In nematodes, maturation of a fraction of mRNAs involves the acquisition of a 22nt spliced leader (SL) sequence from a spliced leader RNA (SL RNA) via intermolecular (trans)-splicing. The long term goal of the proposed research is to understand, in detail, the mechanism an biological significance of this unusual RNA processing pathway. An in vitro system prepared from developing embryos of the parasitic nematode, Ascaris Lumbricoides, catalyzes accurate and efficient trans-splicing using either endogenous or synthetic SL RNA as a trans-splice donor. The nematode SL RNA resembles the U snRNAs required for cis-splicing in that it possesses trimethylguanosine cap structure and Sm binding site. In vitro analysis has shown that the SL RNA functions in trans-splicing only if it is assembled into an Sm snRNP. Further, sequence critical for SL RNA function have been shown to be limited to the splice donor site as well as the Sm binding site and adjacent nucleotides. Function of the latter nucleotides may be imparted by their ability to base pair with U6 snRNA. To better understand the role of the SL RNA in trans-splicing, the SL RNP will be purified using oligoribonucleotide affinity reagents and its protein composition will be defined. These proteins will be compared with proteins which associate with functional and non- functional synthetic SL RNAs in hopes of identifying proteins which correlate with the ability of the SL RNP to function in trans-splicing. Further, the position of an SL RNA/U6 sn RNA interaction (revealed in crosslinking experiments) will be mapped and the functional significance of the interaction will be determined using compensatory mutagenesis and functional reconstitution. To define the U snRNP requirements for trans- splicing, components of the trans-spliceosome will be analyzed by RNA blot and direct RNA sequencing. The direct analysis will be complemented by depletion analysis where specific snRNPs will be selectively removed from the Ascaris extract by affinity chromatography. Finally, functional reconstitution of synthetic U snRNAs will be used to define the sequences and or domains of individual U snRNAs required for cis- and trans-splicing.
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