To determine the cause of the second catalytic step inhibition displayed by certain U5 deletion and insertion mutants, the interactions between such mutants and the 5′ exon were investigated by UV cross-linking. The formation of a cross-link between two molecules indicates that these molecules are in close proximity and may functionally interact. Cross-linking, however, may not detect all interactions, and the absence of a cross-link does not necessarily indicate that two molecules do not interact. We had found previously that deletion of five or more nucleotides from loop 1 prevented the second catalytic step of splicing (O'Keefe et al., 1996). These mutations also abolished cross-link formation between U5 and the 5′ exon, suggesting that inhibition of the second catalytic step resulted from failure to tether the 5′ exon intermediate properly. To determine whether a similar situation brought about the inhibition of the second catalytic step with our current mutants, these mutants were added to U5-depleted extract then splicing was initiated with CYH2 pre-mRNA containing a 4-thiouridine (4-thio-U) at the last nucleotide of the 5′ exon (position −1 in exon 1). RNA isolated from splicing reactions following UV irradiation was deproteinized, and the U5-containing species were captured using a biotinylated oligonucleotide complementary to the U5 snRNA and streptavidin-conjugated paramagnetic beads. As an internal reference for the capturing procedure, a biotinylated oligonucleotide complementary to the U1 snRNA was used simultaneously with the U5 biotinylated oligonucleotide to capture the known U1–pre-mRNA cross-links. UV-irradiated reconstitution reactions containing in vitro transcribed wild-type U5 incubated with U5 and U1 biotinylated oligonucleotides simultaneously captured three cross-linked species (Figure 4A, lane 1). These correspond to the previously characterized cross-links between U5 snRNA and the pre-mRNA, U5 snRNA and the exon 1 splicing intermediate, and U1 snRNA and the pre-mRNA (Newman et al., 1995; O'Keefe et al., 1996). Deletion of the loop 1 sequence from U5 (Del. 1–9) did not affect the U1–pre-mRNA cross-link but prevented formation of the U5–pre-mRNA and U5–exon 1 cross-links (Figure 4A, lane 2), indicating, as observed previously, that this U5 mutation prevents the U5–exon 1 interaction and, therefore, inhibits the second catalytic step of splicing (O'Keefe et al., 1996). Deletions of one nucleotide from different locations within the U5 loop 1 sequence (Del. G1, C2, U4, A8 and C9), which allow both steps of splicing to occur, produced the same cross-linked species found with in vitro transcribed wild-type U5 (Figure 4A, lanes 3–7). Deletions of two nucleotides from loop 1 in two different locations (Del. C2,3 and Del. U4,5), which allow both steps of splicing to occur but have a slight inhibition of the second catalytic step, again produce the same cross-linked species as found with wild-type U5 (Figure 4A, lane 8, and 4B, lane 1). Surprisingly, even when the exon 1 interactions are investigated for the three nucleotide deletions (Del. C2,3,U4 and Del. U4,5,6) that show a substantial inhibition of the second catalytic step, there are still interactions between these U5 mutants and exon 1 before and after the first step of splicing (Figure 4A, lane 9 and 4B, lane 2). When four nucleotides are deleted from loop 1 (Del. C2,3,U4,5 and Del. U4,5,6,7), the U5–pre-mRNA cross-linked species is of very low abundance (Figure 4A, lane 10 and 4B, lane 3), which may explain, in part, the inhibition of the second catalytic step with these mutants. The finding that the deletion mutations still interact with the 5′ exon indicates that the second step defect displayed by some of these mutants is not a result of their inability to interact with exon 1.
Figure 4. Cross-linking between U5 snRNA loop 1 mutants and the 5′ exon. Reconstitution with different U5 snRNA mutants and pre-mRNA containing a 4-thio-U residue at position −1 in exon 1. RNA species containing U5 and U1 snRNAs were selected from UV-irradiated reactions with biotinylated oligonucleotides and streptavidin paramagnetic particles. (A) Reconstitution with in vitro transcribed U5 snRNA (WT) (lane 1), loop 1 deletion (Del. 1–9) (lane 2), one nucleotide loop 1 deletions (lanes 3–7), two nucleotide loop 1 deletion (lane 8), three nucleotide loop 1 deletion (lane 9) and four nucleotide loop 1 deletion (lane 10). pBR322 MspI end-labelled size markers are shown in lane 11. (B) Reconstitution with in vitro transcribed two nucleotide loop 1 deletion (lane 1), three nucleotide loop 1 deletion (lane 2), four nucleotide loop 1 deletion (lane 3), one nucleotide loop 1 insertion (lane 4), two nucleotide loop 1 insertion (lane 5), three nucleotide loop 1 insertion (lane 6), four nucleotide loop 1 insertion (lane 7), three nucleotide loop 1 insertion 5′ (lane 8) and three nucleotide loop 1 insertion 3′ (lane 9). pBR322 MspI end-labelled size markers are shown in lane 10. The various cross-linked species are indicated at the left of each panel. Asterisks indicate background pre-mRNA captured with streptavidin paramagnetic particles.
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Analysis by cross-linking of all the insertion mutations for interaction with position −1 of exon 1 in the pre-mRNA and the exon 1 intermediate revealed that these cross-linked species occur even with the insertions that severely block the second step of splicing (Figure 4B, lanes 4–9). Therefore, the second step defect found with some of these insertion mutations is not due to the inability to interact with the 5′ exon, but to some other defect. In fact there is a dramatic increase in cross-link efficiency/yield with a number of these insertion mutants. Currently it is not clear why, or how, this increase occurs.