The DEAD-Box Protein Rok1 Coordinates Ribosomal RNA Processing in Association with Rrp5 in Drosophila

Abstract

Ribosome biogenesis and processing involve the coordinated action of many components. The DEAD-box RNA helicase (Rok1) is essential for cell viability, and the depletion of Rok1 inhibits pre-rRNA processing. Previous research on Rok1 and its cofactor Rrp5 has been performed primarily in yeast. Few functional studies have been performed in complex multicellular eukaryotes. Here, we used a combination of genetics and developmental experiments to show that Rok1 and Rrp5, which localize to the nucleolus, play key roles in the pre-rRNA processing and ribosome assembly in D. melanogaster. The accumulation of pre-rRNAs caused by Rok1 depletion can result in developmental defects. The loss of Rok1 enlarged the nucleolus and led to stalled ribosome assembly and pre-rRNA processing in the nucleolus, thereby blocking rRNA maturation and exacerbating the inhibition of mitosis in the brain. We also discovered that rrp5^4-2/4-2 displayed significantly increased ITS1 signaling by fluorescence in situ hybridization, and a reduction in ITS2. Rrp5 signal was highly enriched in the core of the nucleolus in the rok1^167/167 mutant, suggesting that Rok1 is required for the accurate cellular localization of Rrp5 in the nucleolus. We have thus uncovered functions of Rok1 that reveal important implications for ribosome processing in eukaryotes.

1. Introduction

Ribosomes are ubiquitously found within organisms, and their primary role within cells is the catalysis of protein synthesis [1]. Proteins are essential in all organisms, as they carry out most of the cell’s essential functions. Therefore, ribosomal function is essential for life. Ribosome-catalyzed protein synthesis requires large amounts of energy and involves very complex processes [1,2,3,4]. Ribosomal RNA synthesis and processing are accompanied by ribosomal protein assembly. Ribosomal protein assembly involves more than 200 proteins. Most of the non-ribosomal proteins involved in this process belong to a variety of energy-consuming enzyme families [3], including the ATPase, RNA helicase, GTPase, kinase, and nuclease families [5]. While pre-rRNA is being transcribed, a variety of assembly factors (AFs), ribosomal proteins, and small nucleolar RNAs (snoRNAs) enter the nucleolus to participate in pre-rRNA folding, processing, and modification [6]. During the biogenesis of eukaryotic ribosomes, AFs and ribosomal proteins assemble with pre-rRNA to form preribosomal particles. After further maturation steps, the particles are transported from the nucleolus into the cytoplasm, where mature 60S and 40S subunits assemble into the 80S ribosomes and subsequently synthesize proteins [5,7].

In studies in yeast, researchers found that Rrp5, a special assembly factor, is involved both in the assembly of the ribosomal large subunit precursor (pre-60S) and the small subunit precursor (pre-40S) [8,9]. During rRNA processing, Rrp5 binds to pre-40S subunits and recruits other assembly factors to pre-40S subunits, including the DEAD box protein Rok1 [10,11]. After the function of Rrp5 in 40S subunit assembly is complete, it remains bound to the pre-60S subunit for its assembly [12,13].

Rrp5 is known to bind specifically to rRNA with the help of DEAD box protein Rok1 [14]. DEAD box proteins are a large class of RNA-dependent ATPases, which participate in various functions including gene expression regulation and RNA metabolism [15]. They carry out functions including unwinding RNA structures, annealing double-stranded nucleotide sequences, binding RNA, and remodeling RNA protein complex structures [16,17,18,19]. Although Rok1 is classified as an RNA helicase, in vitro experiments show that Rok1 does not have obvious RNA helicase activity [14,20]. It does show significant activity in regulating RNA double strand annealing [14]. Sohail et al. discovered the interaction between Rrp5 and Rok1 in rRNA processing in yeast [21]. Experiments in vivo and in vitro have verified that the Rrp5 protein forms a complex by contact with multiple structural regions of the Rok1 protein, found on the domains required for 40S subunit binding. Rok1 protein separates Rrp5 from the pre-40S subunit precursor by hydrolysis of its bound ATP, and transfers Rrp5 to the pre-60S subunit precursor, thus participating in the maturation process of the large subunit precursor [21].

Previous research on Rok1 and Rrp5 has focused mainly on yeast, and few functional studies have been performed using complex multicellular eukaryotes. The loss of Rok1 or Rrp5 leads to cell death in yeast [21], which is a single-cell organism, lacking in developmental processes. Rok1- and Rrp5-dependent effects on survival and development in complex organisms remain to be explored. Here, we use a combination of genetics and developmental experiments to show that Rok1 and Rrp5 exert key functions in the development of D. melanogaster. The accumulation of pre-rRNAs caused by Rok1 depletion leads to the blocking of rRNA maturation and subsequently results in developmental defects. In addition, our results suggest that the loss of Rok1 changes the cellular localization of Rrp5 in the nucleolus of multicellular eukaryotes. We have thus revealed functions of Rok1 that have important implications for ribosome assembly in multicellular organisms, as well as in yeast.

2. Results

2.1. Loss of Rok1 and Rrp5 Enlarges the Nucleolus in D. melanogaster

Mutant alleles of rok1 (rok1^141/141 and rok1^167/167) and rrp5 (rrp5^4-2/4-2 and rrp5^11-1/11-1) were generated by Crispr/Cas9. The molecular lesions in these four alleles are shown (Figure 1A). Two null mutation alleles of rok1 and rrp5 (rok1^141/141 and rrp5^4-2/4-2) were generated with the early termination of translation, and the point mutation allele of rok1^167/167 lost amino acids in the ATP-binding site, while rrp5^11-1/11-1 lost them in a non-functional domain. When we used qRT-PCR to measure the expression level of rok1 and rrp5, we observed significantly reduced expression in rok1^141/141 and rrp5^4-2/4-2 mutants (Figure 1B), as expected, given that Rok1 and Rrp5 are knockouts in null mutants. We noticed that the altered amino acid encoding sites in rok1^167/167 are in the ATP-binding pocket, which is conserved in yeast. We then produced rok1 and rrp5 double mutants using mutant alleles that we had generated. As shown in

Table 1. The lethal phenotypes of rok1 and rrp5 mutations.

Genotype	Lethal Stage	Genotype	Lethal Stage
rok1141/141	2nd instar	rok1167/167; rrp54−2/4-2	2nd instar
rok1167/167	pupa	rok1141/141; rrp54-2/TM6B	Late 3rd instar
rrp54-2/4-2	2nd instar	rok1141/141; rrp511-1/TM6B	2nd instar
rrp511-1/11-1	----	rok1141/TM6B; rrp54-2/4-2	2nd instar
rok1141/141; rrp511-1/11-1	2nd instar	rok1141/TM6B; rrp511-1/11-1	----
rok1141/141; rrp54-2/4-2	2nd instar	rok1167/167; rrp54-2/TM6B	pupa
rok1167/167; rrp511-1/11-1	pupa	rok1167/TM6B; rrp54-2/4-2	2nd instar

This table summarizes the phenotypes of rok1, rrp5 and double mutations. “----“ alive.

, death was expected in homozygous mutants except for rrp5^11-1/11-1, but the stage of lethality was different for each mutant (Table 1). Interestingly, most rok1^167/167 individuals are also lethal during the pupal stage, although the rok1 expression level was not decreased (Figure 1B). To assess larval development in rok1 and rrp5 mutants, we stained the salivary glands with DAPI and discovered that there were obvious “chromatin-poor” regions in the middle of the nucleus (Figure 2A). Compared with rok1¹⁶⁷/TM6B, these areas were significantly larger in the three mutants (Figure 2A), leading to speculation that the nucleolus was enlarged. Finally, Fibrillarin (Fib), a nucleolar marker, was used to confirm that this region was the nucleolus. We observed that the GFP signals were also stronger in rok1 and rrp5 mutants by live fluorescent imaging of salivary glands expressing Fib-GFP (Figure 2B,C).

2.2. Rok1 Is Enriched in the Nucleolus and Responsible for rRNA Maturation

Rok1 is consistently enriched in the nucleoli of yeast cells [22]. We determined whether Rok1 is similarly distributed in the nucleoli of multicellular eukaryotes. To assess the Rok1 protein location within the cell, a Rok1-GFP fusion protein was constructed. As shown in Figure 3A, we detected distinct protein bands representing Rok1 and Rok1-GFP in the embryos rok1-GFP (Figure 3A). We then found that Rok1-GFP is lowly expressed in the tissues, although weak GFP signals in the salivary glands, ovaries, and testes of w¹¹¹⁸ flies were detected. It is worth noting that the prominent enrichment of Rok1 was observed in a “chromatin-poor” region that likely corresponds to the nucleolus (Figure 3B). We also observed that Rok1-GFP accumulates inside the nucleus in oocytes, excluding somatic follicles and nurse cells (Figure 3B). We subsequently investigated the effect of Rok1 and Rrp5 deletions on ribosomal RNA in multicellular eukaryotes. As shown in Figure 3C, qPCR analysis of the mature rRNAs showed that depletion of rok1 and rrp5 significantly decreased the level of 18S, 28S, and 5.8S rRNA in rok1^141/141, rok1^167/167, and rrp5^4-2/4-2 mutants (Figure 3C). In addition, using anti-sense probes for 18S and 28S rRNAs via in-situ hybridization (FISH) experiments, we observed that the probes showed weaker fluorescence in the nucleus, and stronger signals in the cytoplasm (Figure 3D). As expected, rok1^167/167 showed weaker fluorescence for 18S and 28S rRNA compared with rok1¹⁶⁷/TM6B in third-instar larvae (Figure 3D–F).

2.3. Loss of Rok1 Leads to Stalled Ribosome Assembly and Pre-rRNA Processing in the Nucleolus

Rok1′s function in pre-rRNA processing has been extensively studied in yeast [21,22]. Its function in development is less clear. We speculated that abnormal pre-rRNA processing is a key reason underlying nucleolar enlargement. First, we observed that stronger Top1-GFP fluorescence was produced in rok1^167/167 mutants, displaying a larger fluorescent area and higher intensity (Figure 4A). DNA topoisomerase I (Top1) has been proven to mark rDNA loci in larval cells and is responsible for rDNA transcription [23]. Our results suggest that the loss of Rok1 might induce excessive pre-rRNAs in the nucleolus. To further study whether the excessive pre-rRNAs are caused by the accumulation of pre-rRNA intermediates, we determined the relative levels of transcripts containing the external transcribed spacer (ETS) and the first internal transcribed spacer (ITS1) regions. ETS, the first region to be processed, is usually representative of the pre-rRNA transcription rate [15]. The levels of ETS-containing intermediate precursors were significantly increased in Rok1 mutants, suggesting excessive transcription of pre-rRNA in larval cells (Figure 4B). When ITS1 levels were normalized to ETS levels, they also showed approximately 100% and 50% increases in ITS1-containing intermediate precursors in rok1^141/141 and rok1^167/167, respectively. FISH experiments in salivary glands showed ETS- and ITS1-containing pre-rRNAs produced in the nucleolus, and the fluorescence of these pre-rRNAs was significantly increased in the nucleolus of the rok1^141/141 mutant (Figure 4C,D). All these results indicate that there is a stalled ribosome assembly and pre-rRNA processing in the nucleolus.

2.4. Loss of Rok1 Inhibits Mitosis in the Brain

Rok1 has been reported to affect the regulation of the cell cycle in yeast cells [24]. To assess the role of Rok1 in cell division in multicellular eukaryotes, we observed mitosis in the brains of rok1 mutants. By analyzing mitotic chromosomes from the nuclei of rok1 mutations, we discovered that 1.22% of rok1^167/167 chromosomes were in mitosis (n = 12,735) compared with 3.38% in the wildtype (n = 9215). Mitosis was not seen in rok1^141/41 chromosomes (n = 10,195) (Figure 5A,B). Considering the operation error due to chromosome squashing, a commercial anti-phospho-Histone H3 antibody was used to immunostain wing discs in rok1 mutants. Generally, Histone H3 phosphorylation is used as a marker of mitosis [25]. As shown in Figure 5C, H3 fluorescence is significantly reduced in the rok1^167/167 mutant compared with that of the wildtype (Figure 5C,D). Since second-instar larvae are too small for wing disc dissection, the central nervous system (CNS) was observed. No H3 signal was found (data not shown). These results suggest that the depletion of Rok1 inhibits mitosis in multicellular eukaryotes.

2.5. Loss of Rok1 Changes the Cellular Localization of Rrp5 in the Nucleolus

In yeast, Rrp5 binds to pre-40S subunit precursors (SSU), then subsequently binds to the pre-60S subunit precursors (LSU) after release from SSU [21]. We, therefore, speculated that pre-rRNA intermediates were also disrupted in the rrp5 mutant. In our first assay, two anti-sense probes of ITS1 and ITS2 for 18S and 28S precursors, respectively, were used in FISH. As expected, rrp5^4-2/4-2 showed a significant increase in ITS1 fluorescence, and reduced ITS2 fluorescence compared with rrp5^4-2/TM6B in second-instar larvae (Figure 6A,B). In the second assay (Figure 6C), qPCR analysis of ETS and ITS1 showed that deletion of rrp5 significantly increased levels of 18S precursors in the rrp5^4-2/4-2 mutant (Figure 6C). Interestingly, we found that eliminating a copy of rrp5 in the rok1^141/141 mutant could delay the time to lethality in larvae until the late third-instar stage (Table 1), which might be caused by the partial rescue of the increased rrp5 level in the rok1^141/141 mutant (Figure 6D). To further clarify the possible mechanism shown in Figure 6D, the nuclear localization of Rrp5 in the rok1 mutant was studied to assess Rok1 and Rrp5 interaction in multicellular eukaryotes. An mCherry-tagged fusion protein was constructed and detected by Western blotting. Bands were observed for Rrp5 and Rrp5-mCherry at the exact predicted sizes (Figure 6E). An increased signal of rrp5-mCherry was consistently observed in the rok1^167/167 mutant, as expected and in accordance with results in Figure 5D (Figure 6F). Tartakoff et al. found that AFs can relocate between the inner (SSU AF) and outer (LSU AF) layers caused by the inhibition of rRNA processing [26]. We found that in the wildtype, Rrp5 fluorescence was distributed within the nucleolus, indicating co-localization with Rok1. Meanwhile, it was particularly enriched in the core of the nucleolus in the rok1^167/167 mutant, where SSU assembly factors are distributed [26] (Figure 6F), suggesting that tee loss of Rok1 changes the cellular localization of Rrp5 in the nucleolus.

3. Discussion

Rok1, a putative ATP-dependent RNA helicase of the DEAD-box protein family, is required for 18S rRNA synthesis in yeast [28]. Venema et al. found that the GAL:rok1 strain grew slowly after transfer to a glucose medium, when an essential cellular component was depleted gradually [22]. In our study, rok1 null mutant Drosophila larvae died during the second-instar stage (Table 1), which is similar to that observed in yeast. When considering the complex development found in a multicellular eukaryote, the function of Rok1 was deeply exerted in different tissues and stages. We found that the nucleolus of the rok1 mutant was significantly enlarged in Drosophila salivary glands (Figure 2A), and rok1 depletion led to stalled ribosome assembly and pre-rRNA processing in the nucleolus (Figure 4). This effect has not been reported in single-cell organisms that lack the developmental decision-making requirements. RNA polymerase I-mediated rDNA transcription driven by dMyc overexpression also causes nucleolar morphological enlargement due to pre-rRNA overexpression, as has been observed in rok1 and rrp5 mutants [29]. In mammals, Top1 interacts with RNA polymerase I [30,31]. Top1 is specifically inhibited by camptothecin in HeLa cells, resulting in the inhibition of rRNA synthesis [32]. Studies on yeast have found that Top1 depletion leads to the accumulation of many negative supercoils in rDNAs [33]. As shown in Figure 3A, we also observed strong enrichment of Top1 in nucleoli in rok1^167/167 (Figure 4A), suggesting that Top1 is also involved in the pre-rRNA accumulation caused by Rok1 depletion.

Based on previously reported data, a model Rok1 functional cycle during ribosome maturation has been proposed [21]. Ribosome biogenesis is a fundamental cellular process, linked to growth and cell division. Mutation of Rok1 resulted in significant delays in mitotic bud emergence derived from the inhibition of Rok1 expression throughout the cell cycle [24], in accordance with our results seen in Drosophila (Figure 5). The depletion of Rok1 led to abnormal processing of 35S pre-rRNA within ITS1 to produce an abnormal 23S pre-rRNA instead of the mature 18S rRNA, which is subsequently degraded [22]. In our study, qPCR and FISH analysis of the mature rRNAs showed that rok1 depletion significantly decreases levels of 18S, 28S, and 5.8S rRNA in rok1^141/141 and rok1^167/167 mutants (Figure 3C,D). We speculated that some aspects of rRNA processing differ between higher eukaryotes and their yeast counterparts, although the fundamental ribosome biogenesis pathway is conserved.

Previous studies in yeast have shown that Rrp5 is a key assembly factor in rRNA processing [11]. It simultaneously participates in the processing of small subunit and large subunit precursors and bears primary responsibility for the recruitment of other assembly factors [12], which are required for the transfer of Rrp5 from SSU to LSU by the Rok1 protein. Although Rok1 and Rrp5 proteins are conserved, whether their mechanisms during rRNA processing are conserved among eukaryotes (especially multicellular eukaryotes) has not been confirmed. We obtained homozygote mutants of rok1 and rrp5 using the Crispr/Cas9 system (Figure 1A). These mutants showed different degrees of developmental defects. Development in larvae carrying the frameshift mutations rok1^141/141 and rrp5^4-2/4-2 stagnated during the second-instar stage, and development in the rok1^167/167 mutant stagnated in the third-instar stage (Table 1). Compared with the wildtype, pre-rRNA levels increased significantly in the rok1 and rrp5 mutants, while levels of mature rRNAs including 18S, 28S, and 5.8S rRNAs decreased significantly (Figure 3, Figure 4, and Figure 6), indicating that rRNA processing can be blocked by individual loss of Rok1 or Rrp5. In yeast, ATP-bound Rok1 stabilizes Rrp5 binding to 40S ribosomes and is required for the transfer of Rrp5 from SSU to LSU [21]. In our study, rrp5^4-2/4-2 showed a significantly increased fluorescence signal in ITS1, and reduced ITS2 fluorescence by FISH compared with rrp5^4-2/TM6B in second-instar larvae (Figure 6A,B), and qPCR analysis of ETS and ITS1 levels showed that rrp5 depletion significantly increased the level of 18S precursors in the rrp5^4-2/4-2 mutant (Figure 6C). These results suggest that the ITS1 accumulation is caused by Rrp5 depletion. Although pre-rRNA biogenesis stalls in the nucleolus, the loss of Rrp5 subsequently blocks LSU processing, resulting in a significantly reduced ITS2 signal (Figure 6A). Moreover, we observed an obvious shift in Rrp5 localization in rok1 mutants (Figure 6F). Tartakoff et al. found that nucleolar assembly factors (AFs) associated with rRNAs localize to the coaxial layers of the nucleoli in yeast. AFs can relocate between the inner (SSU AF) and outer (LSU AF) layers caused by the inhibition of rRNA processing [26]. Rrp5 has been reported both in the inner and outer layers of nucleoli for SSU and LSU maturation. In rok1^167/167 mutants, Rrp5 was localized and highly enriched in the inner layer of the nucleolus, in which SSU assembly factors are distributed (Figure 6F). We thus assume that the interaction between Rok1 and Rrp5 in eukaryotes is highly conserved.

In our study, we used a combination of genetics and developmental experiments to show that Rok1 and Rrp5, which are localized at the nucleolus, play key functions in ribosome assembly in Drosophila (Figure 7).

4. Materials and Methods

4.1. Drosophila Stocks

Rok1 and Rrp5 mutations were generated as described in Section 4.2. Drosophila stocks were raised on a normal medium containing glucose, corn, sucrose, yeast, and agar under standard laboratory conditions. Stocks including w¹¹¹⁸, nos-Cas9, and Sb/TM6B, were obtained from the Bloomington Drosophila stock center. The Fib-GFP and Top1-GFP stocks were obtained as previously described [23]. The rok1-GFP and rrp5-mCherry stocks were generated as described in Section 4.3.

4.2. Crispr/Cas9-Mediated Mutations of Rok1 and Rrp5

Two mutant alleles of rok1 (CG5589) (rok1^141/141 and rok1^167/167) and rrp5 (CG5728) (rrp5^4-2/4-2 and rrp5^11-/11-1) were generated in a w¹¹¹⁸ background using the CRISPR-Cas9 system. The Cas9 protein and gRNA derived by vasa and U6 promoters were expressed by transgenes inserted into the Drosophila genome, respectively [34]. The target gRNA was designed by the online page: http://tools.flycrispr.molbio.wisc.edu/targetFinder/. The target sequences 5′-CCTGTGCACCCACTGGATC (for rok1) and 5′- AGCTCTTCCAGTTGGGCCG (for rrp5) were chosen and cloned into a pUAST-attB plasmid for phiC31-mediated germline transformation to generate offspring with red eyes (G0). Cross schemes and mutation selections were performed as described in Supplementary Figure S1. PCR primers used for mutation detection are listed in Supplementary Table S1.

4.3. Construction of rok1-GFP and rrp5-mCherry Transgenic Lines

To construct transgenic lines carrying rok1-GFP and rrp5-mCherry, DNA fragments of the whole genomic region containing rok1 and rrp5 were cloned into the pUAST-attB vector by gap repairs based on “Seamless ligation cloning”, and the GFP and mCherry genes were subsequently inserted at the 3′ ends of the Rok1 and Rrp5 coding regions, respectively, upstream of the stop codon by recombineering [35]. Rok1-GFP and rrp5-mCherry fly lines were generated by PhiC31 integrase-mediated germline insertions on chromosome III (86F) and chromosome II (25C), respectively. Because the X chromosome of 86F and 25C strains contains ΦC31, which induces green fluorescence, lines were hybridized with w¹¹¹⁸ to remove this background. PCR primers are listed in Supplementary Table S1.

4.4. Quantitative PCR

Total RNA from thirty second-instar larvae of w¹¹¹⁸, rok1^141/141, rok1^167/167, rrp5^4-2/4-2, rok1^141/141, and rrp5^4-2/TM6B was extracted using TRIzol reagent (Invitrogen, Carlsbad, California, USA). cDNA was generated from 1 µg of total RNA using a PrimeScript^TM RT reagent kit (TaKaRa, Shiga, Japan) for the quantitation of mRNAs including rok1, rrp5, 18S rRNA, 28S rRNA, 5.8S rRNA, ETS, and ITS1 by quantitative real-time PCR. A Bio-Rad CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA) and a KAPA SYBR FAST Universal qPCR Kit (Kapa Biosystems, Boston, MA, USA) were used for quantitative real-time PCR (qPCR). The cDNA standard for each gene was diluted to different concentrations and used as templates for PCR reactions. Act5C (accession number CG4027) was used as an internal control gene. The standard curves of Act5C, rok1, rrp5, 18S rRNA, 28S rRNA, 5.8S rRNA, ETS, and ITS1 were drawn with the logarithm of the copy number of template DNA as the abscissa and the measured CT value as the ordinate. The initial copy number of rok1, rrp5, 18S rRNA, 28S rRNA, 5.8S rRNA, ETS, and ITS1 was calculated from the CT value of each cDNA sample after adjustment by the copy number of Act5C. Each cDNA was displayed for three biological replicates. P-values were indicated using Student’s t-test. The primers are listed in Supplementary Table S1.

4.5. Live Fluorescent Imaging

For rok1-GFP live imaging, salivary glands from third-instar larvae of w¹¹¹⁸, ovaries, and testes from adult flies were dissected in cold PBS. For Fib-GFP live imaging, salivary glands from second-instar larvae of rok1^141/141 and rrp5^4-2/4-2 and third-instar larvae of rok1¹⁶⁷/TM6B and rok1^167/167 were dissected, respectively. For Top1-GFP live imaging, salivary glands from third-instar larvae of rok1¹⁶⁷/TM6B and rok1^167/167 were dissected. For rrp5-mCherry live imaging, salivary glands from third-instar larvae of rok1¹⁶⁷/TM6B and rok1^167/167 were dissected. Thirty biological replicates were detected for each mutation. The tissues were imaged by a Zeiss Axio Image A2 microscope.

4.6. Fluorescence In Situ Hybridization (FISH)

Salivary glands were dissected in PBS from second-instar larvae of rok1¹⁴¹/TM6B and rok1^141/141, and third-instar larvae of rok1¹⁶⁷/TM6B and rok1^167/167. Malpighian tubules were dissected from second-instar larvae of rrp5^4-2/TM6B and rrp5^4-2/4-2 in PBS. Tissues were fixed in 4% paraformaldehyde for 4 h. After washing three times with 2 × SSCT (0.1% Triton X-100), they were rehydrated using an ethanol gradient. After air drying, pre-hybridization was performed in 25% formamide, 5 × Denhards, 5% dextran sulfate, 2 × SSC, 0.2% BSA, and 125 μg tRNA for 2 h at 37 °C. Tissues were incubated at 37 °C overnight with a final probe concentration of 0.5 ng/mL. Post-hybridization washes were performed three times in 0.2 × SSCT for 15 min each. Tissues were then stained with DAPI (0.2 μg/mL in 4 × SSCT) for five min, washed briefly in 4 × SSCT, and allowed to air dry. Fluorescent images were photographed by an Olympus IX83 confocal microscope. Probes are listed in Supplementary Table S1.

4.7. Antibodies

Mouse anti-rok1 (CG5589) and anti-rrp5 (CG5728) sera were raised against 333 amino acid fragments of Rok1 (a.a. 121-453) and Rrp5 (a.a. 151-483) as antigens purified from E. coli. The Rok1 and Rrp5 antibodies were incubated at 1:1000 and 1:2000 on Western blots, respectively. Embryos were collected and homogenized in RIPA buffer, and an anti-mouse secondary antibody (1:5000 dilution, Abcam) was used.

4.8. Immunostaining

Ten wing discs of rok1¹⁶⁷/TM6B and rok1^167/167 from third-instar larvae were dissected in the cold PBS buffer. Then, tissues were fixed with a mixture of 3.7% formaldehyde diluted in PBS for 15 min. Heptane was subsequently added for 30 min at room temperature. After washing three times with 1 X PBST (0.1% Triton X-100), a rabbit anti-phospho-Histone H3 (1:200, Sigma, Saint Louis, USA) antibody was incubated overnight at 4 °C. After washing three times with 1 X PBST (0.1% Triton X-100), Alexa Fluor 555 conjugated goat antirabbit IgG (1:200, Abcam) and DAPI were incubated for 1 h. Fluorescent images were photographed by an Olympus IX83 confocal microscope.

4.9. Mitotic Chromosome Squash

Brains from second- and third-instar larvae of w¹¹¹⁸, rok1^167/167, and rok1^141/141 were dissected in cold PBS buffer following a standard protocol without colchicine treatment [36]. Ten brains from each group were dissected and squashed. Chromosomes were analyzed with a Zeiss Axio Image A2 microscope.