We have been examining the function of the SL1 trans-spliced leader during embryogenesis by characterizing mutants that carry deletions of the SL1 RNA and 5S rRNA gene cluster. These mutations result in early embryonic defects and late embryonic arrest. We reported previously that a gene encoding SL1 RNA is necessary and sufficient to rescue the embryonic lethality associated with these mutations. To further understand the embryonic requirement for SL1, we are examining whether the primary sequence of the leader is important for metabolism and/or expression of trans-spliced messages. Constructs encoding mutant SL1 RNAs containing deletions or substitutions in the leader sequence were tested for their ability to rescue embryonic lethality. We found that constructs containing large deletions or rearrangements of the leader were unable to rescue. However, these mutant leaders may not be efficiently trans-spliced, particularly since the conserved secondary structure of the SL1 RNA appears to be substantially affected. Constructs in which the leader sequence was lengthened, as well as less extensive deletion and substitution mutants, in which secondary structure would not be predicted to be affected, were able to rescue. Therefore, there does not appear to be a stringent length or sequence requirement for either trans-splicing or leader function on a message. These results might indicate that the leader performs a more passive role in mRNA metabolism, perhaps by removing inhibitory sequences in the 5' untranslated region of mRNAs. Testing of additional mutants, as well as analysis of trans-splicing efficiency, is being performed to further analyze leader sequence requirements in vivo.
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