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FIGURE 1. Transgenic WDR79 protein behaves like endogenous WDR79 in Drosophila, and it rescues the WDR79-null phenotype. (A) Diagram of the two major WDR79 constructs used in this study. In each case, the annotated WDR79 transcript is shown in gray. Both constructs include an RFP sequence, one at the carboxyl terminus, the other at the amino terminus. Each includes an upstream UASp sequence for expression under control of GAL4. Both constructs express without GAL4 induction from an endogenous promoter within the cloned WDR79 sequence, although expression is enhanced by GAL4. (B) Endogenous WDR79 (green), detected with an antibody against the protein itself, colocalizes with coilin (red), producing a yellow Cajal body (CB) in the nucleus of a Malpighian tubule cell from a y w fly (arrow). (C) WDR79-RFP protein (green) expressed without GAL4 induction, detected with an antibody against RFP, also colocalizes with coilin (red) in the CB (arrow). (D) Ovarian follicle cells from WDR79-RFP/daGAL4 flies, stained for RFP (red) and coilin (green). Because of UAS-GAL4 variegation, two types of expression patterns are observed in the same tissue. In some cells, WDR79-RFP is overexpressed from the UAS promoter and accumulates in the cytoplasm; in most of these cells, nuclear CBs are not detectable. The remaining cells express WDR79-RFP at a lower level from the endogenous promoter. In these cells, WDR79-RFP accumulates with coilin in the CB in the nucleus. Viability of the cells seems to be unaffected by overexpression of WDR79-RFP. (E–G) Both WDR79-RFP and RFP-WDR79 transgenes can rescue the formation of CBs in WDR79-null flies. Ovarian follicle cells from WDR79-null flies (E) or from WDR79-null flies that express WDR79 from transgenes (F,G), stained for coilin (red) and Lsm11 (green). None of these flies carried a GAL4 driver, so expression is from an endogenous WDR79 promoter. The transgene in F is WDR79-RFP, whose transcript includes the RFP tag; that in G is RFP-WDR79, whose transcript begins downstream from the tag. Cells from WDR79-null flies lack CBs (E). Hence, their nuclei display histone locus bodies (green Lsm11 foci), but CBs are undetectable (no red coilin foci). On the other hand, cells that express either WDR79-RFP (F) or WDR79 alone (G) may have CBs (red coilin foci) in addition to histone locus bodies (green Lsm11 foci). (H) Northern blot analysis of WDR79 mRNA expressed in y w (control) and WDR79MB10832 (mutant) flies. mRNA is expressed from the endogenous gene in y w flies and from transgenes in the mutant background. WDR79 mRNA is undetectable in RNA extracted from WDR79MB10832 mutant flies, suggesting that this is a null mutation.
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FIGURE 2. (A) Gene models of Drosophila WDR79. When we started this project the gene model for WDR79 was based on cDNA clone BT012509, annotated as a full-length mRNA sequence (WDR79-RA). The current gene model is based on RNA-seq analysis (WDR79-RB). Multiple transcription starts that we determined by 5′-RACE are in a good agreement with the current gene model. At the same time, a transcription start corresponding to the earlier annotation was also detected in RNA isolated from males. (Short arrows) Oligonucleotide primers used for RT-PCR analysis to detect different isoforms of WDR79 mRNA. (B) RT-PCR of WDR79 mRNA (using primers shown in A) reveals different isoforms in male and female tissues.
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FIGURE 3. WDR79 transgenic protein interacts with scaRNAs in Drosophila. (A) RT-PCR analysis to demonstrate the specific association of scaRNAs with WDR79 protein. An antibody against RFP was used to immunoprecipitate RNA–protein complexes from flies that expressed RFP-labeled WDR79. The first four columns show RT-PCR analysis for the input extracts, the second four columns for the immunoprecipitate, and the final four columns for the supernatant after immunoprecipitation. The first six rows show that two previously known scaRNAs (U85 and mgU4-65) as well as four previously unknown scaRNA are detectable in the immunoprecipitate. snoRNAs and U4 snRNA are abundant in the input extract but are not found in the immunoprecipitate. None of the tested RNAs are immunoprecipitated from extracts of flies that express RFP alone (columns labeled mRFP). (B) All seven new scaRNAs coprecipitated with WDR79 are detectable by Northern blot analysis in total RNA extracted from wild-type flies (first lane) and from flies that are null for WDR79 protein (second lane). These scaRNAs express at similar levels in mutant and wild-type flies. (C) All seven new scaRNAs are detectable by in situ hybridization in nuclei of Malpighian tubule cells, where they colocalize with U85, a well-established marker for the CB (arrows). The localization of pugU6-40 in the CB (second and third panels in the bottom row) contrasts sharply with the localization of mgU6-47 in the nucleolus (last two panels in the bottom row). Note that the CB is green (U85 signal) in the mgU6-47 panel, whereas it is yellow in the other panels (U85 + new scaRNA).
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FIGURE 4. Postulated base-pairing between novel Drosophila scaRNAs and their substrates: Drosophila spliceosomal snRNAs U1, U2, U5, U6, yeast U2, and two regions in Drosophila 28S rRNA.
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FIGURE 5. Drosophila scaRNAs in RNA modification assays based on Xenopus oocytes. (A) Pseudouridylation of Drosophila U1 snRNA mediated by pugU1-6 in RNA-depleted GV extract. When in vitro–transcribed U1 snRNA was added to the extract along with guide RNA pugU1-6 (blue trace), a prominent peak corresponding to pseudouridine at position 6 was detected compared with the control reaction in which only U1 snRNA was added (black trace). (B) Pseudouridylation of Drosophila U2 snRNA mediated by pugU2-38/40/42 in RNA-depleted GV extract. Addition of the guide RNA and in vitro–transcribed U2 snRNA (red trace) produced a prominent peak corresponding to pseudouridine at position 42, a moderately increased peak at position 38, but no appreciable change at other positions. The black trace shows a control reaction without the guide RNA. (C) Pseudouridylation of Drosophila U2 snRNA injected into Xenopus oocytes. When in vitro–transcribed Drosophila U2 was injected alone as a control, pseudouridines at positions 35, 38, 40, 42, 44, and 45 were clearly detected (black trace). When Xenopus pugU2-34/44 guide RNA was depleted by injecting antisense oligonucleotide αG2, pseudouridylation at positions 44 and 45 was dramatically reduced (red trace). When pre-depleted oocytes were supplemented with Drosophila guide RNA pugU2-35/45, pseudouridylation was restored at positions 44 and 45 (blue trace). In panels A–C the colored dots denote the positions of A (black), C (red), G (blue), and U (green) determined from control sequencing reactions on in vitro–transcribed RNA.
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FIGURE 6.
Modification of U2 snRNA mediated by Drosophila scaRNAs in the yeast S. cerevisiae. (A) Endogenous yeast U2 snRNA is normally pseudouridylated at positions 35, 42, and 44 (RNA from reference strain BY4741). Mutant strains that are deficient for Pus1p, Pus7p, or guide RNA snR81 show the absence of pseudouridylation at position 44 (pus1Δ), position 35 (pus7Δ), or position 42 (snr81Δ). When Drosophila guide RNA pugU2-35/45 is introduced into the pus7Δ strain (pus7Δ +pugU2-35/45), it rescues pseudouridylation at position 35 (star) and additionally induces modification at position 45 (star). The pseudouridylation at position 45 is much more prominent when pugU2-35/45 is expressed in the pus1Δ strain (pus1Δ +pugU2-35/45). Similarly, expression of Drosophila pugU2-38/40/42 in the snr81Δ strain (snr81Δ +pugU2-38/40/42) can rescue pseudouridylation at position 42 (star), and additionally positions 38 and 40 (stars) become modified. Pseudouridylation at position 40 is weak in wild type, pus1Δ (data not shown) and snr81Δ strains but appears more prominent when pugU2-38/40/42 is expressed in the pus7Δ strain (pus7Δ +pugU2-38/40/42). Control primer extension reactions without CMC treatment were run on all RNA samples. They showed no stop signals, as observed for RNA from the BY4741 strain. (B) Only three pseudouridines are detectable in U2 snRNA from BY4741 yeast cells that express either Drosophila pugU2-55 guide RNA (BY4741 + pugU2-55, red trace) or its substrate chimeric yeast–Drosophila U2 (ydU2) snRNA alone (BY4741 + ydU2, black trace). However, an extra peak (star) corresponding to pseudouridine at position 55 in Drosophila U2 snRNA appears when both pugU2-55 guide RNA and its substrate are coexpressed in the same yeast strain (BY4741 + ydU2 + pugU2-55, blue trace). (C) Expression of Drosophila mgU2-48 guide RNA in yeast cells induces 2′-O-methylation at position 48 in coexpressed chimeric yeast–Drosophila U2 snRNA (star, top green trace). Extra stop signals of unknown origin (question mark) were observed in this trace, where the primer extension reaction was performed at a low concentration (LC) of dNTP. No stop signals were produced in control reactions run at high concentration (HC) of dNTPs with the same RNA sample (black trace) or at low concentration with RNA extracted from yeast cells that express only one of the two exogenous RNAs: either mgU2-48 (blue trace) or chimeric yeast–Drosophila U2 snRNA (red trace). Alignment with U2 snRNA sequences is shown with colored dots as in Figure 5 (yeast U2 snRNA at the top of the figure and chimeric yeast–Drosophila U2 snRNA at the bottom).
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FIGURE 7. Modification of artificial substrates mediated by Drosophila scaRNAs in yeast. Artificial RNAs were constructed by inserting the predicted targets for Drosophila scaRNAs into human U87 scaRNA. These were expressed in wild-type yeast cells either alone (blue traces) or along with their corresponding guide RNAs (red traces). When RNA from these transformed yeast strains was tested in primer extension reactions, pseudouridines at the expected positions were detected (Ψ40 in U6, Ψ1960 and Ψ2838 in 28S rRNA). Surprisingly, an additional strong signal was observed at Ψ2842 (star) with pugU1-6. The base-pairing between pugU1-6 and the artificial 28S substrate required for this modification is shown in Figure 4. Sequences are color-coded with dots as in Figure 5. The inserted target sequences from U6 snRNA and 28S rRNA are underlined.
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