Worm Breeder's Gazette 10(1): 67
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Recently, an alternative form of RNA processing (transsplicing) has been shown to occur in C. elegans (Krause and Hirsh, Cell 49, 753-761, 1987). In this RNA processing reaction, sequences from one RNA molecule are joined in an intermolecular reaction to sequences in a second molecule. mRNAs encoded by three of the four actin genes in C. elegans were shown to contain the same 22 nucleotides at their 5' ends as a result of transsplicing. This sequence has been named the splice leader. If the mechanism is trans-splicing, then by analogy with trypanosomes, the two precursor RNA molecules would be joined through a Y-branch intermediate in an intermolecular reaction. Our experimental results indicate that a Y-branch intermediate is involved that can be cleaved with purified debranching enzyme isolated from vertebrate cells; therefore, trans-splicing is indeed the mechanism in C. elegans.We were interested in characterizing other mRNAs that acquire the splice leader. A cDNA library was constructed from poly A+ mRNA and screened using an oligonucleotide complementary to the splice leader. More than 100 clones were isolated and about two dozen have been characterized and sequenced. In an attempt to identify these clones as having homology to known genes, the DNA and deduced amino acid sequences were searched against existing databases. Two clones have strong homology with two different ribosomal protein genes from rat and yeast and are therefore thought to encode ribosomal proteins in the nematode. A second approach has been to examine published nucleotide sequences of C. elegans genes for the presence of a splice acceptor sequence. If such a sequence is present 5' of the initiator ATG codon, and no splice donor sequence is found farther upstream, an oligonucleotide homologous to a portion of the coding sequence is constructed and used for primer extension sequencing of the mRNA. In this way, the mRNAs coding for ubiquitin and glyceraldehyde-3-phosphate dehydrogenase have been shown to contain the splice leader at their 5' ends. Not all mRNAs in C. elegans acquire the splice leader. Two approaches were taken to determine what proportion of the messages in C. elegans acquire the splice leader. First, quantitative dot blots were done using poly A+ mRNA and oligonucleotide probes. Following hybridization, samples were quantitated in a scintillation counter and compared to poly A+ mRNA hybridized to an oligo dT oligonucleotide as a measure of all poly A+ mRNAs. This work suggests that approximately 10% of the poly A+ mRNAs in the worm acquire the splice leader. A second approach asked what proportion of the translated proteins in an in vitro reaction are encoded by mRNAs containing the splice leader at their 5' ends. Poly A+ mRNA was translated in a rabbit reticulocyte in vitro translation system either with or without the addition of an oligonucleotide complementary to the splice leader. The products of the reactions were separated by two-dimensional gel electrophoresis. Approximately 10% of the proteins translated in vitro were arrested upon the addition of the oligonucleotide complementary to splice leader illustrating that these proteins are the products of mRNAs containing the splice leader. Acquisition of splice leader does not appear to be developmentally regulated. Northern analysis of RNA isolated from oocytes (generously provided by Brian Kennedy and Jim McGhee) and from several larval stages suggests that the splice leader is present on mRNAs in general, and on actin mRNAs in particular, throughout development. Is this splice leader present in other organisms? Northern analysis of RNAs from other nematodes (C. elegans var. Bergerac, C. briggsae, Panagrellus revividus, Haemonchus and Ascaris) revealed that these worms contain the same or a very closely related splice leader. However, Northern analysis of RNAs from yeast, trypanosomes, Drosophila, Dictyostelium, Xenopus and human revealed that the evolutionary conservation of this splice leader sequence does not extend to these organisms.