Section V Trans -Splicing in Vitro

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Important information about mechanisms of splicing has been obtained by recreation of the reaction in crude in vitro systems. Nilsen and co-workers have developed an extract from embryos of the nematode Ascaris lumbricoides capable of trans-splicing in vitro and have exploited this system to study mechanistic aspects of the reaction (Hannon et al. 1990a; Maroney et al. 1990a; Yu et al. 1993). They determined that branch-site formation occurs at either of two A residues contained in a sequence with no capability of base pairing with U2 snRNA. So far, no other branch sites have been mapped in nematode introns or outrons. These authors also discovered that the SL sequence itself is not required for the SL snRNP to function in trans-splicing in vitro (Maroney et al. 1991). Even when the first 20 nucleotides of the 22-nucleotide SL are deleted, trans-splicing of the remaining 2-nucleotide exon occurs accurately. In contrast, a region of the “intron” portion of the SL snRNP, including the Sm-binding site and adjacent nucleotides, is critically important for trans-splicing. Subsequent cross-linking experiments demonstrated that this region is involved in a base-pairing interaction with U6 snRNA (Hannon et al. 1992). It seems likely that this interaction is an important aspect of the mechanism of trans-splicing, bringing one of the substrates into contact with critical elements of the spliceosome.

The A. lumbricoides in vitro system has also been exploited to determine which snRNAs are required for trans-splicing (Hannon et al. 1991; Maroney et al. 1996). It has been found that depletion of U2, U4, U5, or U6 by oligonucleotide/RNase H cleavage eliminates both trans- and cis-splicing. However, depletion of U1 inhibits only the latter. This result supports a prediction made by Bruzik et al. (1988) that the part played by U1 in cis-splicing, base pairing with the 5′splice site, is replaced in trans-splicing by the SL exon itself.