Sequence and transcription patterns of 60S ribosomal protein P0, a diapause-regulated AP endonuclease in the flesh fly, Sarcophaga crassipalpis

Gene 255 (2000) 381–388 www.elsevier.com/locate/gene

Sequence and transcription patterns of 60S ribosomal protein P0, a diapause-regulated AP endonuclease in the flesh fly, Sarcophaga crassipalpis Tracy L. Craig, David L. Denlinger * The Ohio State University, Department of Entomology, Columbus, OH 43210-1220, USA Received 26 April 2000; received in revised form 22 June 2000; accepted 3 July 2000 Received by E. Boncinelli

Abstract We have isolated and sequenced a 1308 bp clone from a pupal brain cDNA library of the flesh fly, Sarcophaga crassipalpis, showing 97% amino acid (aa) sequence similarity to Ceratitis capitata 60S acidic ribosomal protein P0 (CcP0) and 93% aa sequence similiarity to Drosophila melanogaster P0 (DmP0). DmP0 is a multifunctional protein necessary for efficient protein translation of the 60S ribosome as well as DNA repair via AP3 endonuclease activity. In this study, we observed that S. crassipalpis P0 (ScP0) is cyclically regulated throughout the fly’s overwintering pupal diapause. Expression of ScP0 cycles out of phase with the 4 day cycles of O consumption: the peak day of O consumption is characterized by low ScP0 expression, while high 2 2 expression is noted during the trough of the O consumption cycle. The O cycles, which are in turn driven by cycles of juvenile 2 2 hormone (JH ), can be eliminated by application of a JH analog (JHA). Pupae rendered acyclic with a JHA application consume O at a constant high rate and ScP0 is consistently downregulated. Our findings thus suggest that the cyclic nature of ScP0 2 regulation during pupal diapause is linked to the JH-mediated metabolic cycles characteristic of this species. © 2000 Elsevier Science B.V. All rights reserved. Keywords: AP endonuclease; Diapause; Hypoxia; Juvenile hormone; P0

1. Introduction Acidic ribosomal protein P0 is a multifunctional protein previously implicated in the association of elongation factors with the ribosome during protein synthesis (Mo¨ller and Maassen, 1986; Rodriguez-Gabriel et al., 1998). Phosphorylation of a serine residue (RodriguezGabriel et al., 1998), located in the highly conserved 17 aa carboxy-tail region of most eukaryotic P0 species (Grabowski et al., 1991), facilitates ribosomal binding (Sanchez-Madrid et al., 1981). Upon dephosphorylation and subsequent dissociation from the ribosome (Sanchez-Madrid et al., 1981), P0 is highly transient Abbreviations: A, adenosine; aa, amino acid(s); AP, apurinic/apyrimidinic; C, cytidine; cDNA, DNA complementary to RNA; G, guanosine; JH, juvenile hormone; JHA, juvenile hormone analog; nt, nucleotide; ORF, open reading frame; UTR, untranslated region(s). * Corresponding author. Tel.: +1-614-292-0165; fax: +1-614-292-2180. E-mail address: [email protected] (D.L. Denlinger)

and easily transported from the ribosome to the nuclear matrix ( Yacoub et al., 1996). The ability of P0 to bind to the nuclear matrix supports its suggested roles in DNA repair ( Yacoub et al., 1996) as well as regulating gene expression (Frolov and Birchler, 1998). Drosophila melanogaster P0 (DmP0) contains an array of DNA repair capabilities including DNase activity for both single- and double-stranded DNA and, most notably, AP3 endonuclease activity ( Yacoub et al., 1996). AP3 endonuclease recognizes and repairs apurinic/ apyrimidinic (AP) sites from compromised DNA (reviewed in Barzilay and Hickson, 1995). A variety of factors have been implicated in AP site formation, including UV irradiation (Boiteux et al., 1985; Kanno et al., 1999) and hypoxia ( Walker et al., 1994; Yao et al., 1994) to name a few. Due to the unusually high frequency of AP sites and their cumulative effects (Lindahl, 1993; reviewed in Barzilay and Hickson, 1995), repair is imperative to prevent impaired DNA replication, blocked transcription and phenotypic mutations (Sauerbrier and Hercules, 1978).

0378-1119/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 0 0 ) 0 0 30 7 - 3

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In this study, we report the isolation and sequence of 60S acidic ribosomal protein P0 from a pupal brain cDNA library of the flesh fly, Sarcophaga crassipalpis, and its transcription patterns during pupal diapause. Diapause is a period of prolonged developmental arrest vital to the overwintering survival of most insects ( Tauber et al., 1986). In S. crassipalpis, development is halted early in the pupal stage (Denlinger, 1972). In a previous study focusing on the isolation of diapausespecific clones, pScD 86, now identified as S. crassipalpis P0 (ScP0), appeared upregulated in diapause (Flannagan et al., 1998), yet the more detailed analysis completed in this study reveals that ScP0 is not continuously upregulated but instead exhibits a cyclic pattern of expression out of phase with cycles of O consumption 2 (Denlinger et al., 1972). Peak days of O consumption 2 are followed by several days of extremely low O con2 sumption. These infradian cycles are in turn driven by cycles of JH activity. JH activity gradually increases during the trough of the O consumption cycle, reaches 2 a threshold, and triggers the next O peak (Denlinger 2 et al., 1984). In this study, we report ScP0 upregulation during the nadir of the O consumption cycle. The 2 application of exogenous JH can be used to elevate O 2 consumption and render the pupae acyclic. We show that this manipulation results in downregulation of ScP0, so ScP0 expression is consistently high during low O consumption and low when O consumption is 2 2 high during diapause. 2. Materials and methods 2.1. Insect rearing Non-diapausing Sarcophaga crassipalpis colonies were maintained at 25°C and long day length (16 h light:8 h dark) as previously described (Denlinger et al., 1972). Diapause-destined colonies were maintained by placing adults with no previous history of diapause at 25°C and short day length (12 h light:12 h dark). The progeny were transferred to 20°C, 12L:12D, where they subsequently entered pupal diapause.

in vivo and cloned in pBluescriptKS(+) prior to sequencing. 2.3. Sequencing Sequence analysis was performed at the University of Georgia Molecular Genetics Facility and The Ohio State University Molecular Sequencing Facility on an ABI 373A DNA sequencer according to the manufacturer’s protocol. Both strands of clone 86 were sequenced, with the initial sequence originating from the pBluescriptKS(+) vector primer sites located at the 3∞ and 5∞ distal regions of clone 86. Upon determination of the initial sequence, 3∞ and 5∞ internal primers (Gibco/BRL) were designed and the clone sequenced in its entirety. The GenBank BLASTA search engine verified the sequence identity of clone 86, and the TFSEARCHA search engine located transcription factor binding sites. 2.4. RNA isolation Poly(A)+ RNA was isolated to monitor the transcription patterns of ScP0 in diapause-destined and nondiapause destined wandering larvae, non-diapausing pupae, and diapausing pupae collected at 10 day intervals throughout the first 60 days of diapause. Samples comprising two or three individuals were placed in TRIzolA reagent (Gibco/BRL), homogenized, and total RNA extracted according to the manufacturer’s protocol (Gibco/BRL). An oligo(dT ) substrate column (Gibco/BRL) was used to isolate poly(A)+ RNA from total RNA, as described by the manufacturer. Poly(A)+ RNA samples were dissolved in 15 ml of formamide and stored at −20°C. Total RNA was suitable for observing the effects of JHA on ScP0 transcription and to view ScP0 RNA levels during troughs and peaks in O consumption. 2 Total RNA samples intended for non-poly(A)+ Northern analysis were dissolved in 50 ml of formamide, and the concentration of RNA was calculated from optical density (OD) readings taken at 260 nm. 2.5. Application of juvenile hormone analog

2.2. Brain cDNA library construction ScP0 was isolated and cloned, utilizing a commercially developed subtractive hybridization technique (Clontech, Palo Alto, CA). The diapause pupal brain cDNA library was screened with cDNA probes originating from non-diapause flesh fly brain mRNA (Flannagan et al., 1998). From the initial screening, 95 clones failed to hybridize to the cDNA library and were isolated. Using diapause and non-diapause poly(A)+ RNA, a secondary screening was performed using Northern blot hybridization. Clone 86, later identified as ScP0, possessed a 2.1 kb transcript that appeared to be upregulated during diapause. Clone 86 was excised

Eighty diapause-destined, third-instar wandering larvae were individually treated with 50 mg/ml of the JHA, methoprene (ZR515, Zoecon Corp., Palo Alto, CA), dissolved in 5 ml of acetone. Topically treated wandering larvae were immediately returned to 20°C, 12L:12D. Diapausing pupae were collected every 10 days for 60 days, and total RNA was extracted for Northern blot analysis. 2.6. Monitoring oxygen consumption Oxygen consumption rates in diapausing pupae were monitored utilizing a Scholander respirometer (Mark

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Co., Brockton, MA) at 20°C. Pupae that had been in diapause for 20 days were sealed in the closed system and allowed 2 days to acclimate. Manometeric measurements were taken daily. Pupae experiencing a peak or trough during the course of the infradian cycle of O 2 consumption (Denlinger et al., 1972) were removed from the respirometer, and total RNA was extracted for Northern blot analysis 2.7. Northern analysis A 1% agarose, 0.41 M formaldehyde gel was used for all Northern blot analyses. For the poly(A)+ Northern, 3 mg of poly(A)+ RNA was loaded into each well, while total RNA Northerns required 20 mg of RNA per well. Ten microliters of RNA loading buffer [2 ml of 10× MOPS, 3.5 ml of formaldehyde, 1.5 ml of SALB, 2 ml of 0.5 mg/ml ethidium bromide, 1 ml of autoclaved 0.1% DEPC ddHOH ] were added to each sample, incubated at 65°C for 10 min, then loaded onto the gel. Gels were electrophoresed at 100 V in 1× MOPS buffer. Electrophoresis was followed by the neutral transfer of RNA onto a MagnaGraph neutral membrane (Osmonics, Westborough, MA). The Turboblotter (Schleicher and Schuell, Keene, NH ) neutral transfer system was used according to the manufacturer’s instructions. Following transfer and neutralization, the membrane was UV-crosslinked (254 nm) for a total of 120 mJ/cm2, wrapped in Saran WrapA and stored at −20°C until hybridization. The ScP0 probe was biotinylated according to the manufacturer’s protocol (New England BioLabs, Beverly, MA). Membranes were prehybridized in hybridization buffer [0.5 M NaCl, 0.1 M NaPO (pH 7.0), 6 mM 4 EDTA, 1% SDS] for 1 h at 68°C. Labeled probe was added to a fresh amount of hybridization buffer in concentrations determined by the protocol (NEBlot Photope Kit, New England BioLabs) and hybridized at 68°C, overnight. The signal was developed according to the manufacturer’s protocol (Photope-Star Detection Kit, New England BioLabs). Membranes were exposed to X-ray film for 3 min at room temperature.

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et al., 1989). TFSEARCHA analysis (Heinemeyer et al., 1998) identifies an E2F transcription factor binding site sequence of TTTGGCGCG located at nt 642–634. What role the E2F binding site plays in ScP0 regulation is unknown. ScP0 has a pyrimidine-rich 5∞ UTR of 72 bp and a 3∞UTR of 285 bp starting at nt 952–1236. The 3∞ UTR contains a poly A signal (AATAAA) at nt 1193– 1198, followed by an oligo(A) tail (nt 1221–1236). 16 BLASTN sequence analysis shows the ScP0 ORF to have 83% nucleic acid identity to CcP0 ( Kouyanou, 1998), 80% identity to DmP0 ( Kelley et al., 1989) and a 66% match to human P0 (Rich and Steitz, 1987). When amino acid (aa) sequences are compared, the match of ScP0 to other species of P0 proteins is even more impressive, with CcP0 ( Kouyanou, 1998), DmP0 ( Kelley et al., 1989), and human P0 (Rich and Steitz, 1987) having 97, 93, and 80% similarity, respectively ( Fig. 2). P0 in eukaryotes possess the highly conserved 17 aa acidic carboxy tail sequence N-(S/E ) (S/E)E(S/E)EE (S/E )D(D/E )DMGFLGLFD-COOH (Grabowski et al., 1991; reviewed in Wool et al., 1995). Within this region, casein kinase I (CKI ) and/or II (CKII ) phosphorylation sites are commonly located ( Rodriguez-Gabriel et al., 1998). The carboxy-tail region of ScP0 contains the CKII phosphorylation sequence N(300)-EESEE-(304)C, with the Ser(302) residue acting as the site of phosphorylation. Phosphorylation of these sites has been shown to initiate and facilitate binding of P0 to the ribosome (SanchezMadrid et al., 1981) where it can mediate binding of elongation factors like EF-2 to the ribosome (Mo¨ller and Maassen, 1986). Dephosphorylation of P0 results in its dissociation from the 60S ribosome (SanchezMadrid et al., 1981). Once dissociated, P0 floats freely in the cytoplasm or is tightly bound to the nuclear matrix ( Yacoub et al., 1996). The propensity of P0 to bind to the nuclear matrix further supports the role of P0 in DNA repair as well as its newly suggested role in the regulation of gene expression ( Frolov and Birchler, 1998).

3.2. ScP0 regulation during S. crassipalpis development 3. Results and discussion 3.1. Sequence analysis of ScP0 ScP0 is a 1308 bp gene, 316 aa protein with 1 ORF of 951 bp present at nt 1–951 ( Fig. 1). A putative second 129 bp ORF is downstream at nt 1046–1174, yet a TBLASTNA search analysis of this region reveals no known sequence similarity, and ScP0’s nearest known sequence matches, Ceratitis capitata P0 (CcP0) ( Kouyanou, 1998) and Drosophila melanogaster P0 (DmP0), do not report such a coding region ( Kelley

In a previous study, ScP0 appeared to be diapauseupregulated, based on a Northern blot analysis of RNA from day 30 diapausing pupae (Flannagan et al., 1998). For the purpose of our study, more detailed Northern blots examining ScP0 expression at 10 day intervals for the duration of diapause were performed. As Fig. 3 illustrates, non-diapause destined wandering larvae, non-diapausing pupae, and diapause-destined wandering larvae showed significant expression of the 2.1 kb ScP0 transcript. Once pupae entered diapause, ScP0 expression became cyclic in nature. All three replicates of the experiment demonstrated a similar cyclic pattern

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Fig. 1. Nucleic acid sequence of ScP0 cDNA isolated from a Sarcophaga crassipalpis brain cDNA library (GenBank Accession No. AF220939). The P0 ORF start codon (ATG) is located at nt positions 1–3, and the stop codon ( TAA) is located at nt positions 949–951. A 129 bp putative coding region is found between nt 1046 and 1174. The P0 ORF is underlined and underscored by the aa sequence. A complete 5∞-UTR (nt −72 to −1) and a 285 bp region 3∞ of the primary P0 ORF stop codon are present. The 3∞ UTR possesses a polyadenylation signal at nt 1193–1198 and an oligo(A) tail from nt 1221 to 1236. 16

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Fig. 2. DIALIGN 2 (Morgenstern, 1999) protein sequence alignment of 60S acidic ribosomal P0 from various eukaryotic species. The 316 aa sequence of Sarcophaga crassipalpis P0 (ScP0), 317 aa Ceratitis capitata P0 (CcP0), 317 aa Drosophila melanogaster P0 (DmP0), and 317 aa human P0 were compared. Areas where aa sequence identity was conserved are noted with an asterisk, while ‘:’ denotes aa sequence similarity

of expression. To help explain why ScP0 transcription is cyclic during pupal diapause, we looked to the inversely related cycles of oxygen consumption and JH metabolism (Denlinger et al., 1972, 1984) already observed in diapause.

3.3. Correlation of ScP0 expression with O consumption 2 cycles One possible explanation for the cyclic expression of ScP0 during diapause is that expression correlates with

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Fig. 3. Northern blot analysis of ScP0 expression during various stages of S. crassipalpis development. Numbers represent days in pupal diapause. For each RNA sample, 3 mg of poly(A)+ RNA were loaded onto a 0.41 M formaldehyde, 1% agarose gel, run at 100 V, gravimetrically transferred to nylon membrane and hybridized at 65°C for 12 h. The membrane was exposed to X-ray film overnight at −70°C.

the infradian cycles of O consumption (Denlinger et al., 2 1972; Sla´ma and Denlinger, 1992). To test this possibility, pupae in different phases of the O consumption 2 cycle were examined for expression of ScP0. Northern blot analysis showed ScP0 upregulation during troughs in the O consumption cycle and downregulation during 2 the O consumption peak ( Fig. 4). This pattern was 2 observed in duplicate samples. This suggests a strong correlation between periods of low O consumption and 2 ScP0 upregulation. Interestingly, other acidic P0 proteins and AP endonucleases, including the human AP endonuclease, Ape, are known to be induced by hypoxic stress ( Walker et al., 1994; Yao et al., 1994). Quite possibly, the periods of low O consumption observed 2

in diapausing fly pupae evoke a physiological response similar to hypoxia. 3.4. Altering the O cycles and ScP0 expression with a 2 JH analog A topical application of JHA to diapause-destined wandering larvae alters the O consumption cycles of 2 the pupae (Denlinger et al., 1984). Pupae treated in this manner have an elevated rate of O consumption, and 2 O consumption is no longer cyclic. In this experiment, 2 we demonstrated that eliminating the troughs of the O cycle also eliminated the cyclic upregulation of ScP0 2 ( Fig. 4). Expression of ScP0 was consistently low in the

Fig. 4. Low, acyclic expression of ScP0 during pupal diapause after topical application of JHA. Third-instar wandering larvae were treated with 50 mg of JHA each and allowed to enter diapause. Numbers represent the number of days in diapause, post-treatment. Untreated pupae in days 40, 50 and 60 of diapause were used as controls to illustrate a normal cycle of ScP0 regulation. Twenty micrograms of total RNA were loaded onto a 0.41 M formaldehyde, 1% agarose gel for each time point, run at 100 V and transferred onto a nylon neutrally charged membrane. Hybridization at 68°C for 12 h was followed by exposure to X-ray film for 2 min at room temperature.

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Fig. 5. Upregulation of ScP0 expression during the trough of the infradian cycle of O consumption. Day 20 diapausing pupae were 2 monitored at 20°C for 2–4 days. For both samples, 20 mg of total RNA were loaded onto a 0.41 M formaldehyde, 1% agarose gel, run at 100 V and transferred onto a nylon neutrally charged membrane. Hybridization at 68°C for 12 h was followed by exposure to X-ray film for 2 min at room temperature.

duplicate samples subjected to Northern analysis. Thus, the expression of ScP0 is consistently downregulated during periods of elevated O consumption, either in 2 naturally occurring O consumption peaks, as shown in 2 Fig. 5, or when O consumption is artificially elevated 2 by the use of JHA, as shown in Fig. 4. Whether the response of ScP0 is in direct response to the changes in JH titer or a response to altered rates of O consumption promoted by changes in the JH titer 2 is unknown. Certain hormones, such as thyrotropin, the thyroid stimulating hormone, are known to induce 60S acidic ribosomal protein P0 RNA in rats ( Ikeda et al., 1991). 3.5. No ScP0 upregulation in response to physical wounding or cytoxic events Previous studies of P0 proteins have shown that these multifunctional proteins possess AP3 endonuclease activity as well as an assortment of other DNA repair capabilities ( Yacoub et al., 1996). In an attempt to upregulate ScP0 transcription by eliciting latent AP endonuclease activity, diapausing pupae were subjected to one of four DNA damage-inducing treatments: physical wounding with a pin to the thorax, hypo- or hyperthermia (−10°C for 1 h, or 43°C for 2 h, respectively), and UV-C irradiation (240 mJ/cm ). ScP0 transcription 2 remained unchanged 0, 1, 3, 6, 9 and 12 h after wounding, 1 h after hypothermia, immediately after hyperthermia, and 0, 1, 4, 8 and 16 h after UV treatment when compared to untreated controls (data not shown). 3.6. Conclusions

1. The complete nucleotide sequence of the 1308 bp gene, Sarcophaga crassipalpisP0 (ScP0), including 1 ORF and complete 5∞ and 3∞UTR regions was attained.

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2. An E2F transcription factor binding site is located in the ScP0 minus strand. 3. The ScP0 amino acid sequence shows high identity to Ceratitis capitata (97%), Drosophila melanogaster (93%) and human (80%) P0 proteins. 4. ScP0 is upregulated in-phase with cycles of low O 2 consumption during pupal diapause. 5. When JH is used to elevate the O consumption rate, 2 ScP0 is downregulated, thus providing further evidence of linkage between the ScP0 expression pattern and cycles of O consumption. 2 Acknowledgements This work was supported in part by a grant from the US Department of Agriculture (Grant No. 9835302-6659). We appreciated critical reviews of the manuscript by Drs. J.P. Rinehart and S. Diakoff.

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