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SL1 Trans-Soliced Leader RNA Performs an Essintial Embrvonic Function in Caenorhabditis Elegans That Can Also Be Supplied by SL2 RNA

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SL1 Trans-Soliced Leader RNA Performs an Essintial Embrvonic Function in Caenorhabditis Elegans That Can Also Be Supplied by SL2 RNA Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press 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 Although the trans-splicing process and certain struc- leader sequence onto the 5' end of mRNAs is a mecha- tural features of the SL RNAs are conserved in organisms nism of mRNA maturation that occurs in lower eukary- that perform trans-splicing, the functions of trans-splic- otes, including trypanosomes, nematodes, trematodes, ing are not fully understood. In some cases, however, and Euglena (for review, see Agabian 1990; Nilsen 1993). trans-splicing is known to result in the production of The spliced leader RNA (SL RNA), the precursor RNA functional mRNAs. For example, trans-splicing in trypa- containing the trans-spliced leader sequence, appears to nosomes apparently serves to process polycistronic participate in trans-splicing as an SL ribonucleoprotein mRNAs into monocistronic units; as a result, all (RNP)(Bruzik et al. 1988; Thomas et al. 1988; Van Doren mRNAs receive a spliced leader (Walder et al. 1986; and Hirsh 1988; Maroney et al. 1990; Michaeli et al. Agabian 1990).Similarly, in the nematode Caenorha bdi- 1990; Palfi et al. 1991). The nematode SL RNP protein tis elegans, processing of polycistronic messages derived components include the Sm proteins, which also associ- from the fraction of transcription units that are orga- ate with most U snRNAs involved in cis-splicing (Bruzik nized into operons appears to occur by trans-splicing of et al. 1988; Mattaj 1988; Thomas et al. 1988; Van Doren the downstream messages to the minor spliced leader and Hirsh 1988; Maroney et al. 1990). Other RNP com- SL2 (for review, see Blumenthal 1995). ponents known to participate in cis-splicing, such as the Although it is clear that trans-splicing acts to process U2 and U6 snRNPs, are also required for trans-splicing polycistronic messages, it is also likely to perform other in both trypanosomes and nematodes (Tschudi and Ullu functions. Only -10% of all mRNAs in C. elegans re- 1990; Hannon et al. 1991; Watkins and Agabian 199 1). ceive 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 'Corresponding author. not been found to contain operons or an SL2-like leader GENES & DEVELOPMENT 10:1543-1556 0 1996 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/96 $5.00 1543 Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Eerguson et al. RNA; mRNAs from most of these species are trans- mally SL1 -trans-spliced mRNAs produce functional spliced to a leader that is identical to the major trans- products even in the absence of zygotic SL1. spliced leader, SL1, in C. elegans (Krauseand Hirsh 1987; Nilsen 1993). Although the function of SL1-trans-splic- ing is not known, -60% of C. elegans messages are Results trans-spliced to this leader (Zorio et al. 1994))implying Identification of mutations that delete the rrs-1 gene that SL1 performs other functions beyond the processing cluster and isolation of rescuing clones of operons. In addition to SL1 and SL2, other spliced leaders have been characterized recently in C. elegans In an effort to identify zygotically transcribed genes es- (Ross et al. 1995). These novel spliced leaders are most sential for early stages of embryonic development in C. similar to SL2, but their functions are not generally un- elegans, we isolated two allelic loss-of-function muta- derstood (Ross et al. 1995).Given the existence of these tions (e2482 and wl ) that lead to early defects in embryo- other SL RNAs, it is conceivable that there may be ad- genesis and late embryonic arrest. To investigate the ditional roles for trans-splicing in C. elegans. function of the corresponding genomic region, we iden- Although the trans-splicing specificity of some of the tified molecular clones that rescued these mutations. SL RNAs has not been well characterized, mechanisms e2482 and wl were localized to an -500-kb interval by clearly exist to discriminate between SL1 and SL2-trans- mapping them relative to restriction site polymorphisms splicing. SL2 is not found on the most upstream mRNAs (Fig. lA,B). Pools of cosmid clones corresponding to this derived from polycistronic messages, and SL1 is not gen- interval were transformed into e2482/+ animals and erally found on downstream mRNAs of such operons tested for their ability to rescue embryonic lethality. (Spieth et al. 1993; Zorio et al. 1994).In addition, unlike From the one rescuing pool (pool 2, Fig. 1B; Table l), a SL2, SL1 is found on many monocistronic messages. The single cosmid (K07H12)was identified that is sufficient only requirement for trans-splicing of SL1 onto a pre- for rescue (Table 1). Rescue was indicated by a decrease mRNA appears to be the presence of an outron, an AU- in the fraction of arrested embryos produced by trans- rich sequence followed by a splice acceptor site but con- formed heterozygotes and the presence of viable e2482 taining no splice donor site (Conrad et al. 1991, 1993, homozygous larvae (Table 1). As expected, rescue was 1995).In contrast, splicing of SL2 onto downstream mes- never complete, as the transformed DNA assembles into sages in operons may be coupled to polyadenylation of an extrachromosomal array that is inefficiently trans- the upstream mRNA in the polycistronic message mitted during cell division (Mello et al. 199 1).Some res- (Spieth et al. 1993). cued animals survived to adulthood, however, and it was A number of observations suggest that the SL1 leader possible to propagate homozygous mutants for many might facilitate the expression of trans-spliced mRNAs. generations that were transgenic for the rescuing se- For example, in many cases, SL1 is spliced close to the quences. initiation codon of the protein coded by a trans-spliced Fragments derived from K07H12 were further tested mRNA (Bektesh et al. 1988), and it has therefore been for rescue, and an - 1-kb fragment (1-kblK07H12; Table proposed that SL1 may serve to promote efficient trans- 1)was found to rescue both e2482 and wl. The sequence lation of these messages (Bektesh et al. 1988; Spieth et al. of a 985-bp subclone of this fragment (not shown) was 1993). Consistent with this notion, recent studies have found to be nearly identical to the published sequence of shown that the SL1 leader can enhance translation in a 980-bp repeat from the 110-kb rrs-1 cluster (Nelson and vitro (Maroney et al. 1995).Additional studies have be- Honda 1985). Previous analysis of repeats from the rrs-1 gun to identify the RNA sequences and protein compo- cluster showed that each contains one gene encoding 5s nents required for trans-splicing in vitro and in vivo (for rRNA and one encoding SL1 RNA, a 105-nucleotide review, see Agabian 1990; Nilsen 1993; Blumenthal RNA that includes the 22-nucleotide SL1 leader (Nelson 1995); however, the requirement for neither trans-splic- and Honda 1985; Krause and Hirsh 1987)(Fig. 1C).There ing nor a trans-spliced leader have been demonstrated were 21 bp that differed between the 985-bp sequence of directly. our rescuing subclone and the published sequence of the We describe the characterization of mutations that 980-bp repeat. Significantly, the 5s rRNA and SL1 RNA eliminate the tandemly duplicated copies of the SL1 coding regions were identical between the two se- RNA gene and demonstrate that SL1 RNA is essential quences. These observations suggest that the 985-bp sub- for normal embryonic development and viability. Fur- clone that rescues e2482 and wl does not encode an ac- thermore, we show that the essential function of SL1 tivity that is unique from other rrs-1 repeats, consistent RNA can be provided by SL2 RNA.
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