Protein hPrp31 requires stem I and stem II of the 5′ stem-loop of U4 for binding

stem-loop of U4 for binding

The next question investigated was that of which of the structural elements of U4 snRNA that were found to be protected from hydroxyl radical attack by protein hPrp31 in the footprinting experiment were required for stable binding of protein hPrp31 to the

15.5K-U4 snRNA binary complex. For this more detailed analysis, a series of mutant RNA oligonucleotides was prepared, and these were assayed by EMSA for ternary-complex formation. To ease solubilisation of hPrp31, a fusion protein comprising the maltose-binding protein (MBP) fused to hPrp31 was used (Figure 4-19 A). This behaves in a manner identical to non–tagged hPrp31 (data not shown). In all experiments, the binary 15.5K-U4 snRNA complex (B in Figure 4-19 B) was first assembled with recombinant 15.5K protein and radiolabelled RNA oligonucleotide. Formation of the ternary complex (T in Figure 4-19 B) was then initiated by adding MBP-hPrp31 protein. Complexes were then analysed directly by native PAGE at 4 °C.

Since the single-stranded 5′ region of the U4 snRNA was protected by the protein, this segment was deleted first (U4-2, Figure 4-19 B). No reduction in the amount of ternary complex formed (Figure 4-19 B; lanes 3 and 7) was observed compared with the longer deletion mutant U4 snRNA (U4-1), which binds in a manner identical to that of the wild-type U4 snRNA (Nottrott et al., 2002). Hence, the contact between the single-stranded 5′ end of the U4 snRNA and protein hPrp31 is not required for stable binding. However, the deletion of five base pairs from stem I severely depressed the formation of stable ternary complex (U4-3;

Figure 4-19 B, lanes 3 and 11). Residual ternary complexes (in yields of approximately 20%

compared with the U4-2 or U4-1 RNA) were unstable, resulting in an ill-defined smear migrating above the binary 15.5K-U4 snRNA complex. Binding activity could not be restored by extending the truncated three-base-pair-long stem I of mutant U4-3 by a C-G base pair (U4-4, SL1 in reference Nottrott et al. (2002), Figure 4-19 B). Furthermore, changing the sequence of the upper part of stem I while leaving the number of base pairs intact, or deleting the bulged-out A, did not reduce the yield of ternary complex (oligonucleotides U4-10 and U4-11; data not shown). Therefore, it is possible to reach the conclusion that the length of stem I is critical for stable binding of protein hPrp31 to the binary 15.5K–U4 snRNA complex, and that this double-helical RNA structure, irrespective of sequence, forms part of the binding determinant.

Figure 4-19 Binding of hPrp31 to the U4 snRNP requires the complete stems I and II of the 5' stem-loop of the U4 snRNA.

A. SDS PAGE of MBP-hPrp31. MBP-hPrp31 was stained with Coomassie blue. B. The gels show EMSA of the interaction between protein hPrp31 and the 15.5K–U4 snRNA deletion mutant complex. The binary complex was first assembled with protein 15.5K and mutant U4 snRNA s (top panels), and ternary-complex formation was then initiated by adding protein hPrp31. Complexes were separated on a 6% native polyacrylamide gel. The positions of the protein-RNA complexes are indicated on the right: R, free RNA; B, binary complex consisting of the RNA and 15.5K; T, ternary complex, consisting of the RNA, 15.5K and protein hPrp31. The sequence and structure of the respective U4 snRNA deletion mutant used is indicated above each panel.

The hydroxyl-radical footprint showed protection of the entire penta-loop in the ternary complex (Figure 4-18 A and B). Next, a number of different penta-loop mutants were tested, and their effect on stable ternary-complex formation in the experimental system outlined above was assayed. The U4-2 mutant (Figure 4-19 B), which has wild-type properties while being closest in length to the other mutants, was used as a reference with which to compare the mutants. Replacement of the penta-loop by an unrelated sequence did not impair complex formation (U4-5, Figure 4-20).

Its replacement with a tetranucleotide loop (UGAA in U4-6, Figure 4-20; other loops tested were UUCG and GCAA) likewise had no effect (Figure 4-20, lanes 3 and 11 and data not shown). Therefore, a more dramatic change in the penta-loop's backbone was introduced by opening it up. An RNA duplex with dangling ends was created by annealing the 5′ (20–37)

and 3′ (38–52) halves of the U4 snRNA 5′ stem-loop (U4-A, Figure 4-20). Unexpectedly, this duplex still allowed efficient ternary-complex formation (Figure 4-20, lanes 3 and 15).

Finally, the loop was simply deleted, by annealing 5′ and 3′ halves lacking the penta-loop sequence entirely (U4-B, Figure 4-20).

Figure 4-20 Binding of hPrp31 to the U4 snRNP does not depend on sequence and structure of the terminal penta-loop.

EMSA analysis of the interaction between protein hPrp31 and the 15.5K–U4 snRNA complex containing mutant RNAs was performed as described in Figure 4-19. U4-A and U4-B duplexes were generated before the assay by annealing the radiolabelled 5′ and the unlabelled 3′ half of the 5′ stem-loop. A schematic representation of the sequence and the structure of each loop mutation in the U4 5′ stem-loop (corresponding to oligonucleotide U4-2) is indicated above the respective panel. Numerous other penta– and tetranucleotide loops were tried out; all with the same result (see text).

This duplex's stability was dramatically impaired (EMSA; data not shown); it no longer supported the formation of significant amounts of stable binary 15.5K–U4 snRNA complex (Figure 4-20, lane 18), as compared with the reference U4-2 (lane 2) and with all other mutants tested (lanes 6, 10, 14). However, this duplex did support the efficient formation of stable ternary complex upon addition of protein hPrp31 to the binary complex assembly mixture (lane 19). Protein hPrp31 therefore appears to stabilise transient interactions of protein 15.5K with U4 snRNA in the ternary complex. Therefore, it may be inferred that the penta-loop per se is not essential for protein hPrp31 binding to the 15.5K–U4 snRNA binary complex. In addition to the penta-loop, protein hPrp31 had also been found to be in contact with the adjacent short stem II (Figure 4-18A and B). Since the penta-loop was not

required for stable ternary-complex formation, the function of stem II in ternary-complex formation was next investigated.

Figure 4-21 Elongation of stem II is critical for hPrp31 binding to the U4 snRNP.

EMSA analysis of the interaction between protein hPrp31 and the 15.5K–U4 snRNA complex containing mutant RNAs was performed as described in Figure 4-19. A schematic representation of the sequence and the structure of each stem II mutation tested is given above the respective panel.

As a first step, stem II was extended by a single C-G base pair (U4-7, Figure 4-21).

Strikingly, this mutant no longer supported ternary-complex formation in the assay system used (Figure 4-21, lanes 3 and 7; approximately 90% reduction of ternary complex formation when quantified by Phosphoimager analysis). Extension by a U-A base pair (U4-8, Figure 4-21) had a similar effect (Figure 4-21, lane 11). Not surprisingly, identical results were obtained by increasing the length of the stem to 5 or 7 base pairs (Figure 4-21, U4-12 and U4-9; lane 19 and 15 respectively). Furthermore, deletion of the penta-loop with retention of the three-base-pair-long stem II (U4-C, Figure 4-21) was similarly detrimental to ternary-complex formation (Figure 4-21, lane 23). The inhibitory effect of this additional base pair was thus independent of the presence of the penta-loop. A shortening of stem II was not deemed feasible because the already weak binding of 15.5K to the penta-loop deletion (mutant U4-3) would be further compromised by interfering with the 15.5K binding site (Reuter et al., 1999; Vidovic et al., 2000).

Taken together, these data show that the length of stem II is important for the association of protein hPrp31 with the 15.5K–U4 snRNA binary complex. Since protein hPrp31 is known to interact also with the 15.5K-U4atac snRNA binary complex (Nottrott et al., 2002), while the U4atac snRNA has a different stem II sequence and the same length, the conclusion may be strengthened: the determining factor is apparently the length, and not the sequence.

So far, these results show that both stem I and stem II are required for stable binding of protein hPrp31 to the 15.5K–U4 snRNA binary complex, to form the ternary hPrp31-15.5K–U4 snRNA complex. While stem I could not be shortened without loss of binding activity, stem II – unexpectedly – could not be elongated.

4.3.3. The Nop domain is necessary and sufficient for