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The SL1 trans-soliced leader RNA performs an essintial embrvonic function In Caenorhabditis elegans that can also be supplied by SL2 RNA Kimberly C. Ferguson, Paul 1. Heid, and Joel H. ~othman '

Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706 USA

Covalent joining of leader RNA exons to pre-mRNAs by trans-splicing has been observed in protists and invertebrates, and can occur in cultured mammalian cells. In the nematode Caenorhabditis elegans, -60% of mRNA species are trans-spliced to the 22-nucleotide SLI leader, and another -10% of mRNAs receive the 22-nucleotide SL2 leader. We have isolated deletions that remove the rrs-1 cluster, a gene complex that contains -110 tandem copies of a repeat encoding both SL1 RNA and 5s rRNA. An SL1-encoding gene alone rescues the embryonic lethality caused by these deletions. Mutations within the Sm-binding site of SL1 RNA, which is required for trans-splicing, eliminate rescue, suggesting that the ability of the SL1 leader to be trans-spliced is required for its essential activity. We observe pleiotropic defects in embryos lacking SL1 RNA, suggesting that multiple mRNAs may be affected by the absence of an SL1 leader. We found, however, that SL1-receiving messages are expressed without an SL1 leader. Surprisingly, when overexpressed, SL2 RNA, which performs a distinct function from that of SL1 RNA in wild-type animals, can rescue the lethality of embryos lacking SL1 RNA. Moreover, in these mutant embryos, we detect SL2 instead of SL1 leaders on normally SL1-trans-spliced messages; this result suggests that the mechanism that discriminates between SL1 and SL2-trans-splicing may involve competition between SL1 and SL2-specific trans-splicing. Our findings demonstrate that SL1 RNA is essential for embryogenesis in C. elegans and that SL2 RNA can substitute for SL1 RNA in vivo.

[Key Words: trans-splicing; C. elegans; RNA processing; embryogenesis; ribosomal RNA]

Received March 28, 1996; revised version accepted April 24, 1996.

Trans-splicing of a small 22- to 39-nucleotide RNA leader sequence onto the 5' end of mRNAs is a mechanism of mRNA maturation that occurs in lower eukaryotes, including trypanosomes, nematodes, trematodes, and Euglena (for review, see Agabian 1990; Nilsen 1993). The spliced leader RNA (SL RNA), the precursor RNA containing the trans-spliced leader sequence, appears to participate in trans-splicing as an SL ribonucleoprotein (RNP) (Bruzik et al. 1988; Thomas et al. 1988; Van Doren and Hirsh 1988; Maroney et al. 1990; Michaeli et al. 1990; Palfi et al. 1991). The nematode SL RNP protein components include the Sm proteins, which also associ- ate with most U snRNAs involved in cis-splicing (Bruzik et al. 1988; Mattaj 1988; Thomas et al. 1988; Van Doren and Hirsh 1988; Maroney et al. 1990). Other RNP components known to participate in cis-splicing, such as the U2 and U6 snRNPs, are also required for trans-splicing in both trypanosomes and nematodes (Tschudi and Ullu 1990; Hannon et al. 199 1; Watkins and Agabian 199 1).

'Corresponding author.

Although the trans-splicing process and certain structural features of the SL RNAs are conserved in organisms that perform trans-splicing, the functions of trans-splicing are not fully understood. In some cases, however, trans-splicing is known to result in the production of functional mRNAs. For example, trans-splicing in trypanosomes apparently serves to process polycistronic mRNAs into monocistronic units; as a result, all mRNAs receive a spliced leader (Walder et al. 1986; Agabian 1990). Similarly, in the nematode Caenorha bditis elegans, processing of polycistronic messages derived from the fraction of transcription units that are organized into operons appears to occur by trans-splicing of the downstream messages to the minor spliced leader SL2 (for review, see Blumenthal 1995).

Although it is clear that trans-splicing acts to process polycistronic messages, it is also likely to perform other functions. Only -10% of all mRNAs in C. elegans receive SL2 and only -25% of all C. elegans genes appear to be organized into operons (Zorio et al. 1994; Ross et al. 1995). Nematode genera other than Caenorhabchtis have not been found to contain operons or an SL2-like leader

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Eerguson et al.

RNA; mRNAs from most of these species are trans- spliced to a leader that is identical to the major trans- spliced leader, SL1, in C. elegans (Krause and Hirsh 1987; Nilsen 1993). Although the function of SL1-trans-splicing is not known, -60% of C. elegans messages are trans-spliced to this leader (Zorio et al. 1994)) implying that SL1 performs other functions beyond the processing of operons. In addition to SL1 and SL2, other spliced leaders have been characterized recently in C. elegans (Ross et al. 1995). These novel spliced leaders are most similar to SL2, but their functions are not generally understood (Ross et al. 1995). Given the existence of these other SL RNAs, it is conceivable that there may be additional roles for trans-splicing in C. elegans.

Although the trans-splicing specificity of some of the SL RNAs has not been well characterized, mechanisms clearly exist to discriminate between SL1 and SL2-trans- splicing. SL2 is not found on the most upstream mRNAs derived from polycistronic messages, and SL1 is not generally found on downstream mRNAs of such operons (Spieth et al. 1993; Zorio et al. 1994). In addition, unlike SL2, SL1 is found on many monocistronic messages. The only requirement for trans-splicing of SL1 onto a pre- mRNA appears to be the presence of an outron, an AU- rich sequence followed by a splice acceptor site but containing no splice donor site (Conrad et al. 1991, 1993, 1995). In contrast, splicing of SL2 onto downstream messages in operons may be coupled to polyadenylation of the upstream mRNA in the polycistronic message (Spieth et al. 1993).

A number of observations suggest that the SL1 leader might facilitate the expression of trans-spliced mRNAs. For example, in many cases, SL1 is spliced close to the initiation codon of the protein coded by a trans-spliced mRNA (Bektesh et al. 1988), and it has therefore been proposed that SL1 may serve to promote efficient translation of these messages (Bektesh et al. 1988; Spieth et al. 1993). Consistent with this notion, recent studies have shown that the SL1 leader can enhance translation in vitro (Maroney et al. 1995). Additional studies have begun to identify the RNA sequences and protein components required for trans-splicing in vitro and in vivo (for review, see Agabian 1990; Nilsen 1993; Blumenthal 1995); however, the requirement for neither trans-splicing nor a trans-spliced leader have been demonstrated directly.

We describe the characterization of mutations that eliminate the tandemly duplicated copies of the SL1 RNA gene and demonstrate that SL1 RNA is essential for normal embryonic development and viability. Fur- thermore, we show that the essential function of SL1 RNA can be provided by SL2 RNA. Elimination of zygotic SL1 RNA results in the inappropriate trans-splicing of the SL2 leader onto normally SL1-trans-spliced mRNAs, suggesting that the mechanism that discriminates between SL1 and SL2-trans-splicing may involve competition between the two RNAs for trans-splicing machinery. This observation may explain our finding that whereas the lack of zygotic SL1 RNA affects many aspects of embryonic development, a number of nor-

mally SL1 -trans-spliced mRNAs produce functional products even in the absence of zygotic SL1.

Results

Identification of mutations that delete the rrs-1 gene cluster and isolation of rescuing clones

In an effort to identify zygotically transcribed genes essential for early stages of embryonic development in C. elegans, we isolated two allelic loss-of-function mutations (e2482 and wl ) that lead to early defects in embryogenesis and late embryonic arrest. To investigate the function of the corresponding genomic region, we identified molecular clones that rescued these mutations.

e2482 and wl were localized to an -500-kb interval by mapping them relative to restriction site polymorphisms (Fig. lA,B). Pools of cosmid clones corresponding to this interval were transformed into e2482/+ animals and tested for their ability to rescue embryonic lethality. From the one rescuing pool (pool 2, Fig. 1B; Table l ) , a single cosmid (K07H12) was identified that is sufficient for rescue (Table 1). Rescue was indicated by a decrease in the fraction of arrested embryos produced by transformed heterozygotes and the presence of viable e2482 homozygous larvae (Table 1). As expected, rescue was never complete, as the transformed DNA assembles into an extrachromosomal array that is inefficiently trans- mitted during cell division (Mello et al. 199 1). Some rescued animals survived to adulthood, however, and it was possible to propagate homozygous mutants for many generations that were transgenic for the rescuing sequences.

Fragments derived from K07H12 were further tested for rescue, and an - 1-kb fragment (1-kblK07H12; Table 1) was found to rescue both e2482 and wl . The sequence of a 985-bp subclone of this fragment (not shown) was found to be nearly identical to the published sequence of a 980-bp repeat from the 110-kb rrs-1 cluster (Nelson and Honda 1985). Previous analysis of repeats from the rrs-1 cluster showed that each contains one gene encoding 5s rRNA and one encoding SL1 RNA, a 105-nucleotide RNA that includes the 22-nucleotide SL1 leader (Nelson and Honda 1985; Krause and Hirsh 1987) (Fig. 1C). There were 21 bp that differed between the 985-bp sequence of our rescuing subclone and the published sequence of the 980-bp repeat. Significantly, the 5s rRNA and SL1 RNA coding regions were identical between the two sequences. These observations suggest that the 985-bp subclone that rescues e2482 and wl does not encode an activity that is unique from other rrs-1 repeats, consistent with our finding that other repeats from the cluster were also able to rescue these mutations (not shown). These results also suggested that wl and e2482 might be deletions that remove some or all of the rrs-1 repeats.

To examine whether wl and e2482 are deletions of the rrs-1 cluster, genomic DNA prepared from homozygous mutant embryos was analyzed. Southern blot analysis showed that few or no rrs-1 repeats were present in either mutant (Fig. 2A-C). Moreover, analysis of genomic

1544 GENES & DEVELOPMENT




C. elegans SL1 RNA is essential for embryogenesis

VR unit Figure 1. Genetic and physical maps of him-5 unc-76 rrs-1 dpy-21 par4 - - - - the region to which e2482 and wl map. (A) . 0 . . Genetic map of a portion of the right arm

0 . . . 0 . . .

0 . of linkage group V. e2482 and wl map ap- . . . . . proximately halfway between the genes unc-76 and dpy-21. The location of the

R 1 2 B 9 - rrs-1 cluster was determined previously by W04GlO

rrs-1 cluster - F16GS - 5 0 k b mapping of the position of a polymorphism - that is immediately adjacent to the cluster

-K07H12 I u - I (Nelson and Honda 1985). (B) Physical map W P ~ ,I '\ stplos W P ~ of a segment of the region shown in A.

/ \

,/ SL1 5 s rRNA \ \ RESCUE

4-- + \

1 SI;I site 985

A SmA3 AATGG - Sm Site AATTTTGG

b ~ r n - ~ l u AA GACGCGTC TGG -

wP1, stP108, wP3, and wP2 are polymorphism~ used to map e2482 and wl. The cosmid clones contained in the rescuing pool 2 (K07H12, F16G5, W04G10, and R12B9) and the position of the 110-kb rrs-1 cluster are shown above the line. (C) Map of the 985-bp rescuing subclone derived from cosmid K07H12. The fragment is ar- bitrarily numbered from nucleotide 1, the first nucleotide of the left BamliI site, to nucleotide 985, the last nucleotide of the right BamHI site. The location (boxes) and direction of transcription (arrows) of the SL1 (nucleotides 127-231) and 5s rRNA

(nucleotides 408-526) transcription units, the position of the 435-bp SLl+, 5s-rescuing fragment, and the position of the 740-bp SL1 -, 5s + fragment are shown. (B) BamHI; (Bm) BsmI; (Br) BseRI; (T) ThaI. The table (right) indicates whether the clones shown in C and D are able to rescue the embryonic lethality of e2482. ( D ) Sequences of the wild-type Sm-binding site in SL1 and the mutant Sm sites in the constructs SmA3 and Sm-Mlu. Shadowing indicates extra nucleotides derived from ligation of the MluI linker.

DNA digested with a restriction enzyme that releases sufficient quantities of maternally supplied 5s rRNA to the rrs-1 cluster as high molecular weight fragments de- complete embryogenesis and hatch. In contrast, a 740-bp tected no remnants of the rrs-1 cluster in either wl or fragment containing the 5s rRNA gene alone (SL1- / e2482 embryos (Fig. 2D). Thus, both mutations appear to 5s +, Fig. 1C) did not rescue embryonic lethality (Table delete the entire rrs-1 cluster. 1). In addition, the development of 12 homozygous mu-

tant embryos from these transgenic lines was followed and none of the mutant phenotypes appeared to be de-

The SL1 RNA-encoding gene is necessary to rescue tectably altered by the presence of the 5 s rDNA (Fig. the embryonic lethality of deletions of the rrs-1 cluster 3BID). Thus, zygotic 5S rRNA is neither necessary nor To test whether the embryonic lethality caused by these sufficient to rescue these mutants. rrs-1 deletions results from the lack of SL1 RNA, 5s rRNA, or both, constructs encoding each of these RNAs were tested individually. A construct containing a 435- bp sequence from the subcloned rrs-1 repeat of K07H12, including the entire SLl gene and only 25 nucleotides of the 5s rRNA coding region (SL1 + 15s - , Fig. 1 C), was found to be sufficient to rescue the embryonic lethality of both alleles (Table 1). Embryonic lethality was rescued with approximately the same efficiency as with the entire 1-kb rrs-1 repeat (Table 1). Unlike the animals rescued with the 1-kb repeat, however, most of the defi- ciency homozygotes rescued with the SL1 gene alone died early in larval development with no other obvious phenotypes, and none survived to adulthood. Of 10 rescued embryos followed throughout embryogenesis, all embryos looked similar to wild-type embryos and under- went normal differentiation and morphogenesis, demon- strating that the SL1 gene rescues all conspicuous embryonic defects (Fig. 3A,C). This observation also demonstrates that zygotic 5s rRNA is not required until postembryonic stages, implying that embryos contain

The Sm binding site in SLI RNA is essential for embryonic viability

SL1 RNA contains a highly conserved Sm binding site that binds nematode and mammalian Sm proteins (Bruzik et al. 1988; Thomas et al. 1988; Van Doren and Hirsh 1988). The Sm binding site is present in many trans-spliced leader RNAs and in most snRNAs that direct cis-splicing (Bruzik et al. 1988; Mattaj 1988; Nilsen et al. 1989; Ross et al. 1995). The Sm site, and the proteins that bind to this site, appear to be required for various aspects of small nuclear RNP (snRNP) assembly, localization, and stability (Mattaj 1988; Jones and Guth- rie 1990; Rymond 1993; Roy et al. 1995). Mutation of the Sm-binding site in the SL RNA of the nematode Ascaris lumbricoides abolishes trans-splicing in vitro (Maroney et al. 1990). Additional experiments demonstrated that two elements in the Ascaris SL RNA are sufficient for trans-splicing activity in vitro; one of these elements includes the Sm-binding site (Hannon et al. 1992). We

GENES & DEVELOPMENT 1545




Ferguson et al.

Table 1. Transformation rescue experiments - - - - -- . - - -

Percent rescue of embryonic lethality No. of rescued lines1

Clone tested total no. of lines e2482 (no.)" wl (no.)" Embryonic rescueb

None Pool 2 K07H12 1 kbIK07H12 SL1+ 15s - SLl - /5S + SmA3 Sm-M l u

N.A. 212 212

10113 415 01 7 0120 018

0 (1688) N.D. N.D.

36 (2287) 35 (1476)

N.D. N.D. N.D.

N.A. + + + + -

Heterozygous e2482 or wl animals were used for transformation experiments (see Materials and methods). The average percentages of arrested embryos for the parental lines were 25.3% for e2482 and 25.9% for wl, as expected of heterozygous animals carrying a recessive lethal mutation. Percent rescue was calculated using the formula lOO[l - (average percent arrested embryos observed1 average percent arrested embryos from the parental strain)]. (N.A.) Not applicable; (N.D.) not determined. "Total number of progeny counted, including arrested embryos and larvae. b~mbryonic rescue was scored as + if the average percentage of arrested embryos was below 20%, and arrested elongated embryos andlor extra Unc-76 larvae (significantly greater than the -2% Unc-76 recombinants usually observed in the parental strain) were observed (see Materials and methods). "Lines that showed no apparent rescue with the 1-kblK07H12 and SL1+ 5s fragments were not used in the calculation of percent rescue. d~ l t hough the 740-bp fragment used in this transformation experiment does not by itself rescue the embryonic lethality of e2482, it appears to provide sufficient levels of 5s rRNA to rescue the absence of 5s rRNA in the deletion mutants. This was demonstrated by transforming the 740-bp fragment together with the 435-bp SL1 fragment. In all seven lines obtained from transformation of the two fragments, transformants gave rise to extra fertile Unc-76 adults. These animals proved to be rescued homomzygous mutants, as they produced a higher percentage (>40%) of arrested e2482 embryos, than do the Unc-76 recombinants (-25% arrested embryos).

created two mutations in the Sm binding site of the C. elegans SL1 RNA (Fig. ID) to test whether this conserved sequence is required for rescue of the rrs-1 deletion mutants. One mutation is a small deletion that re- moves 3 of the 8 bases comprising this site (SmA3); the other is a substitution of these 3 bases with an 8-base sequence (Sm-Mlu) (Fig. ID). We found that both SmA3 and Sm-Mlu abolished rescue in many lines tested (Ta- ble 1). These results imply that SL1 RNA is the relevant rescuing activity within the 435-bp fragment and demonstrate that the Sm site is required for its function, consistent with the notion that trans-splicing of the SL1 leader to mRNAs is an essential process. Thus, SL1 RNA appears to be both necessary and sufficient for rescue of the embryonic lethality of rrs-1 deletions.

The SLI leader is absent from normally SL1 -trans- spliced messages in mutants lacking zygotic SLI RNA

The two rrs-1 deletions appear to remove all SLl-encoding genes and presumably eliminate all zygotically supplied SL1 RNA. There appears to be a substantial pool of maternal SL1 RNA in early embryos (G. Seydoux, pers. comm.), however, and it is conceivable that this pool might be sufficient to sustain some SL1-trans-splicing of zygotically transcribed messages. To determine whether SL1-trans-splicing occurs in embryos lacking zygotic SL1 RNA, we investigated whether the SL1 leader was present on messages that are normally SL1-trans-spliced.

The products of the ges-1, elt-1, hlh-1, and myo-3

genes were selected for these studies. The messages encoded by each of these genes are normally trans-spliced to SL1 (Krause et al. 1990; Spieth et al. 1991; Kennedy et al. 1993; Okkema et al. 1993), and each is embryonically transcribed (Waterston 1989; Krause et al. 1990; Spieth et al. 1991; Kennedy et al. 1993). Moreover, there is no evidence of a maternal requirement for any of these gene products. Thus, analysis of these messages should reveal whether zygotic messages are SL1-trans-spliced. The fate of each message was followed in mutant embryos by use of a PCR-based assay that detects SL1-trans-spliced messages. We found that although SL1 was always detected on each of these messages in wild-type RNA samples, in most samples of mutant embryo RNA tested, SL1 was not detected on any of these messages (Fig. 4; Table 2). Thus, the absence of zygotic SL1 RNA correlates with the absence of an SLl leader sequence from at least four normally SL 1 -trans-spliced mRNAs.

Absence of zygotic SLI RNA leads to pleiotropic defects during embryonic development

The finding that SL1-trans-splicing does not appear to occur in rrs-1 deletion mutants suggested that the function of zygotic messages requiring SL1-trans-splicing for their proper expression may be reflected in the phenotype of embryos carrying these deletions. Consistent with the prevalence of messages that are trans-spliced to the SL1 leader in C. elegans [-60% of all mature mRNAs (Zorio et al. 1994)], we observe widespread defects in mutant embryos lacking the SL1-encoding genes





C elegms SL1 RNA is essential for embryogenesis

Figure 2. Analysis of the rrs-I cluster in wl, e2482, and wild- type animals. (A] Southern blot of genomic DNA probed with the 985-bp rescuing repeat sequence. Genomic DNA from animals of the indicated genotype [wl, e2482, N2 (wild-type)] was digested with BamHI, which fragments the rrs-1 cluster into -1-kb repeats (arrow). Quantitation of the amount of 1-kb repeat from each mutant indicated that the wl DNA preparation contained -2% of the wild-type level of 1-kb repeat, and the e2482 preparation contained -8% of the wild-type level (not shown). The presence of rrs-l sequences in the mutant DNA is attributable to contaminating wild-type DNA in the mutant preparations. (B] A different region of the blot shown in A ex- posed for a longer period to allow detection of another (low-copy number) genomic sequence that hybridizes to the repeat sequence. This low-copy sequence was used as a control to determine whether any or all of the repeats detected in A derive from contaminating wild-type DNA in the mutant embryo DNA preparations. Shown is a representative control fragment (2.8 kb, arrow) that flanks the cluster, as determined by Southern analysis of flanking cosmids (not shown). The amount of this fragment in the mutant DNA preparations corresponded to much less than one copy per diploid genome (not shown). This indicates that this sequence is deleted in both mutants and can therefore be used as a control for the amount of contaminating wild-type DNA in the mutant DNA preparations. Quantita- tions showed that the ratio of the amount of this fragment in the mutant DNA to that in wild-type was comparable to the ratio seen for the 1-kb rrs-l repeat. This finding indicates that both mutants contain very few, or no, rrs-1 repeats. Positions of molecular weight markers are shown at the left in kilobases. (C] The blot shown in A and B was stripped and reprobed with cosmid ZC130, which contains sequences from chromosome I. This provided a control that was used to normalize for the total amount of DNA loaded in each lane. (Dl Analysis of wl, e2482, and wild-type (N2) DNA preparations digested with PstI, which does not cut within a typical rrs-1 repeat. Positions of molecular weight markers are shown at the left in kilobases. Small amounts of wild-type high molecular weight fragments were detected after longer exposures (not shown); this is attributable to contaminating wild-type DNA. Because neither allele contained any smaller rrs-1 -hybridizing fragments detectable even after long exposures, the rrs-1 cluster appears to be deleted com- pletely in both mutants.

Figure 3. Phenotypes of wild-type embryos, arrested mutant embryos, and embryos rescued with an SL1 RNA-encoding construct. (A) Wild-type (N2] pretzel stage embryo. This embryo has elongated into a worm that will hatch shortly after this stage. (B) Homozygous e2482 terminally arrested embryo. A number of differentiated cell types can be seen. (Arrows) Epi- dermal nuclei; (arrowhead] programmed cell death corpse. The phenotypes of wl embryos are not distinguishable from those of e2482 embryos (not shown]. (C) e2482 embryo rescued with the 435 bp SL1+ 15s - fragment. The embryo shown is at the pretzel stage, as in B. This embryo hatched and showed an Unc-76 phenotype, implying that it carried e2482, which is marked with the closely linked unc-76(e911) mutation. After hatching, this animal died early in larval development, as expected of an e2482 homozygote rescued with the SL1 gene, but without the 5s rRNA gene. (Dl A representative e2482 embryo produced by an e2482 heterozygote transgenic for the 740-bp SL1- 15S+ construct. It was not practical to determine whether this particular embryo carries the transformed DNA, as the lethal phenotype precludes scoring of the transgenic marker (rol-6) gene. However, like the untransformed parent strain, the transformed heterozygotes produced -25% arrested embryos (Table 1), and all of the arrested embryos showed terminal phenotypes that were indistinguishable from the phenotypes of nontransformed e2482 embryos. Bar, - 10 pm.

(Figs. 3B, 5, and 6). The earliest phenotypes that we dis- cern in both e2482 and wl mutants include defects in cell migrations associated with gastrulation (Fig. SA-F] and premature cell division in the early endoderm lineage. Both phenotypes appear reproducibly at the onset of gastrulation (-28-cell stage), indicating an early requirement for zygotic SL1 RNA. Interestingly, it is at approximately this stage in embryogenesis that the maternal pool of SL1 RNA begins to decrease significantly (G. Seydoux, pers. comm.). We also observe a defect in compaction during mid-embryogenesis: In mutant embryos, blastomeres fail to adhere properly at the stage in which they normally become tightly packed in wild-type embryos (Fig. SG,H). In addition, defects in specification of certain cell types are observed: Some cells (e.g., the locomotory muscles of the body wall] are made at reduced numbers, and others (e.g., muscles of the feeding organ, or pharynx] are often absent entirely (Fig. 6A-Dl. Finally, the mutant embryos uniformly fail to undergo





Ferguson et al.

controls wt mut

RT I + +'++I r= I + + I

hlh- 1 I

Figure 4. RT-PCR analysis of SLl -trans-splicing of embryonic messages in wild-type (N2) and e2482 mutant embryos. For each message, two representative samples are shown for each genotype. A + or - sign above the blot indicates whether a reverse transcription was performed. - RT indicates RNA was used directly in a PCR reaction. The same RNA samples were used for the + RT and - RT reactions. Controls in which only E. coli was used to make the extracts are indicated as - emb. RT-PCR was then performed on these mock extracts (see Materials and methods). For elt-1 and myo-3, arrows point to the positions of the expected products; the origin of the other product in each case is not known. [Lanes 1, 2) Wild-type SL1; (lanes 3 4 , e2482 SLl; (lanes 5,6) wild-type SL1, - RT controlsi (lanes 7,8) e2482 SL1, - RT controls, (lanes 9,10) no embryonic RNA added, + RT-PCR controls. Positions of molecular weight markers of the indicated size in base pairs are shown at the right.

normal morphogenesis later in embryogenesis and arrest as unelongated masses of differentiated cells that are not fully enclosed by epidermal tissue (Fig. 3BJ. This range of phenotypes is consistent with an essential role of zygotic SL1 in the expression or metabolism of multiple embryonic mRNAs.

Although embryos lacking the SL1-encoding gene cluster show severe developmental defects, some of the phenotypes of e2482 and wl embryos suggest that expression of a subset of mRNAs normally receiving SL1 may not be profoundly affected in these mutants. Some cell types, such as the germ-line precursors and the intestine, appear to be properly specified in e2482 and wl embryos (e.g., Fig. 6E-HI. In addition, programmed cell deaths, which require the zygotically expressed ced-3 gene product (Ellis and Horvitz 1986), occur in e2482 and

wl embryos [e.g., Fig. 3BJ, even though ced-3 mRNA is normally SL1-trans-spliced (Yuan et al. 1993). These results indicated that the SL1 leader may not be essential for the expression of some messages that are normally SL 1 -trans-spliced.

Normally SLI -trans-spliced messages are expressed in the absence of the SL1 leader

As reported above, at least some zygotic messages that are trans-spliced to SL1 in wild-type embryos do not receive SL1 in mutants lacking the SLl-encoding genes. To test whether these messages are translated in the absence of the SLl leader, we analyzed for the presence of the hlh-I, myo-3, and ges-1 gene products in mutant embryos. High levels of immunoreactive protein encoded by the hlh-1 and myo-3 genes were seen in the appropriate cells [Fig. 7A-D). Moreover, the gut esterase encoded by the ges-1 gene (Edgar and McGhee 1986) was observed at high levels in the intestine (Fig. 7E,F). These results indicate that the lack of zygotic SL1 RNA does not dra- matically attenuate the expression of these mRNAs.

The selectivity of SL2-trans-splicing is altered in mutants lacking zygotic SLl RNA

The ability of the ges-1, myo-3, and hlh-1 messages to be

Table 2. RT-PCR analysis of SLI -trans-splicing in wild-type and mutant embryos

No. of samples positive for SL1-trans-splicingltotal no. samplesa

mRNA tested N2 (wild type) e248zb

ges-l 515 014 hlh-1 4/4 0/9 myo-3 12/12 116' elt-ld 8/8 2/1OC

"For each wild-type and e2482 sample listed, a portion of the same RNA sample was used in a control PCR reaction in which the reverse transcriptase (RT) step was omitted ( - RT lanes, Fig. 4; see Materials and methods). In addition, two control reactions were performed for each message, in which a portion of a mock extract containing no embryonic RNA was added to the RT-PCR reaction ( - emb lanes, Fig. 4; see Materials and methods). A product was not detected in any of these controls. b ~ o r each e2482 sample in which an SL1 product was not detected, a product was found to be amplified either with primers specific for an internal portion of the messages (not shown), or with SL2 (Fig. 8; Table 31, confirming that these messages were present in the samples. 'The SL1 products detected in the e2482 samples for elt-1 and myo-3 messages may reflect infrequent SL1-trans-splicing of these messages or might instead be the result of contaminating wild-type embryos in the selected population (see Materials and methods). *1n one set of experiments for elt-1 (218 wild-type reactions and 2/10 e2482 reactions), DNase was not added. In subsequent experiments, products were detected in the -RT control reactions for some other messages, so the DNase step was added (see Materials and methods].






Alternatively, another trans-spliced leader, such as SL2, might substitute for SL1 in its absence. Although SLl and SL2 are spliced to the same acceptor site consensus sequence, SL2 is thought to be specifically involved in processing downstream cistrons from polycistronic messages; it is not found on the 5' end of the first mRNA derived from a polycistronic message (Huang and Hirsh

Figure 5. Migration and adhesion defects associated with the lack of zygotic SL1 RNA. (A-D] Migration of the endoderm progenitor cells. The daughters of the endoderm progenitor cell (Ea and Ep] and the germ-line precursor (P,] have begun migrat- ing into the interior of a wild-type embryo (A) but remain on the surface of an e2482 embryo (B], -100 min postfertllization. (C] A wild-type embryo -120 min postfertilization after Ea and Ep have divided; all four granddaughters of E have moved into the interior of the embryo. (D) An e2482 embryo at the same stage as shown in C; all E granddaughters remain on the surface of the embryo (Eaa and Epa are out of the plane of focus]. (E,F] Tracings of positions of the muscle cell progenitor D and its descendants (Da, Daa, Daaa, and Daaap], during development from 100-225 min after first cleavage (wild-type] and 10W00 min after first cleavage (~2482). The largest circle represents the largest cell (Dl; the smallest circle represents the smallest cell (Daaap). Ar- rows indicate the general direction of cell movements. (El Mi- gration of D cells in a wild-type embryo; as shown, the cells move anteriorly and also into the interior of the embryo. (F] D cell migration in an e2482 embryo; the movement of the cells appears largely random, and cells remained on the surface of the embryo throughout most of the period followed. This migration defect was observed in all eight e2482 embryos examined. (G,H) Cell adhesion defects. (G) Cells on the surface of wild-type embryos at -180 min postfertilization appear to undergo a compaction process. (H) Cells on the surface of e2482 embryos at about the same time as in G appear uncompacted and rounded up. Bar, -10 pm.

expressed in the absence of an SL1 leader may indicate that trans-splicing is not required for their expression.

Figure 6. (A-H) Analysis of cell-type specific markers. [(A,C,E,G] wild-type embryo; (B,D,F,H) terminally arrested c2482 embryo]. JA,B] Embryos stained with antibody NE814C6, which recognizes all body-wall muscle cells. Muscles are organized into rows at this stage in wild-type embryos. Reduced numbers of body-wall muscle cells are observed in mutant embryos, and cells are located primarily in the posterior region of the embryo. (C,D] Embryos stained with antibody 3NB12, which recognizes a subset of pharyngeal muscle cells. Few or no 3NB12-positive cells are observed in mutant embryos; two are seen in this embryo (arrows; Dl. (E,F] Embryos stained with antibody 1CB4, which recognizes all 20 intestinal cells, intestinal-rectal valve cells (arrow), pharyngeal gland cells (not shown), and a few neurons (not shown). Approximately the proper number of intestinal cells are observed in mutant embryos, however, morphogenesis of the intestine is aberrant. In- testinal-rectal valve cells are present. (G,H) Embryos stained with antibody OIClD4, which stains the two germ-line precursors (arrows). Although these precursors are present in mutant embryos they do not migrate to their proper location. Bar, -10 km.

GENES &. DEVELOPMENT 1549




Eerguson et al.

bryo, muscle cells are clustered in the posterior, presumably because of cell migration defects {cf. Fig. 5F). The mutant embryos express fewer than the wild-type number of hlh-I-positive muscle cells. On the basis of similar results with other markers (e.g., Fig. 6B), this defect does not appear to be specific for the hlh-I product. (C,D] Expression of the myo-3 product in a wild-type comma stage embryo and a terminally arrested e2482 embryo, respec- tively. The muscles are clustered at the posterior of the mutant embryo, and fewer than normal numbers are seen, as described above. ( E , F ) Embryos stained for the gut esterase activity of the ges-l product. Expression is seen in a wild-type comma stage embryo and a terminally arrested e2482 embryo. Bar, - 10 pm.

1989; Spieth et al. 1993; Zorio et al. 1994). In contrast, SL1 is spliced onto a larger subset of messages but is infrequently spliced onto downstream messages in operons (Spieth et al. 1993; Zorio et al. 1994). Thus, a selection mechanism can apparently discriminate between SL1 and SL2-trans-splicing, depending on the location of the splice site in the pre-mRNA sequence (Spieth et al. 1993). The mechanism of this discrimination process is unknown.

To determine whether the discrimination of SL2- trans-splicing is altered in cells lacking SL1, we examined messages that lack SL1 for the presence of the SL2 leader, using the PCR-based assay described above. Pre- vious studies have found that although SL1 is detectable on ges-1, hlh-1, elt-I, and myo-3 messages in wild-type (N2) embryos, SL2 is not detected on these messages (Krause et al. 1990; Spieth et al. 1991; Kennedy et al. 1993; Okkema et al. 1993) (Fig. 8; Table 3; data not shown). Surprisingly, however, we found that SL2 is fre- quently detectable on the elt-1 and myo-3 messages (Fig. 8; Table 3) in embryos lacking zygotic SL1 RNA. We also detected SL2 on the hlh-I and ges-1 messages, albeit less consistently (data not shown]. The finding that the SL2 leader is trans-spliced inappropriately in rrs-1 deletion mutants indicates that the selection mechanism that prevents trans-splicing of SL2 onto the 5' end of unproc- essed transcripts can be overridden in the absence of SL1 RNA.

SL2 can substitute for SL1 when expressed from a multicopy extrachromosomal array

It is conceivable that functions normally supplied by SL1

are instead provided by SL2 in its absence, thereby allowing the expression of the aberrantly SL2-trans-spliced messages. The embryonic lethality associated with deletion of the SL1-encoding genes, however, demonstrates that although SL2 is trans-spliced onto messages that

Figure 8. RT-PCR analysis of SL2-tmns-splicing in wild-type (N2) and e2482 mutant embryos. For each message, two representative samples are shown for each genotype. The arrows point to the correct size product; the origin of the other products in each case is not known. The reverse transcription reactions were the same as were used for the SL1 analysis (Fig. 4). (See Table 3 for an explanation of the controls.) (Lanes 1,2) wild-type SL2; (lanes 3,4) e2482 SL2. Positions of molecular weight markers of the indicated size in base pairs are shown at the left.





C. eleguns SL1 RNA is essential for embryogenesis

Table 3: RT-PCR analysis of SL2-trans-splicing in wild-type and mutant embryos

No. of samples positive for SL2-trans-splicingltotal no. samplesa

mRNA tested N2 (wild type) e2482

"The same RT reactions used to generate the SL1 data in Table 2 and Fig. 4 were used in these analyses. The - RT controls for these experiments were performed using the SL1 primer and appropriate downstream primer as described in Table 2. Two controls were performed for each message in which a portion of a mock extract containing no embryonic RNA was added to the RT-PCR reaction using the SL2 primer and the appropriate downstream primer; a product was not detected in any of these controls (see Materials and methods]. b ~ h e detection of SL2 in two of the samples might be attributable to rare trans-splicing of SL2 onto myo-3. This has not been reported previously (Okkema et al. 1993); however, such a rare trans-splicing event might be detectable in our experiments because of the large number of samples analyzed. In two additional wild-type samples, a product was detected; however, the signal was not reproducible in multiple PCR attempts. There- fore, these samples were excluded from the above data. 'For each e2482 sample in which a product was not detected, control amplifications were performed using primers specific for an internal portion of the messages to confirm the presence of the elt-1 messages in the samples (not shown).

normally receive only SL1, SL2 cannot substitute fully for SL1 when expressed at normal levels. Because SL2 appears to be trans-spliced less efficiently onto some messages such as hlh-1 andges-1, it is possible that some messages may not receive enough SL2 to be properly expressed in rrs-1 deletion mutants. Unlike the SL1-encoding genes, which are present in >lo0 tandemly re- peated copies, there are few copies of the SL2 gene in the C. elegans genome (Huang and Hirsh 1989; Ross et al. 1995); the failure of SL2 RNA to substitute for SL1 RNA might simply reflect lower levels of the former. Alterna- tively, since SL2 is only -45% identical to SL1 (Huang and Hirsh 1989), the differences between the sequences of these two leaders might account for the inability of SL2 to substitute for SL1 in rrs-1 deletion mutants. To investigate further whether SL1 RNA supplies a function that cannot be provided by SL2 RNA, we asked whether an SL2-encoding gene could rescue deletions of the rrs-1 cluster when expressed at elevated levels from an extrachromosomal array. When exogenous DNA is introduced into worms by transformation, it is generally in- corporated into an extrachromosomal element carrying multiple copies of the DNA; transgenes are presumably overexpressed from such extrachromosomal arrays.

A construct encoding the 110-nucleotide SL2 RNA was introduced into e2482/+ animals and stable transgenic lines established. Transgenic lines were tested for rescue of embryonic lethality. Unexpectedly, we found that the SL2-encoding gene rescued the embryonic le-

thality of e2482 embryos with approximately the same efficiency as the SLI-encoding fragment (Fig. 9; Table 4). As with the SL1 gene alone, most rescued animals arrested as early larvae. Primer extension analysis of RNA prepared from one rescuing transgenic line confirmed that SL2 is overexpressed from the extrachromosomal array (data not shown). In addition, when transgenic lines containing the SL2-encoding construct, as well as the 740-bp 5s rRNA fragment, were established, a fraction of the rescued animals reached adulthood; these adults were invariably sterile. These findings imply that SL2 RNA can substitute functionally for SL1 RNA during embryonic and most of postembryonic development when expressed at higher than normal levels. The ability of SL2 to substitute for SL1, however, appears to be in- complete; unlike an SL1-encoding transgene, the SL2 gene apparently cannot provide the function required for fertility.

Discussion

We report five major findings: (1) Zygotic SLl is essential for a number of events in embryogenesis and for embryonic viability in C. elegans; (2) 5s rRNA is not required zygotically in the embryo but is required for postembryonic development; (3) some messages that are normally SL1-trans-spliced are expressed even without an SL1 leader; (4) in the absence of SL1 RNA, SL2 is inappropriately trans-spliced onto messages that are normally trans-spliced only to SLl; and (5) SL2 can substitute functionally for SL1.

Trans-splicing of SLI appears to be essential for embryogenesis

We have demonstrated that zygotic SL1 RNA performs an essential function beginning early in C. elegans embryogenesis. An SL1 RNA-encoding clone is necessary and sufficient to rescue the embryonic lethality of deletions that encompass the entire 110-kb rrs-1 cluster. Our findings also show that the maternal contribution of 5s rRNA to the oocyte appears to be sufficient to sustain embryonic development to completion.

Figure 9. Rescue of e2482 mutant embryos with an SL2-encoding construct. (A) An e2482 embryo rescued with the SL2 RNA-encoding construct. The embryo shown is at the pretzel stage as in Fig. 3, A and C. This embryo hatched, exhibited an Unc-76 phenotype, and died early in larval development (c.f. Fig. 3C). (B] Terminally arrested e2482 embryo, shown for compar- ison. Bar, -10 pm.





Ferguson et al.

Table 4: SL2 transformation rescue experiments

Percent rescue of Clone No. of rescued lines/ e2482 embryonic Embryonic tested total no. of lines lethality (no.] rescue

None N.A. 0 (2039) N.A. SL2 15/22 42 (4769) +

--

Scoring of embryonic rescue was performed and percent rescue was calculated as described in Table 1. (N.A.) Not applicable.

It is possible that unspliced SL1 RNA may perform an essential function distinct from its role in SL1-trans- splicing per se, as has been shown to be the case for the U1 snRNP, which participates in cis-splicing (Wassar- man and Steitz 1993). Our results and previous data, however, are consistent with the view that trans-splicing of the SL1 leader onto embryonic mRNAs is necessary for its essential function. We have shown that rescue of embryonic lethality depends on a functional Sm- binding site. This site is well-conserved in all nematode SL RNAs and in U snRNAs from fungi to vertebrates (Bruzik et al. 1988; Guthrie and Patterson 1988; Mattaj 1988; Huang and Hirsh 1989; Nilsen et al. 1989) and is required for trans-splicing in vitro (Maroney et al. 1990; Hannon et al. 1992). Previous studies have determined that -70% of mRNAs receive a spliced leader (Zorio et al. 1994); we have shown that at least some messages fail to be trans-spliced to SL1 in rrs-1 deletion embryos. Therefore, our findings demonstrate that trans-splicing does not occur properly in rrs-l deletion embryos, likely accounting for the phenotypes that we observe.

SL1 vs. SL2-trans-splicing

In rrs-1 deletion embryos, SL2 is spliced onto messages that normally receive only SL1 (Fig. 8). The selection process that governs SL1 versus SL2-trans-splicing in wild-type animals may involve competition between SL1 RNPs and SL2 RNPs, the former having a greater affinity for the 5'-most splice acceptor site of a primary transcript. Our results indicate that when SL1 is absent, the SL2 RNP is able to recognize splice acceptor sites in the context of sequences to which the SL1 RNP is normally recruited; this observation is consistent with such a competition model.

The finding that SL2 can be spliced onto messages that are normally SL1-trans-spliced may account in part for its ability to substitute functionally for SL1 in the embryo when overexpressed, despite the divergence in the sequence of the two spliced leaders. We found, however, that the SL2-rescued mutants grow more slowly than those rescued with SL1 (K. Ferguson et al., unpubl.) and that adults from the rescued lines are always sterile. Therefore, SL2 may not be capable of performing all of the functions normally provided by SL1. Alternatively, overexpression of SL2 from an extrachromosomal array may not provide sufficient levels of SL2 RNA to allow

trans-splicing onto all normally SL1-trans-spliced messages, or SL2 may not recognize SL1 splice acceptor sites efficiently, as suggested by our observations that SL2 is found only sporadically on some messages in rrs-1 deletion mutants (not shown).

Zygotic SLI RNA is necessary for multiple events in em bryogenesis

We found that multiple processes during embryogenesis are affected in mutants lacking zygotic SL1 RNA. For example, some cell types, such as epidermis (not shown) and body-wall muscle (Fig. 61, are made at fewer than normal numbers as determined by analysis of cell fate- specific markers. In the case of body-wall muscle, at least three muscle-specific markers, including the products of the SL1-trans-spliced hlh-I and myo-3 mRNAs, are expressed; however, there are uniformly too few muscle cells in mutant embryos. Experiments in which individual muscle cell precursors were laser ablated indicated that this failure to make muscles is preferen- tially restricted to particular muscle cell lineages [J.H. Rothman, unpubl.). Approximately normal numbers of muscle cells are made from the C and D muscle precursors in these mutants; however, the MS muscle progenitor, which normally gives rise to 28 muscle cells, pro- duces very few muscles in mutant embryos. Schnabel (1995) reported that cellular interactions are required to specify muscles arising from MS. Thus, there may be a SL 1 -trans-spliced message required in this signaling process that is affected by the absence of SL1 in rrs-1 deletion mutants. Other phenotypes that we have characterized, such as cell migration and cell adhesion defects, suggest that cell adhesion molecules may not be properly expressed in these mutants. Although SL1 has not been shown directly to regulate a cell-type-specific event, it is noteworthy that another trans-spliced leader in C. elegans, SL4, is expressed specifically in the epidermis (Ross et al. 1995), perhaps indicative of a cell-type-specific regulatory process.

Our findings that SL1 is required for many aspects of C. elegans embryogenesis are consistent with the widespread occurrence and apparent conservation of the trans-splicing process. RNA trans-splicing occurs natu- rally in trypanosomes, Euglena, nematodes, and trematodes (for review, see Agabian 1990; Nilsen 1993), and mammalian cells are also competent to perform trans- splicing when provided with the appropriate substrates (Bruzik and Maniatis 1992). Certain secondary structural elements are common to many known SL RNAs (Bruzik et al. 1988; Nilsen et al. 1989; Nilsen 1993), and all known trans-spliced leaders in nematodes are identical to the C. elegans SL1 leader (Nilsen 1993), with the exception of the leader from the nematode Meloidogyne incognita (which differs from SL1 at one position), C. elegans SL2, and a family of SL2-related spliced leaders in C. elegans (Huang and Hirsh 1989; Ray et al. 1994; Ross et al. 1995). The observation that multiple processes occurring during embryogenesis are affected in mutants lacking zygotic SL1 RNA is consistent with a






requirement for SL1 in the metabolism of multiple embryonic messages; the conservation of trans-splicing suggests that this requirement is likely to be true for other species.

SL1 is not required for expression of some SL1- trans-spliced messages

Given that -60% of messages receive SL1, it is not sur- prising that the lack of SL1 RNA causes severe pleiotropic defects in the C. elegans embryo. Not all aspects of embryogenesis, however, are affected. Although mutant embryos develop more slowly than wild-type embryos, they arrest after the normal number of cell divisions have occurred and appear healthy many hours after their arrest (not shown). In addition, many cell types appear to be properly specified in these mutants (Fig. 6). Thus, differentiation and other cellular functions, such as cell division and programmed cell death, are not generally blocked. In addition, we have shown that products of some normally SL1-trans-spliced messages are expressed.

There are several possible explanations for our findings that normally SL1 -trans-spliced messages appear to be expressed in rrs-1 deletion mutants. First, it is conceivable that some normally trans-spliced messages, like the 30% of mRNAs that are not trans-spliced, may be expressed efficiently without a trans-spliced leader, as has been observed for a modified version of the normally SL1-trans-spliced rol-6 mRNA (Conrad et al. 1993)) or the absence of the SL1 leader may cause subtle defects in expression of these messages that cannot be detected by our methods. Although zygotic SL1 is essential, there is as yet no direct evidence that any SL1-trans-spliced message has an absolute requirement for a trans-spliced leader to be expressed in vivo; analysis of additional gene products in the rrs-1 deletion mutants may identify messages whose expression requires an SL1 leader. Second, some messages may be properly expressed in these mutants by receiving SL1 from the maternal pool of SL1 RNA; however, this pool is not detectable in somatic nuclei past the 50-cell stage (G. Seydoux, pers. comm.). Finally, the inappropriate trans-splicing of SL2 onto normally SL1-trans-spliced messages, such as myo-3 and elt-1, in rrs-1 deletion mutants may allow them to be expressed.

What is the essential role of SL1!

Although no clear role for SL1 has been demonstrated, our results show that it is essential for viability. What function does the SL1 leader contribute to trans-spliced mRNAs? Trans-splicing of SL1 onto messages may provide cis-acting sequences that actively control the stability, localization, or translation of messages. Our findings that SL2, which is quite divergent from the other nematode trans-spliced leaders, can substitute for SL1 during embryogenesis, suggests that there may not be a rigid structural requirement for the primary sequence of the leader per se. Recent evidence suggests that the SL1

leader sequence may improve translatability of a message (Maroney et al. 1995). Alternatively, trans-splicing per se might function to remove inhibitory sequences in the 5'-untranslated region of primary transcripts, thereby allowing them to function. The availability of mutants lacking zygotic SL1 will make it possible to examine the sequences required for the proper function of the leader and to determine what role i t might have in metabolism or translation of a message in vivo.

Materials and methods

Worm culture, strains, and anti bodies

The mutations described here were isolated from C. elegans var. Bristol strain N2 (Brenner 1974). Culturing, mutagenesis, and genetic manipulation of C, elegans were as described by Brenner (1974). Nematodes were grown at 20°C in all experiments. Methods for mounting and viewing C, elegans embryos by Nomarski microscopy have been described previously (Sulston et al. 1983). Embryos were fixed and stained for immunofluorescence as described (Sulston and Hodgkin 1988). Mutant embryos were collected for Nomarski microscopy and immunofluorescence -14-18 hr after being laid, unless otherwise indicated. Wild-type siblings have hatched by this time. Antibodies NE814C6 (Goh and Bogaert 199 1 ), 3NB12 (Priess and Thomson 1987), and 1CB4 (Okamoto and Thomson 1985), were obtained from the MRC-Cambridge collection. OIClD4 (Strome and Wood 1982) was a gift from Susan Strome. Antibody 5-6.1.1.1, which recognizes the myo-3 gene product (Miller et al. 1983), was provided by David Miller 111, and the hlh-1 antibody (Krause et al. 1990) was a gift from Michael Krause. Anti-rabbit and anti-mouse fluorescein-conjugated antibodies were obtained from Sigma. Embryos were stained for gut esterase activity, indicating the presence of the ges-1 gene product, as described previously (Edgar and McGhee 1986). The following mutations on linkage group V were used for mapping and balancing the rrs-1 deletion mutations: sma-1 (e30); rol-4(sc8); unc-61 (e228); unc-76(e911); dpy-21 (e428).

Isolation and mapping of the mutations

e2482 was isolated in a general, genome-wide screen for ethyl- methanesulfonate (EMS)-induced zygotic embryonic lethal mutations that affect early events in embryogenesis. e2482 was mapped between unc-76 and dpy-21 by standard three-factor recombination experiments (Sulston and Hodgkin 1988). Re- combinants between unc-76 and dpy-21 placed e2482 14/30 of the distance from unc-76 to dpy-21. w l was isolated in a targeted embryonic lethal screen designed to identify EMS-induced lethal mutations in the vicinity of the e2482 locus. In this latter screen, L4-stage rol-4(sc8) unc-61 (e228)/sma-I (e30) unc- 76(e911) hermaphrodites were mutagenized with 50 mM EMS, and F, progeny were scored for the absence of either the RolUnc or SmaUnc phenotypes. Absence of either marker indicated the presence of a zygotic lethal mutation. A total of -1 1,000 hap- loid genomes was screened. This screen identified 209 zygotic lethal mutations on the right arm of chromosome V. Comple- mentation tests against e2482 were performed, and one of the 209 mutations ( w l ) was found to be allelic to e2482.

e2482 and w l were mapped relative to the Tcl polymorphism stP108 (Williams et al. 1992), which is specific for the Bergerac (Bo) strain. Heterozygotes containing a Bristol (N2) chromosome in trans to a Bergerac (Bo) chromosome were constructed,





Ferguson et al.

and recombinants between unc-76 and the lethal mutation and dpy-21 and the lethal mutation were mapped by PCR analysis as described elsewhere (Williams et al. 1992). All 269 such recombinants carried the Bo stPIO8 pattern, indicating tight linkage of the lethal mutations to that polymorphism. Additional restriction site polymorphisms were identified by Southern blot analysis of N2 and Bo genomic DNA digested with various restriction enzymes. Several cosmids in the region were used as probes, and three cosmids were found to detect three Hind111 polymorphisms (wP1, wP2, and wP3). A portion of the recombinants described above was analyzed for each polymorphism. Of 38 unc-76 recombinants, 37 carried the Bo wP1 pattern; of 19 dpy-21 recombinants, 17 carried the Bo wP3 pattern; and of 21 dpy-21 recombinants, 17 carried the Bo wP2 pattern. These results placed the e2482 lesion between wP1 and wP3. wl mapped to an interval consistent with this location (wl recombinants were not mapped with respect to wP1).

Plasmid constructions

The 985-bp BamHI fragment from K07H12 was subcloned into the Stratagene Bluescript I1 SK vector. One subclone was chosen for sequence analysis and transformation experiments. Of the 21 bp that differ between this subclone and the published repeat sequence, 20 of these differences are clustered in the spacer region between the 3' ends of the 5s rRNA and SL1 RNA coding regions. The other difference is located upstream of the 5s rRNA and SLl RNA coding regions. The 985-bp subclone was digested with BsmI and XmnI (a site within the vector sequence) to yield a fragment containing 435 bp of the 985-bp insert (as shown) plus an additional 1945 bp from the vector, which was gel purified. The 740-bp fragment was purified from a ThaI di- gest of the 985-bp subclone insert. The Sm mutations were constructed by digestion of the 985-bp subclone with BseRI, which cuts in the middle of the Sm site. The 3' protruding ends were converted to blunt ends by digestion with T4 polymerase and ligated to create SmA3. Alternatively, the ends were ligated to an 8-bp MluI linker, and the ligation mixture was digested with MluI and religated to form the Sm-Mlu mutant. The sequence alterations of the mutations were confirmed by sequence analysis.

Isolation of genomic DNAs and Southern analysis

Genomic DNA was prepared from a mixed population of wild- type animals as described previously (Pilgrim 1993). Arrested homozygous wl and e2482 embryos were isolated based on previously described procedures (Shamu 1989). Briefly, animals heterozygous for each mutation were grown in liquid cultures. Worms were allowed to settle, and the pellet was resuspended in M9 buffer. The worms were treated with 1.6 N NaOH, 6% NaOC1, to kill the adults but not the embryos. The embryos were pelleted by centrifugation and washed three times in M9 buffer. After 24 hr, the embryos were pelleted by centrifugation, resuspended in M9 buffer, and treated with 0.25 M KOH, 1.2% NaOC1, to dissolve hatched larval worms and adult carcasses, leaving arrested embryos. Embryos were pelleted by centrifugation and washed in M9 buffer. Genomic DNA was prepared, and -3 pg of each DNA was digested and loaded onto a 1% agarose gel (for analysis of the rrs-1 repeat) or a 0.6% agarose gel (for analysis of the high molecular weight fragments). Gels were blotted and filters probed with the rrs-1-rescuing subclone that was labeled with [ C Y - ~ ~ P ] ~ A T P by random priming with the Prime-a-Gene kit from Promega. Quantitation was performed on a Molecular Dynamics PhosphorImager model 425E.

Microin jection techniques

Microinjection of DNAs was performed as described elsewhere (Mello et al. 1991). Constructs used in the transformation experiments are described in Figure 1, with the exception of the SL2 construct, which is a subclone of a 3.8-kb BamHI fragment containing one copy of the gene encoding SL2a RNA. One to 10 pglml of the indicated construct was coinjected with 100 pg/ ml of rol-6(su1006) DNA (a dominant marker used to select transformants) into unc-76(e911) e2482/unc-61 (e228) adult hermaphrodites. F, progeny that showed a Rol phenotype were cloned onto individual plates. F, broods that contained Rol animals, indicating stable transmission of the injected DNA, were used to establish transgenic lines. To test each of the indicated constructs for rescue of the wl allele, extrachromosomal arrays from two of the rescued e2482 lines were crossed into wl heterozygotes. For each line, the number of arrested embryos and the total number of progeny were scored from at least 10 adult animals in the F, or later generations. For each experiment in which rescue of embryonic lethality was observed, animals ex- hibiting an Unc-76 phenotype were also observed [as is expected of rescued animals because the unc-76(e911) mutation is closely linked to e2482 in the strains assayed for rescue]. This provided additional evidence that homozygous mutant embryos were rescued. For each construct tested, the average percentage of arrested embryos was calculated by determination of the percentage of arrested embryos produced by each line, and averag- ing these percentages. For all cases in which rescue was observed, these percentages were significantly different from that observed for the parental strain, and in most cases, the percentages were highly statistically significant (X0.002).

Isolation of embryonic RNA and RT-PCR analysis

Total RNA isolation and RT-PCR was performed essentially as described elsewhere (Conrad et al. 1991; Spieth et al. 1993), with the following modifications or additions: For preparation of embryonic RNA, -10-30 elongating wild-type embryos or -10- 30 homozygous mutant embryos -8-10 hr postfertilization were hand-picked into 50 p1 of sterile water. Mutant embryos were identified under the dissecting microscope as those embryos that had not elongated 8-10 hr postfertilization; this method has potential limitations, as it is difficult to confirm that all embryos picked are homozygous rrs-1 deletion mutants. Embryonic extracts and total RNA were prepared, and RNA was resuspended in 1 x DNase buffer. One unit of RQ1 DNase (Promega) was added and samples incubated at 37°C for -10 min. After phenol-chloroform extraction, RNA was precipi- tated with two volumes of ethanol. The DNase step and second phenol-chloroform extraction were found to be important in some cases to prevent spurious signals that were detected in the - RT controls (see below) when these steps were omitted. One- eighth of the RNA was used directly in a control PCR reaction ( - RT), performed as described below, and one-half of the sample used in an annealing reaction, followed by reverse transcription ( + RT). Additional controls ( - emb) were performed by preparation of extracts as above containing Escherichia coli alone (which is normally transferred along with the embryos), addition of one-half of the sample to an annealing reaction, and performance of RT-PCR as described below. Annealings were performed at 50°C for 75 min with 200 ng of the appropriate downstream primers (described below). One-half of each annealing reaction was added to a reverse transcription reaction mix consisting of 33 mM Tris-HC1, at pH 8.3, 40 mM NaC1, 6.7 mM DTT, 20 mM magnesium acetate, 6.7 mM of each dNTP, 20 units of RNAsin ribonuclease inhibitor (Promega), and 8 units






of AMV reverse transcriptase (Promega). Reactions were incubated at 42°C for 30 min. Reactions were diluted to 50 p1 with water, and 5 p1 was used in a PCR reaction. PCR was performed with 50 ng of the appropriate upstream and downstream primers (described below) for 35 cycles under the following conditions: 94°C for 30 sec, 50°C for SL1 reactions and 45°C for SL2 reactions for 1 min, and 72°C for 1 min. Products were separated on 1.3% agarose gels, gels blotted, and filters probed with [a-32P]dATP-labeled probes corresponding to an internal portion of the messages tested, which were generated by PCR. To generate the probes, %o of an initial PCR reaction, performed ac- cording to the conditions described below and either -2 pg of mixed stage genomic DNA or 5 ng of cloned hlh-1 cDNA (a gift from Michael Krause) as the original template, was used in a subsequent PCR reaction in a standard buffer provided by the manufacturer (Perkin Elmer) with 50 ng of each primer (described below), 25 pCi of [a-32P]dATP, and 2.5 units of Taq polymerase (Perkin Elmer). PCR was performed for 20 cycles under the following conditions: 94°C for 30 sec, 48°C for 1 min, and 72°C for 1 min. After hybridization, blots were washed twice with 2 x SSPE, 0.1 % SDS, at room temperature, and twice with 1 x SSPE, 0.1 % SDS, at 65°C. The following oligonucleotides were used as downstream primers in the reverse transcription and PCR (numbers correspond to complementary positions of the GenBank sequences): ges-l 4900-4876 (exon 3), (Kennedy et al. 1993); hlh-1 53 13-5292 (exon 3), (Krause et al. 1990); myo-3 4230-421 1 (exon 2), (Dibb et al. 1989); elt-1 1052- 1034 (exon 2) (Spieth et al. 1991). Oligonucleotides corresponding to the first 20 nucleotides of either the SL1 or SL2 leader were used as upstream primers (Spieth et al. 1993). The following oligonucleotides were used to generate the probes for South- ern analysis of the RT-PCR products [numbers indicate positions in the GenBank sequence (upstream primers) or complementary positions (downstream primers)]: ges-1 upstream 33 14-3329, downstream 3366-335 1 (exon 1); hlh-1 upstream 503 1-5056, downstream 5 180-5 164 (exon 2); myo-3 upstream 2971-2987, downstream 3036-30 16 (exon 1); elt-1 upstream 740-757, downstream 857-841 (exon 1).

Acknowledgments

J.H.R. is grateful to Dr. John White for providing space in his laboratory at the Medical Research Council (MRC), Cambridge, UK, during initial phases of this work, for encouragement, and for stimulating discussions. We thank Phil Anderson, Dave Brow, Judith Kimble, Michael Terns, and Marv Wickens, as well as members of the Rothman laboratory, for comments on a previous version of the manuscript. We thank Tom Blumenthal and Kevin Van Doren for the SL2 construct, the Blumenthal laboratory for primers, protocols, and helpful discussions, and Jim McGhee and Michael Krause for protocols and primers. Some of the strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the Na- tional Center for Research Resources of the National Institutes of Health. K.C.F. and P.J.H. were supported by a Cell and Mo- lecular Biology Predoctoral Training Grant from the National Institutes of Health (NIH). This work was supported by a grant from the NIH (GM 48137), a Searle Scholars Award from the Chicago Community Trust, and a Shaw Scientists Award from the Milwaukee Foundation to J.H.R.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

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K C Ferguson, P J Heid and J H Rothman RNA.function in Caenorhabditis elegans that can also be supplied by SL2 The SL1 trans-spliced leader RNA performs an essential embryonic

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