endoplasmic reticulum calcium ATPase (SERCA) are essential in Caenorhabditis elegans

Gene 261 (2000) 211±219

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Two isoforms of sarco/endoplasmic reticulum calcium ATPase (SERCA) are essential in Caenorhabditis elegans Jeong Hoon Cho, Jaya Bandyopadhyay, Jiyeon Lee, Chul-Seung Park, Joohong Ahnn* Department of Life Science, Kwangju Institute of Science and Technology, Kwangju, 500-712, South Korea Received 3 August 2000; received in revised form 17 October 2000; accepted 7 November 2000 Received by D. Court

Abstract SERCA (Sarco/Endoplasmic Reticulum Calcium ATPase), a membrane bound Ca 21- /Mg 21- dependent ATPase that sequesters Ca 21 into the SR/ER lumen, is one of the essential components for the maintenance of intracellular Ca 21 homeostasis. Here we describe the identi®cation and functional characterization of a C. elegans SERCA gene (ser-1). ser-1 is a single gene alternatively spliced at its carboxyl terminus to form two isoforms (SER-1A and SER-1B) and displays a high homology (70% identity, 80% similarity) with mammalian SERCAs. Green ¯uorescent protein (GFP) and whole-mount immunostaining analyses reveal that SER-1 expresses in neuronal cells, bodywall muscles, pharyngeal and vulval muscles, excretory cells, and vulva epithelial cells. Furthermore, SER-1::GFP expresses during embryonic stages and the expression is maintained through the adult stages. Double-stranded RNA injection (also known as RNAi) targeted to each SER-1 isoform results in severe phenotypic defects: ser-1A(RNAi) animals show embryonic lethality, whereas ser-1B(RNAi) results in L1 larval arrest phenotype. These ®ndings suggest that both isoforms of C. elegans SERCA, like in mammals, are essential for embryonic development and post-embryonic growth and survival. q 2000 Elsevier Science B.V. All rights reserved. Keywords: C. elegans; Sarco/endoplasmic reticulum calcium ATPase; Isoforms; Green ¯uorescent protein; RNA-mediated interference

1. Introduction Calcium pumps, along with Ca 21 release channels, are essential components in maintaining intracellular Ca 21 homeostasis in muscle and non-muscle cells. Sequence analysis and biochemical investigation of several Ca 21pumping ATPases have classi®ed these proteins into two major classes: 1) Plasma membrane Ca 21-ATPase (PMCA) (Carafoli et al., 1996), and 2) Sarco (endo) plasmic reticulum Ca 21- ATPase (SERCA) (Wu et al., 1995; MacLennan et al., 1997). Both PMCA and SERCA have the highest af®nity for Ca 21 mobilization from the cytoplasm and together maintain the resting cytoplasmic Ca 21 concentrations. SERCA is an intracellular membrane-bound Ca 21-/ Mg 21-dependent ATPase that sequesters Ca 21 into the SR/ Abbreviations: C. elegans, Caenorhabditis elegans; C-terminus, carboxyl terminus; dsRNAs, double-stranded RNAs; GFP, green ¯uorescent protein; PCR, polymerase chain reaction; SER-1, C. elegans homologue of mammalian SERCA; SERCA, Sarco/Endoplasmic Reticulum Calcium ATPase; TBS-T, Tris-buffered saline with Tween-20; 3 0 UTR, 3 0 untranslated region; RNAi, RNA-mediated interference * Corresponding author. Tel.: 182-62-970-2488; fax: 182-62-970-2484. E-mail address: [email protected] (J. Ahnn).

ER (intracellular Ca 21 stores) and is known to play an important role in various cellular processes (reviewed by Grover, 1985; Misquitta et al., 1999). SERCA proteins are encoded by three differentially expressed genes in mammals, namely, SERCA1, 2, and 3 (Wu et al., 1995). SERCA 1 gene products are expressed mostly in fast-twitch skeletal muscles. SERCA 2a protein is expressed at high levels in cardiac and slow-twitch striated muscles while SERCA 2b is ubiquitously expressed and, hence, is described as a housekeeping protein. SERCA 3 is alternatively spliced and produces mRNAs, encoding three protein isoforms. It is expressed in some non-muscle tissues, including intestine, thymus, cerebellum, and lymph nodes. Mutations or impaired function and expression of SERCA have shown that the protein is associated with several diseases (Just, 1996; Odermatt et al., 1996; Varadi et al., 1999; Sudbrak et al., 2000). Thus SERCA is known to occupy a central role in signal transduction, and regulate a wide range of cellular processes, including cell proliferation and apoptosis, cardiac contractility, calcium waves, and neuronal functions (see review Misquitta et al., 1999). The nematode Caenorhabditis elegans is a popular model for developmental and genetical studies. Approximately 40% of the sequenced proteins have their homologues in

0378-1119/00/$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(00)00536-9

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J. Hoon Cho, J. Bandyopadhyay / Gene 261 (2000) 211±219

other organisms (reviewed by Ahringer, 1997; The C. elegans Sequencing Consortium, 1998). A putative SERCA-type pump is found in the C. elegans genome located on chromosome III (cosmid K11D9) and is alternatively spliced to produce two isoforms. Since C. elegans represents a unique model for calcium homeostasis studies (Baylis et al., 1999; Dal Santo et al., 1999; Kraev et al., 1999; Reiner et al., 1999; Rongo and Kaplan, 1999), we undertook a functional study of the nematode SERCA. Here we report the expression pattern of C. elegans SERCA and analyzed the phenotypic effects by doublestranded RNA-mediated interference (RNAi) for the two spliced variants. Double-stranded RNAs targeted to speci®c isoforms resulted in embryonic lethality or L1 larval arrest, indicating that SERCA is indispensible during embryogenesis, and plays an important role in the growth and survival of C. elegans. 2. Materials and methods 2.1. C. elegans strains and a cosmid clone Wild type C. elegans, the Bristol strain (N2) were obtained from the Caenorhabditis Genetics Center (CGC) at the University of Minnesota, USA. A cosmid clone, K11D9, was obtained from A. Coulson (The Sanger Center, UK). Breeding of C. elegans was carried out according to Brenner (1974). 2.2. Construction of SERCA gene and reporter gene fusion Green ¯uorescent protein (gfp) reporter gene was used to construct ser-1::gfp fusion constructs. The 5 0 upstream promoter region of the ser-1 gene containing approximately 2.56 kb from the initiation (Met) codon was cloned by PCR from the cosmid K11D9 DNA, and fused to a promoterless gfp vector, pPD95.75 (C. elegans expression vector kit was provided by A. Fire) lacking the nuclear localization signal (NLS) (Fire et al., 1998a). Expression plasmids together with plasmid pRF4 (dominant rol-6) as a transformation marker were microinjected into wild-type animals to obtain germline transformants as described by Mello and Fire (1995). 2.3. Antibodies and Immuno¯uorescence microscopy Monoclonal (mouse) anti-SERCA1 ATPase antibody (Catalog Number MA3-911, Af®nity Bioreagents, Inc., Golden, CO, USA) produced from puri®ed rabbit skeletalform SERCA was used to stain wild type animals. Caenorhabditis elegans was immunostained as described (Ahnn and Fire, 1994). Brie¯y, worms were transferred to a poly-L lysine subbed slide, permeabilized by freeze-cracking, and ®xed in 2208C methanol for 3 min. After stepwise incubation in methanol-water mixtures [75, 50, 25% (v/v) methanol, each for 2 min] and washing with TBS-T [50 mM Tris-Cl (pH 7.8), 150 mM NaCl, 0.1% Tween 20], animals were

incubated with anti-SERCA antibodies diluted 1:200 in TBS-T at room temperature for 6 h. Samples were washed three times, each for 2 min in TBS-T, incubated for 6 h with goat anti-mouse secondary antibody (rhodamine-conjugated) and then washed as above. Slides were mounted in 80% glycerol with 1% n-propyl gallate and observed under a ¯uorescence microscope (Olympus BX50). 2.4. In vitro transcription and RNA-mediated interference (RNAi) The C. elegans genome database predicts two isoforms for SERCA, a larger SER-1A and a shorter SER-1B. Two DNA fragments were prepared by PCR ampli®cation of the cosmid K11D9 genomic DNA corresponding to the downstream coding sequences and 3 0 UTRs of the respective SERCA isoforms and subcloned into the pBluescript vector (Fig. 5). A 531 bp of the ser-1A DNA fragment (amino acid residues 995±1059 and 336 bp of 3 0 UTR) and a 426 bp of the ser-1B DNA fragment (amino acid residues 995±1004 and 396 bp of 3 0 UTR) were used to prepare double-stranded RNAs (dsRNAs) to target the two SERCA isoforms speci®cally. Double-stranded RNA targeted to ser-1A or ser-1B was injected alone or together. In addition, pPD79.44 GFP vector was used to prepare dsRNAs to speci®cally target GFP in a ser-1::gfp line and detect expressions in certain tissues. Double-stranded RNAs were prepared using an in vitro transcription kit (Promega) as described (Fire et al., 1998b; Montgomery et al., 1998; Timmons and Fire, 1998). RNA injections (400 ng/ml) were carried out in the body cavity of the adult hermaphrodites as described (Fire et al., 1998b). After recovery in bacteria-seeded worm agar plates, the injected worms were transferred to fresh worm plates at 24-h intervals. The progeny of injected animals were observed for phenotype or GFP expression. 2.5. Analysis of phenotypes in animals subjected to RNAi Interference with the two SERCA isoforms was assayed in a wild-type (N2) background. Phenotypes analyzed included viability, pharyngeal pumping, and moving speeds. The number of unhatched embryos (Ahnn and Fire, 1994) or embryos that incompletely hatched were counted as dead or arrested embryos. ser-1-de®cient and control animals were examined under low magni®cation stereomicroscope, as well as under DIC microscope. 3. Results and discussion 3.1. Identi®cation of single SERCA gene with two alternatively spliced variants in C. elegans In order to investigate SERCA in C. elegans, we searched the worm genome database for SERCA-like sequences. Only one SERCA homologue (SER-1) was found on the cosmid K11D9 physically mapped to chromosome III

J. Hoon Cho, J. Bandyopadhyay / Gene 261 (2000) 211±219

(LGIII) (Fig. 1A). The gene (ser-1) is alternatively spliced at the C-terminus to yield two variants, ser-1A and ser-1B. BLAST search (Altschul et al., 1990) and the Intronerator, a web-based program to study C. elegans genes (Kent and Zahler, 2000), reveal that both the proteins differ at their C-terminal ends. SER-1A isoform is predicted to consist of 7 exons encoding 1059 amino acids while SER-1B isoform is predicted to consist of 8 exons encoding 1004 amino acids (Fig. 1B). In SER-1B, the terminal aspartic acid of exon 7 at position 994 is spliced to a valine residue at the start of exon 8 (amino acid position 995), a region that corresponds to an extended 3 0 untranslated region (UTR) of SER-1A. To further con®rm the existence of these isoforms, we sequenced ser-1 exons at their C-terminal ends including 3 0 UTR's from a mixed stage worm cDNA library. Brie¯y, isoform-speci®c primers were designed to conduct nested PCR in the C-termini including the regions where the two isoforms differed in their sequences (amino acid positions 800 to 1059 and 288 bp of 3 0 UTR of SER-1A; amino acid positions 800 to 1004 and 326 bp of 3 0 UTR of SER-1B). The PCR products of both isoforms were directly sequenced and con®rmed with the gene prediction (data not shown). The deduced amino acid sequences for both isoforms showed approximately 78±80% similarity (68±70% identity) to SERCA1, 2 or 3 proteins in other organisms (Fig. 2), and the homology existed throughout the entire lengths of the proteins excepting the C-terminal ends where no homology was observed for either isoform. High sequence similarity for SERCA between C. elegans and other organ-

Fig. 1. Molecular nature of ser-1gene. (A) Genetic and physical maps of the putative ser-1 region. The position of ser-1 relative to the nearby genetic markers and regions covered by the yeast arti®cial chromosomes (Y74E5 and Y39A1), on the LG III is shown. ser-1 is mapped to the cosmid K11D9 (Accession No. Z92807) (shown in bold line). The neighboring cosmids are indicated by horizontal lines. (B) The predicted ser-1gene is composed of two alternatively spliced isoforms (ser-1A and ser-1B). The larger (SER1A) consists of 7 exons encoding 1059 amino acids and the shorter (SER1B) is composed of 8 exons encoding 1004 amino acids. Splicing occurs at the C-terminal end in exon 7. Exon 8 of the SER-1B isoform corresponds to an extended region of the 3 0 UTR of SER-1A isoform. Exons of both isoforms are indicated by black boxes separated by introns.

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isms suggests that the protein is evolutionarily conserved even in distant organisms. Furthermore, high degree of homology between SER-1A and SER-1B also suggests that all SERCA pumps are similar in their conformations. As already mentioned, ser-1 is alternatively spliced at the Cterminus to yield two isoforms, a phenomenon also observed in mammals (Ozog et al., 1998). Alternative splicing of SERCA gene transcripts produces mRNAs encoding proteins with different C-termini and different 3 0 untranslated regions (UTRs) which gives rise to differences in translational ef®ciencies and post-translational modi®cations (Mertens et al., 1995). Thus, a similar role may also be ascribed to the two SER-1 isoforms in C. elegans, which arise as a result of alternative splicing of the same transcript at the 3 0 end. 3.2. SER-1 is expressed in various tissues in C. elegans To visualize the expression of ser-1gene, we used the gfp reporter gene (see Section 2.2). Transgenic animals carrying the ser-1::gfp fusion constructs showed expressions of GFP in various tissues during all developmental stages. Green ¯uorescent protein expressed throughout the body, but more strongly in body-wall muscles from early embryonic stages and persisted through adult stages (Fig. 3A±C). Fluorescence was also detected in several neuronal cells. The ventral nerve cord with distinct cell bodies (Fig. 3G) and excretory cells extending antero±posteriorly along the body also showed strong GFP expression (Fig. 3H). Using dsRNAs targeted to GFP in a ser-1::gfp transgenic line (see Section 2.4), tissue-speci®c GFP expression was observed in vulval muscles and in the isthmus and terminal bulb regions of the pharynx, and neuronal cells (Fig. 3D±G). On the other hand, GFP expression in body-wall muscle was completely reduced (Fig. 3D). Thus, vulval muscles, pharyngeal muscles, and neuronal cells were resistant to gfp RNAi, a phenomenon already observed elsewhere where tissue-speci®c interference was observed and nonstriated vulval muscles were shown to be resistant to RNAi (Fire et al., 1998b). However, the underlying mechanism for such tissue-speci®c interference is not known. Taken together, GFP expression pattern of SER-1 suggests that the protein is expressed widely in C. elegans as observed in mammals (Wu et al., 1995). Additionally, SERCA pumps in C. elegans may play important roles in neuronal activities and muscle functions. To further localize SER-1 in situ, we stained wholemount wild-type C. elegans with anti-SERCA antibodies (see Section 2.3). Distinct staining was observed in pharyngeal muscles and vulva epithelial cells (Fig. 4A,B). Reduced but prominent signals were also observed in excretory cells extending antero-posteriorly along the whole body (Fig. 4C). Hence, these staining patterns were in conformity with the GFP expression patterns observed earlier. However, no body-wall muscle or vulval muscle stainings were observed even though strong GFP expressions were

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Fig. 2. Amino acid sequence alignment of C. elegans SERCA (SER-1A and SER-1B) and vertebrate SERCAs. The predicted protein sequences of SER-1A and SER-1B are aligned with rabbit skeletal SERCA (rabbit SERCA1fast, Accession No. P11719), mouse SERCA2a (Accession No. CAA11450), mouse SERCA2b (Accession No. CAB72436) and human SERCA3 (Accession No. AAC24525). Shaded boxes indicate the regions of identity among the different SERCAs.

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Fig. 3. gfp expression under the control of the ser-1 promoter. Transgenic animals expressing ser-1::gfp were observed under a ¯uorescence microscope and photographed. (A) An embryo inside the eggshell shows strong and widespread GFP expression. Expression is stronger in certain areas and along the body-wall muscles. (B) A larva showing body-wall muscle cells, nerve cord, and pharyngeal muscles expressing GFP. (C) An adult animal showing GFP expressing in body-wall muscles (shown by an arrow) from anterior head (left) to posterior tail (right). The twist of the body is due to the roller phenotype of this transgenic animal. (D) An adult progeny of ser-1::gfpline after injection of gfp dsRNA showing residual expression in vulval and pharyngeal muscles. Body-wall muscle expression has been completely interfered by RNAi. Magni®ed views of pharyngeal expression (E), and vulval muscle (arrow) and epithelial cell expression (arrow head) (F). (G) Residual expression of GFP in neuronal cells, particularly in the ventral nerve cord (arrow) of ser-1::gfp transgenic line after gfp RNAi. (H) Magni®ed view of the mid-body region of a transgenic showing excretory cell expression (arrow). Each scale bar indicates 50 mm.

already detected in these tissues (Fig. 3A±F). Probably, C. elegans SERCA isoforms are also tissue speci®c, as seen in mammals. Distinct tissue-speci®c expressions of SER-1 in whole-mount animals can be achieved when isoform-speci®c C. elegans antibodies are generated.

3.3. SER-1 is essential during embryogenesis, larval growth and survival In order to assess the function of the SER-1 isoforms directly, we performed dsRNAi (see Sections 2.4 and 2.5)

Fig. 4. Immunostaining of wild-type animals with mammalian SERCA antibody. Wild-type adult worms stained with anti-SERCA antibodies show staining in (A) pharyngeal muscles, (B) vulva epithelial cells (arrow head), and (C) excretory cells extending along the antero-posterior length of the body (arrow). Bar, 50 mm.

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Fig. 5. Genomic structure of SER-1 isoforms depicting the regions used for RNAi experiments in this study. Black boxes indicate the lengths of DNA (ser-1A and ser-1B) ampli®ed by PCR from the cosmid K11D9 and used as templates for in vitro transcription and RNAi (see Section 2.4). These DNA fragments are unique sequences speci®c for each isoform and hence, they were used for RNAi experiments to examine possible isoform-speci®c effect.

and examined the effects of interference at the cellular level. It is already known in C. elegans that dsRNAs interfere with gene expression of a target gene in a sequence-speci®c manner (Fire et al., 1998b; Montgomery et al., 1998; Timmons and Fire, 1998). Double-stranded RNAs targeted to speci®c C-terminal sequences of each isoform were prepared from genomic DNA by in vitro transcription (Fig. 5, see Section 2.4). Injections were carried out in adult body cavity and phenotypes of F1 progeny were observed. As a control, gfp dsRNAs were injected into wild type animals. As shown in Table 1, wild-type worms receiving no injection or gfp dsRNAs produced progeny with little or no obvious defects. On the other hand, hermaphrodites injected with dsRNAs targeted to each SER-1 isoform showed severe phenotypes (Fig. 6A±F and Table 1). First, we examined the survival / lethality rate in all progeny of ser-1A (RNAi) worms. Majority of the embryos (approximately 65% of the total embryos) showed lethal phenotype from late gastrulation and hence designated as `dead embryos' (Fig. 6B,D). A fraction of the remaining hatched embryos (approximately 29%) showed early larval (L1) lethality (Table 1 and data not shown). The eggshells around the dead RNAi embryos were readily apparent as observed in wild-type embryos (Fig. 6A,C). Although it was evident that the RNAi embryos underwent a number of cell divisions (late gastrulation stage containing more than 200 embryonic nuclei), they failed to undergo any obvious morphogenesis. These

embryos were mostly arrested as balls of cells and in some cases showed aggregated appearance (Fig. 6B,D). The remaining embryos that hatched and developed as L1 larvae were found to be completely paralyzed with no signs of pharyngeal pumping or locomotion and hence designated as `dead larvae' (data not shown). Under the Nomarski differential interference and polarized light microscopy, the dead larvae showed signs of highly disorganized internal body parts including the developing embryos, body-wall muscle cells, and the intestine (data not shown). The worm body further showed a ¯attened appearance with signs of complete paralysis. Wild type embryos or wild-type adult worms were found at very low frequencies in these plates indicating a highly penetrant phenotype for ser-1A (Table 1). We then checked the phenotypes in the progeny of ser1B(RNAi) worms. In contrast to ser-1A(RNAi) worms, progeny of ser-1B (RNAi) animals showed very low frequencies of arrested or dead embryos (Table 1). Though the majority of the eggs hatched, these larvae showed no signs of growth beyond the L1 stage and exhibited very slow pharyngeal pumping. Such a phenomenon was observed for a large number of these progeny even after 6 days of egglaying by the parents and hence designated as `arrested' (Table 1). These L1 arrested larvae also moved slowly and the movement was often kinky (Fig. 6F). The body cavity of these arrested larvae were further characterized by the appearance of several vacuoles indicative of cellular degeneration (compare Fig. 6F with Fig. 6E).

Table 1 Effects of SERCA RNAi on wild type animals a

WT gfp RNAi ser-1A RNAi ser-1B RNAi Ser-1A 1 1B RNAi

Total number

Dead embryos

Dead L1

Arrested L1

WT adults

485 472 434 493 456

12 (2.5%) 39 (8.3%) 284 (65.4%) 44 (9%) 280 (61%)

0 0 128 (29.5%) 28 (5.7%) 161 (35%)

0 0 0 402 (81.5%) 0

473 (97.5%) 433 (91.7%) 22 (5%) 19 (3.8%) 18 (4%)

a Double-stranded RNAs corresponding to gfp, ser-1A, ser-1B or ser-1A 1 ser-1B were injected to the wild-type hermaphrodites at a ®nal concentration of approximately 400 ng/ml. The Fl progeny of the injected parents were examined for phenotypes (see Sections 2.5 and 3.3). The unhatched embryos or the embryos that incompletely hatched were designated as `dead embryos'. These dead embryos were mostly arrested as balls of cells. The L1 larvae which were completely paralyzed and showed no signs of movement or pharyngeal pumping were considered as `dead L1'. The L1 larvae which showed no signs of growth beyond the L1 stage and exhibited very slow pharyngeal pumping were designated as `arrested L1'.

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Fig. 6. Phenotypes of ser-1A (RNAi) worms (see Section 2.5). (A) Wild-type embryo at late gastrulation, and (C) during comma stage. (B, D) Dead embryos during late gastrulation resulting from ser-1A RNAi. These embryos showed little or no signs of obvious morphogenesis and were mostly arrested as balls of cells. (D) A dead embryo at comma stage showing aggregated appearance of cells from ser-1A RNAi. Compare (B) and (D) with the wild- type embryos in (A) and (C). In all cases, the eggshells were apparent. (E) A wild-type L1 larva. (F) A growth-arrested L1 larva from ser-1B RNA injected parents showing a kinky movement. ser-1B (RNAi) animals were characterized by the presence of a number of vacuoles within the body cavity (shown by arrows). Compare (F) with the wild-type L1 larva shown in (E). Bar, 20 mm

Next we examined the effect of dsRNAs targeted to both isoforms simultaneously. When dsRNAs of ser-1A and ser1B were co-injected, the resulting phenotype such as embryonic or larval lethality similar to ser-1A dsRNA injection alone was observed (Table 1). However, no arrested larvae at the L1 stage were observed in these experimental groups. Interestingly, when dsRNAs targeted to an approximately 760 bp region in the exon 5 (amino acid residues 375± 627) were injected, terminal phenotypes similar to ser-1A (RNAi) or coinjection of two isoforms were observed (data not shown). From the results achieved from RNAi experiments in the present study suggest a possible role for SER-1A during embryogenesis and survival of the worms. On the other hand, severe defects resulting from RNAi can also be explained by the differences in the amount of coding sequence chosen for the purpose. It is worth noting that the terminal phenotypes resulted from sequence corresponding to the last 65 amino acids of SER-1A, whereas the L1 larval-

arrest phenotype resulted from sequence corresponding to the last ten amino acids of SER-1B. Thus it is possible that the differences in the phenotypes are re¯ected in the amount of coding sequence and that ser-1B (RNAi) re¯ects a weaker RNAi effect. Since both the isoforms differed at the C-terminal ends only, it was impossible to select additional coding sequences for SER-1B. Taken together, our results indicate that SER-1 plays an important role during embryogenesis, survival and growth of the worms. Moreover, SER-1A seems to be very essential during the early stages of embryogenesis and survival of the animals, while SER-1B seems to have a more essential role during growth and development of the worms. Thus, both isoforms seem to display a developmental stage-speci®c function in C. elegans. Since SERCA was shown to express in body-wall muscles and pharyngeal muscles of C. elegans, we sought to investigate its role in these tissues. Locomotory and pharyngeal pumping activities were carefully examined in ser-

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1A and ser-1B (RNAi) animals and compared with wild-type animals. Since ser-1A (RNAi) animals resulted in early larval or embryonic lethality, the phenotypes were dif®cult to assess in these animals. As already mentioned earlier in the text, ser-1B (RNAi) animals that were arrested at L1 larval stages exhibited very slow moving speeds when compared to wild-type (data not shown). When pharyngeal pumping rate was recorded for these animals, an abnormally reduced pumping (18 ^ 6 times/min, n ˆ 8) was observed in comparison to wild-type L1 larvae (pumping rate was recorded 195 ^ 12 times/min, n ˆ 8) and wild-type adult animals (pumping rate was recorded 228 ^ 11 times/min, n ˆ 8). Thus, it is probable that SER-1B isoform could be essential in body-wall muscle and pharyngeal muscle contractions. Although pharyngeal muscles were already known to escape the effect of RNAi (Fig. 3E; Fire et al., 1998b), it is still possible that ser-1 RNAi may be effective to some degree in the pharyngeal muscles thereby reducing the pumping rate in the animals. At the same time, the fact that the reduced pumping in L1 arrested larvae was likely a consequence of arrested growth cannot be ruled out. In C. elegans, calcium channels, like inositol triphosphate (IP3) receptor or ryanodine receptors (Ryr) are known to regulate intracellular Ca 21 levels (Maryon et al., 1998; Baylis et al., 1999). Reports have also shown that these channels demonstrate a high degree of structural and functional conservation from nematodes to mammals (Baylis et al., 1999). Moreover, calcium channels and calcium pumps (like SERCA) are the two opposing factors that control the cytosolic Ca 21 transients (Camacho and Lechleiter, 1993). Amongst several functions implicated for SERCA in higher animals, the generation of Ca 21 waves by the SERCA pumps in various cell types have been well studied (Montero et al., 1997). For example, overexpression of SERCA 1 and 2b causes increased frequency of IP3- induced Ca 21 waves (Camacho and Lechleiter, 1993, 1995), and overexpression of calreticulin, a lectin chaperone located in the ER, modulates IP3-iduced Ca 21 release by targeting SERCA 2b (Camacho and Lechleiter, 1995; John et al., 1998). Hence, SERCA, as already discussed elsewhere (Misquitta et al., 1999), occupies one of the pivotal roles in signal transduction. The majority of the proteins that directly or indirectly associate with SERCA and have been studied extensively in higher animals have also been identi®ed and characterized in the nematode C. elegans (The C. elegans Sequencing Consortium, 1998). Thus, C. elegans SERCA pump may also occupy a similar role in the nematode system implicating an evolutionary conserved function for the protein. 3.4. Conclusions 1. A single SERCA gene (ser-1), alternatively spliced to form two isoforms, was identi®ed in the C. elegans genome. Sequence homology shows that the two isoforms, SER-1A and SER-1B are highly conserved when compared with other organisms.

2. The expression pattern of SER-1 using the gfp reporter gene and immuno¯uorescence indicate a widespread expression in C. elegans. It expresses in neuronal cells, body-wall muscles, pharyngeal and vulval muscles, vulva epithelial cells, and excretory cells. 3. RNA-mediated interference with isoform-speci®c sequences of ser-1 showed severe phenotypic defects in RNAi worms. Double-stranded RNAs targeted to SER1A isoform resulted in terminal phenotypes, such as embryonic lethality, suggesting its essential role during embryogenesis and survival of the worms. On the other hand, ser-1B RNAi resulted in L1 larval arrest rather than early lethality suggesting that the isoform is essential for the growth of the worms. Taken together, these data suggest that the two isoforms may have developmental stage-speci®c functions in C. elegans, in addition to other essential functions. 4. C. elegans SERCA may have an important role in signal transduction, similar to higher animals, suggesting a possibility of a well-conserved mechanism of action of the protein even in a distantly related organism.

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