endogenous w gene. The dosage of the X chromosome-linked Adh-w transgene is noted. Similar expression in males and females with different copy number is a reflection of dosage compensation. The number of w-Adh transgenes is noted. Increasing dosage caused diminished transcription of Adh-w. Various doses of w-Adh were achieved using w-Adh2 on chromosome 2 and w-Adh3 on 3 (Pal Bhadra et al., 1999). (F) Graphical representation of the means (± SD) of triplicate run-on measurements of Adh-w transcription in the presence of zero, one, two, or four copies of w-Adh in males and females. The means (± SE) of triplicate measurements from Northern analysis data (from Pal Bhadra et al., 1999) of a single (and thus with sexually dimorphic expression) X-linked Adh-w with zero to four w-Adh transgenes in both males and females with increasing w-Adh dosage is presented for comparison. Molecular Cell 2002 9, 315-327DOI: (10.1016/S1097-2765(02)00440-9)

Figure 3 Formation of 21–25 Base-Pair RNAs during Silencing (A) Autoradiogram of a blot using low molecular weight RNA of adult females showing accumulation of 21–25 nt RNAs. RNA was extracted from flies carrying one to ten copies of full-length Adh transgenes. The small antisense (as) Adh RNAs were detected by hybridization with a 3′ Adh sense RNA. Probing with an antisense Adh RNA for small sense RNAs revealed a similar pattern (not shown). Base-pair size markers are shown at the left. (B) Autoradiogram of a blot probed for 21–25 nt Adh RNAs using low molecular weight RNA from one to six w-Adh copies. The dosage and sex from which each sample was derived are noted. Molecular Cell 2002 9, 315-327DOI: (10.1016/S1097-2765(02)00440-9)

Figure 4 Threshold Induced Silencing of Adh Transgenes (A) Structure of the hsp70 promoter-Adh reporter construct. The arrow on the top indicates the transcriptional orientation. The arrow at the bottom indicates the hsp70 promoter from +198 to −68 bp. (B) Autoradiograms of Northern blot analysis of Adh mRNA. The copy number of each transgene is noted. The transcripts of each male genotype were examined in three different circumstances: no heat, heat shock (37°C for 45 min), and 20 hr after heat shock. The relative amount of Adh transcripts (Adh/β-tubulin ratios ± SE) from triplicate measurements is presented in the bar diagrams. β-tubulin acts as a gel loading control. All stocks are homozygous for the Adhfn6 endogenous allele. The heat-shocked sample of hsp70-Adh plus four Adh transgenes shows a significant reduction of Adh RNA. (C) The presence of 21–25 nt RNAs was examined in the pulsed threshold silencing conditions by hybridizing with a sense Adh probe as described in Figure 3. NH, no heat shock; H, heat shock. Transgene dosage is noted at the bottom. Small RNAs are only present in the class containing four Adh transgenes plus the hsp70-Adh construct following heat shock. Molecular Cell 2002 9, 315-327DOI: (10.1016/S1097-2765(02)00440-9)

Figure 5 Effect of piwi on Posttranscriptional Silencing (A) Autoradiograms of Northern analysis showing the effect of representative piwi (piwi1/piwi2) alleles on threshold-induced Adh silencing are presented. The level of Adh transcripts in adult male flies that were heterozygous with normal or homozygous (heteroallelic) for piwi was examined. Each of these classes possesses one copy of hsp70-Adh and either two or four copies of Adh genes. The resulting four types of flies were examined in two different circumstances, untreated and heat shocked. The copy number of each construct or allele is noted. The mean ratios of Adh/β-tubulin ± SE from triplicate measurements is presented in the bar diagrams. β-tubulin acts as a gel loading control. (B) Segregating genotypes for piwi were examined for the presence of 21–25 bp RNAs as described in Figure 3. The silencing class with four Adh genes and an induced hsp70-Adh has a high accumulation when piwi is heterozygous. In the same class, but with piwi homozygous, the formation of the small RNAs is diminished. Base-pair size markers are shown at the left. Molecular Cell 2002 9, 315-327DOI: (10.1016/S1097-2765(02)00440-9)

Figure 6 Effect of piwi on Transcriptional Silencing (A) Eye color of Adh-w/Y flies carrying one copy of the w-Adh transgene that are heterozygous with normal or heteroallelic mutant for piwi. All flies have a w− background. The mutations and transgene copy number are noted. (B) Northern and transcriptional run-on analyses of piwi. At the top is a Northern transfer showing the effect of heteroallelic piwi (piwi1/piwi2) on white transcripts of the Adh-w transgene in the presence of zero to two copies of the reciprocal w-Adh construct in males. To determine the effect of piwi on w-Adh/Adh silencing, each Northen blot was also probed for Adh. The copy number of each construct or allele is noted below. The relative amount of white and Adh RNA (white/β-tubulin ± SE and Adh/β-tubulin ± SE) from triplicate measurements of each male genotype is represented by the bar diagrams. Run-on transcriptional analysis of the same genotypes is shown below. A graphical representation of the means (± SD) of triplicate experiments illustrates the relief of silencing in the heteroallelic piwi1/piwi2 mutant flies. Molecular Cell 2002 9, 315-327DOI: (10.1016/S1097-2765(02)00440-9)

Figure 7 Effect of piwi on Transcriptional Silencing Histograms are presented showing the relative effect of piwi homozygous mutants (mut/mut) versus heterozygotes (mut/+) on Adh-w alone, Adh-w plus one w-Adh, and Adh-w plus two w-Adh transgenes (for piwi2 and piwi1/piwi2 only). Values are mean ratios ± SE of three different assays. The amount of radioactive label for each RNA on Northern blots was measured using a Fuji Bas 2000 phosphorimager. All values were corrected for loading differences by rehybridizing each blot with the β-tubulin probe. The mutant homozygotes were compared to the heterozygotes in a ratio to determine the influence of the homozygous mutations on silencing. Ratios greater than the nonsilenced control (Adh-w) indicate the degree to which silencing is diminished. The values marked with an asterisk are significantly different from the Adh-w genotype (P < 0.05). Molecular Cell 2002 9, 315-327DOI: (10.1016/S1097-2765(02)00440-9)