(Rasche et al, 2012) have shown (by crosslinking and chemical structure probing) that in isolated, catalytically active spliceosomes, Cwc2 contacts various elements of RNA, such as introns and U6 snRNA in the internal stem loop (ISL) and its conserved ACAGAGA sequence. Characteristics of Cwc2 such as the accessibility of the Zinc Finger domain (ZnF) and the RNA recognition motif (RRM) in its structure and the positively charged connector element and depression, suggest Cwc2 might crosslink with RNA at multiple points on its surface.
Using the UV-Irradiation method (as described in the techniques section), the UV caused covalent Protein-RNA bonds to form. The sites at which these bonds form are cross linking sites. These sites can then be identified using mass spectrometry and thus the regions of Cwc2 that interact with RNA can be found. Binary U6 snRNA – Cwc2 complexes that were formed in vitro were used because U6 snRNA is the only snRNA found crosslinked to Cwc2 during splicing in yeast extracts or in purified catalytically active spliceosomes. U4 snRNA was also used for comparison (more later).
They first needed to find whether or not a stable and defined complex could be reconstituted between Cwc2 and U6 snRNA in solution (as described in the techniques section).
This identified crosslinked peptides, which were then analysed under MS to narrow the crosslinked regions from peptides to single residues (fragment ions containing this amino acid are shifted by the mass of the crosslinked RNA or fragments on spectra). 7 residues were found to crosslink with RNA, F47, C87, Y120/ R121, Y138, K152 and C181. Only two of which (Y120 and R121) are together in sequence and space, the others are distributed on the same side of the positively charged surface of the protein, see figures 1 and 2. Using the UV-Irradiation method (as described in the techniques section), the UV caused covalent Protein-RNA bonds to form. The sites at which these bonds form are cross linking sites. These sites can then be identified using mass spectrometry and thus the regions of Cwc2 that interact with RNA can be found. Binary U6 snRNA – Cwc2 complexes that were formed in vitro were used because U6 snRNA is the only snRNA found crosslinked to Cwc2 during splicing in yeast extracts or in purified catalytically active spliceosomes. U4 snRNA was also used for comparison (more later).
They first needed to find whether or not a stable and defined complex could be reconstituted between Cwc2 and U6 snRNA in solution (as described in the techniques section).
 |
Figure 1. The location of the cross linking residues F47, C87, Y120, R121, Y138, K152 and C181 (as labelled) showing that they are not grouped together but in fact located rather separately from each other in both sequence and space. It also shows that they are also all on the same “side” of the protein. |
 |
Figure 2. The electrostatic potential shows the overall positively charged surface of the molecule and the general locations of the cross linked residues (just to give an idea). |
The Torus domain (F47) is adjacent to the RNA-binding side of the RRM domain. The fact that there are crosslinking sites in the two canonical RNA binding domains confirms that they bind RNA in Cwc2. There are cross linking residues on the same side of the protein as the RRM domain (i.e. F47 and K152 (see figure 3)), this could mean that F47 and K152 are also be part of the RRM-based RNA-binding site of Cwc2.
 |
Figure 3. The K152 residue is exposed to the solvent, however, the side chain of the crosslinked F47 is buried in the Cwc2 core. Thus the RNA crosslinked at this position either contacts the backbone of F47, or the side chain moves to the surface of Cwc2 when RNA is bound due to a conformational change. |
 |
Figure 4. The two crosslinking residues on the connector element – Y120 and R121, show that the connector element most probably also aids in U6 snRNA-Cwc2 interactions. |
So Far : Three distinct regions in the structure that are capable of contacting RNA have been found, consistent with a possible role for Cwc2 in accommodating several RNA elements from the spliceosomal RNA–RNA network
(McGrail et al, 2009) have shown that Cwc2 binds RNA non-specifically, so the next thing is to see if all of the previously identified crosslinking residues bind RNA with any preference or not. As mentioned earlier, comparative crosslinking assays were performed with U6 snRNA-Cwc2 and U4 snRNA-Cwc2 complexes. Whilst the two RRM crosslinking residues did not show any net difference in binding, crosslinks involving the ZnF (C87), the connector element (Y120) and the Torus domain (F47) were much more frequently identified and validated (by MSMS) with U6 snRNA-Cwc2 compared with Cwc2 crosslinked to U4. Thus the latter 3 domains do have a binding preference for U6.
To find which regions of U6 snRNA bind to which regions of Cwc2 they used a crosslinking assay with a binary U6 snRNA ISL-Cwc2 (bases 50-90) complex. After dissociation of U1 and U4 snRNAs from the spliceosome, U6 snRNA rearranges and forms an internal stem–loop (ISL), which plays a central role in the catalysis of splicing. U6-ISL was crosslinked frequently to both RNP2 and RNP1, and to a lesser extent (compared with U6) to the ZnF and connector element, while no crosslinks were found for the Torus domain. Therefore in binary complexes, different Cwc2 sites can distinguish between distinct regions of U6 snRNA. The RRM binds the ISL and the ZnF and the connector element contact other U6 snRNA regions.
To further support the idea that Cwc2 is a multipartite RNA-Bp, a point mutation assay to show how each identified cross linking residue affects binding was used. The only cross linking residue that could not undergo mutational assay was C87 as the zinc finger is essential for Cwc2 stability and the C87 has a structural role that affects the zinc finger's capability to stabilise the Cwc2 protein (McGrail et al, 2009). Single mutations (as shown in Figure 5) did not change RNA binding affinity for U6 snRNA suggesting that the presence of multiple interactions between Cwc2 and RNA means that they can compensate for the loss of any one crosslinked residue (except C87) . However mutations of Y138 caused a faster migrating RNP complex than wild type suggesting that the conformation of the RNP complex changed, perhaps due to a change in interaction between the RRM region and the U6 snRNA.
When double point mutations were introduced however, there were changes in RNA binding affinity. The double point mutations that had effects were mutations of Y138 and Y120 or C181; these mutations resulted in a decrease in the U6 snRNA that shifted in the gel from free U6 snRNA to the complex. This indicates C181 and Y120 are important for binding of RNA. No difference was observed between Y138-F47/Y138-K152 mutants and the Y138 single point mutant, so it can be assumed that these are not essential for RNA binding. Also the Y120-F47 mutants were comparable to the wild type, which implies that mutations in the connector and torus regions are not sufficient to change RNA binding.
When double point mutations were introduced however, there were changes in RNA binding affinity. The double point mutations that had effects were mutations of Y138 and Y120 or C181; these mutations resulted in a decrease in the U6 snRNA that shifted in the gel from free U6 snRNA to the complex. This indicates C181 and Y120 are important for binding of RNA. No difference was observed between Y138-F47/Y138-K152 mutants and the Y138 single point mutant, so it can be assumed that these are not essential for RNA binding. Also the Y120-F47 mutants were comparable to the wild type, which implies that mutations in the connector and torus regions are not sufficient to change RNA binding.
 |
Figure 5: Electrophoretic mobility shift analysis of the interaction of recombinant Cwc2 mutants with yeast U6 snRNA. Equal amounts of [32P]-50-labelled U6 snRNA were incubated in the presence of increasing concentrations of purified full-length Cwc2 (wt), single point Y138 mutant, or one of five double point mutated proteins. Resulting RNP complexes and free U6 RNAwere resolved by native 6%PAGE. The identity and concentrations of the proteins used are indicated above the gel. Migration positions of RNA–protein complexes and unbound U6 snRNA is shown on the right. |
Overall this indicates that both the RRM and connector sequences are important for RNA binding, thus supporting the Cwc2 multipartite protein theory.
References:
References:
Rasche N, Dybkov O, Schmitzova´ J, Akyildiz B, Fabrizio P, Lu¨hrmann R (2012) Cwc2 and its human homologue RBM22
promote an active conformation of the spliceosome catalytic centre. EMBO J 31: 1591–1604
McGrail JC, Krause A, O’Keefe RT (2009) The RNA binding protein Cwc2 interacts directly with the U6 snRNA to link the nineteen complex to the spliceosome during pre-mRNA splicing. Nucleic Acids Res 37: 4205–4217
