No abstract

Several different mRNAs from Caenorhabditis elegans contain the same 22-nucleotide leader sequence at their 5' ends that is acquired in a trans-splicing reaction. About 10 to 15% of the major proteins are translated from mRNAs that contain the spliced leader, among them two ribosomal proteins, ubiquitin, GAPDH, a heat shock protein (hsp70a), and three actins. The same spliced leader sequence is present in mRNAs isolated from nematodes from several different genera; but it is not present in mRNAs from other organisms. The spliced leader is encoded as a spliced leader (SL) RNA about 100 nucleotides long. The gene for the SL RNA is located in the 5S rDNA repeat in C. elegans; however, this association with the 5S repeat is not preserved in other genera. The 22-nucleotide spliced leader sequence is conserved in three genera of nematodes. [Key Words: Caenorhabditis elegans; spliced leader; trans-splicing]Received April 25, 1988; revised version accepted August 18, 1988. Krause andHirsh (1987) reported finding a common RNA leader sequence on the 5' ends of three different actin mRNAs in the nematode Caenorhabditis elegans.The 22-nucleotide leader sequence is spliced onto the actin mRNAs from a separate 100-nucleotide precursor RNA, and this 22-nucleotide sequence became known as the spliced leader (SL), and its precursor, the spliced leader RNA (SL RNA). The mechanism by which the SL is transferred onto the mRNAs appeared to be through trans-splicing because (1) the SL and actin genes are widely separated in the genome, (2) a consensus splice donor sequence is present in the SL RNA, and (3) These observations raise questions regarding the generality of the SL. Do other mRNAs in C. elegans contain the SL or is it a peculiarity of the three actin genes in which it was originally described? Further, is the SL present in other organisms? As shown here, other mRNAs in C. elegans acquire the SL, as do mRNAs from other nematodes. Although we could not find the C.elegans 22-nucleotide SL sequence in RNA from other more distantly related organisms, trans-splicing may occur, but with different SL sequences. Results mRNAs in addition to actin contain SLThe possibility that mRNAs other than actins-1, -2, and -3 contain SL derives from an earlier observation. Some RNAs of higher molecular weights hybridize to a sequence complementary to SL in a Northern blot (Krause and Hirsh 1987). However, at that time the specificity of the hybridization was not established. We show here by additional Northern blot analyses of C. elegans RNAs that other mRNAs contain SL. We used a synthetic oligodeoxynucleotide complementary to the SL sequence as the hybridization probe (Fig.

Maturation of some messenger RNAs in the nematode Caenorhabditis elegans involves the acquisition of a 22-base leader at their 5' ends. This 22-base leader, called the spliced leader (SL), is derived from the 5' end of a precursor RNA of 90-100 bases, called spliced leader RNA (SL RNA). SL RNA is transcribed from a 1-kilobase DNA repeat which also encodes the 5S ribosomal RNA. A subset of mRNAs in C. elegans acquire SL from SL RNA by a trans-splicing mechanism. SL behaves as a 5' exon in the trans-splicing reaction. Using antisera against the Sm antigen that is associated with small nuclear ribonucleoprotein particles (snRNPs), we precipitated SL RNA from extracts of C. elegans, indicating that it is bound by the Sm antigen in vivo. SL RNA also possesses the unique trimethylguanosine (m32,2,7G) cap characteristic of most small nuclear RNAs. Therefore, SL RNA is a chimaeric molecule, made up of an snRNA attached to a 5' exon and is a constituent of a snRNP.

The origin-defective simian virus 40 (SV40) mutant 6-1 has been useful in transforming human cells (Small et al., Nature [London] 296:671-672, 1982; Nagata et al., Nature [London] 306:597-599, 1983). However, the low efficiency of transformation achieved by DNA transfection is a major drawback of the system. To increase the efficiency of SV40-induced transformation of human fibroblasts, we used recombinant adenovirus-SV40 virions which contain a complete SV40 early region including either a wild-type or defective (6-1) origin of replication. The SV40 DNA was cloned into the adenovirus vector in place of early region 1. Cell lines transformed by viruses containing a functional origin of replication produced free SV40 DNA. These cell lines were subcloned, and some of the subclones lost the ability to produce free viral DNA. Subclones that failed to produce free viral DNA were found to possess a mutated T antigen. Cell lines transformed by viruses containing origin-defective SV40 mutants did not produce any free DNA. Because of the high efficiency of transformation, we suggest that the origin-defective chimeric virus is a convenient system for establishing SV40-transformed cell lines from any human cell type that is susceptible to infection by adenovirus type 5.Cells range from fully permissive to fully nonpermissive for simian virus 40 (SV40) infection. Permissivity results in full expression of the viral genome, replication of viral DNA, and production of infectious virions. Nonpermissive infection results in expression of the early viral genes, but no viral DNA is synthesized or virions produced. Nonpermissive cells can be transformed, and the viral genome can be integrated into the host cell genome. Human cells are semipermissive for SV40. Virus production occurs to ca. 1% the level produced by permissive monkey kidney cells (12). The majority of viral DNA replication occurs in ca. 1 to 2% of the cells, where it reaches levels equivalent to wild-type infection (18). A portion of the population can become transformed and integrate viral DNA into the host cell genome (18); however, the efficiency is quite low. The frequency of transformation can be enhanced by transfection of SV40 origin-defective mutants (13). Because DNA transfection is an inefficient process, the frequency of transformation is still not very high. We wanted to determine whether we could increase the efficiency of SV40-induced tranformation after infection with recombinant viruses. We were also interested in determining whether having a functional versus a defective SV40 origin of replication would affect the frequency of transformation by recombinant viruses.The chimeric viruses contain the SV40 early region which encodes T antigen (HpaII-BamHI fragment) (16) cloned into the helper independent adenovirus vector, AE1/X (17). The SV40 origin of replication is either wild type or the orimutant 6-1, having six nucleotides deleted at the BglI site (4, 5). The SV40 DNA is cloned into the adenovirus vector in place of early regions la and lb. ...

Approximately 10% of the mRNAs in the nematode Caenorhabditis elegans mature through a trans-splicing mechanism that involves the transfer of a 22-nucleotide spliced leader to the 5' end of the pre-mRNA. The spliced leader RNA exists as a small nuclear ribonucleoprotein particle and has the trimethylguanosine cap that is characteristic of eucaryotic small nuclear RNAs. We found that the trimethylguanosine cap present on the spliced leader RNA was transferred to the pre-mRNA during the trans-splicing reaction. Thereafter, the trimethylguanosine cap was maintained on the mature mRNA. This is the first example of eucaryotic cellular mRNAs possessing a trimethylguanosine cap structure.Eucaryotic mRNAs possess modified 5' termini of the form m7G(5')ppp(5')N (monomethylguanosine). This modification, known as a cap, is important in binding factors involved in the initiation of translation (for reviews, see references 19 and 20). The cap influences splicing of premRNA and contributes to the stability of mRNA (4,5,10,11,16,17).Small nuclear RNAs (snRNAs) are an important component of the splicing machinery in eucaryotes (for reviews, see references 7 and 21). These RNAs are complexed with proteins into small nuclear ribonucleoprotein particles (for reviews, see references 14 and 15). snRNAs have a trimethylguanosine cap [m32 2.7G(5')ppp(5')N] at their 5' ends (for a review, see reference 18) and are not translated.Approximately 10% of the mRNAs in the nematode Caenorhabditis elegans mature through an unusual mechanism in which the 5' 22 nucleotides of the mRNA are acquired through a trans-splicing reaction (1,12). This reaction involves a unique small nuclear ribonucleoprotein particle containing spliced leader (SL) RNA (2,22,23). SL RNA provides the 5' exon (the SL) in the trans-splicing reaction and is thought to act as its own Ul snRNA during the trans-splicing reaction (2). SL RNA also possesses the trimethylguanosine cap that is characteristic of eucaryotic snRNAs (22,23).Because of the importance of caps in mRNA maturation and translation, we examined whether the trimethylguanosine cap on the SL RNA was transferred to and retained on trans-spliced mRNAs in C. elegans. RNA purified from a mixed population of C. elegans containing all developmental stages was immunoprecipitated by using the monoclonal antibody recognizing trimethylguanosine (as described by Van Doren and Hirsh [23] (Fig. 1A, lanes 6 and 7). Protection of the antisense SL RNA probe yielded a fragment of approximately 90 bases. The actin 1 gene is one of four C. elegans actin genes. Actin 1 mRNA acquired the SL through trans-splicing and was immunoprecipitated by the monoclonal antibody, showing that it has a trimethylguanosine cap (Fig. 1B, lanes 6 and 7). Actin 1 mRNA will protect 220 bases of the antisense actin 1 RNA probe. The actin 4 gene is the only member of the actin gene family that does not undergo the trans-splicing reaction. The fragment of the antisense RNA probe protected by actin 4 mRNA was 83 bases. In contrast to actin 1 mRNA, actin 4...