Cwc2 as a folding unit
The RNA binding domains are found in the N-terminal region of the polypeptide. The C-terminal region (residues 237-339) interacts with WD40 domain of Prp19 protein, which provides scaffold for the NTC protein complex. The Cwc2 tertiary structure could not be crystallised initially due to the disordered C-terminal region preventing the formation of the required, stable structure.
To determine whether this C-terminal domain is in fact essential for the catalytic mechanism, limited proteolysis with GluC enzyme yielded a Cwc2 fragment of residues 1-240, purified through size-exclusion chromatography, named Cwc2ΔC :
To determine whether this C-terminal domain is in fact essential for the catalytic mechanism, limited proteolysis with GluC enzyme yielded a Cwc2 fragment of residues 1-240, purified through size-exclusion chromatography, named Cwc2ΔC :
- A standard yeast splicing extract depleted in endogenous Cwc2 was achieved through the expression of Cwc2, TAP-tagged at it's c-terminus.
- A TAP tag consists of CBP, TEV protease cleavage site and Protein A, which binds tightly to IgG –sepharose purification column, creating a depleted extract.
- Western Blotting confirmed the depletion, and it showed a very low splicing activity (possibly due to residual Cwc2), illustrating its essential role in splicing.
- Addition of this fragment to the depleted extract restored the cells splicing capacity, suggesting the C-terminal is not an essential domain, and the proteolytic-resistant fragment is the catalytic core.
To determine whether this fragment is still able to interact with vital protein components of the spliceosome in the same manner as the full length fragment:
Figure 2: Polyacrylamide gel, visualising protein fragments using autoradiography.
Residual levels of mRNA calculated (%): 16, 109, 97, 16, 50, 21, 58, 69 corresponding to lanes 3-10 respectively.
An electrophoretic mobility assay further confirmed that the generated fragment still retained its RNA binding ability. Equal amounts of U6 snRNP were added to increasing concentration of the wild-type full length Cwc2, Cwc2ΔC, and then just the RRM domain:
- Radio-labelled pre-mRNA was incubated with a yeast whole extract, either mock extract and or with Cwc2 depleted extract. The mock was used as a reference for 100% relative to amount of mRNA calculated.
- Addition of either wild type Cwc2 or mutated Cwc2 extract were added.
- The RNA was then analysed, after incubation, on a polyacrylamide gel through autoradiography.
- The residual levels of mRNA were calculated and they fluctuated depending on the type of residue mutated were recovered in the mutagenesis studies. Mutation of Y120A produces a very low residual mRNA level, suggesting it must be an essential residue.
- The results revealed that the Cwc2 fragment could bind to U6 with similar affinity to the U6 snRNP as the full length protein.
- It also illustrated that the RRM domain could not bind to the U6 snRNP efficiently, stressing the importance of the other RNA binding domain: ZnF.
The Completed Crystal Structure and its RNA binding Domains
The Crystallisation of Cwc2
- The structure of Cwc2ΔC was solved by single analogous dispersion (SAD).
- Crystallisation was carried out at 20 ºC using the sitting-drop vapour-diffusion method.
- The data was collected around the zinc K-edge, obtaining a structure with a 2.4A resolution, and refined R/Rfree = 22.3/25.5 [ R= R factor ;Rfree = Free R ]
- The purified, well-define stable structure turned out to be composed of residues 3-226, excluding its unstable C-terminal domain, enabling it to have with good stereochemistry.
Figure 3: The crystal structure reveals a compact, globular structure with nine a-helices, five 310-helix and four B-strands. The RNA binding domains previously discovered are easily recognisable, and are explained in more detail later on. It is shown as an asymmetric unit composed of two molecules.
Comparison to the human homologue Rbm22
- Roles of NTC and NTC-associated proteins are evolutionary conserved across species.
- The human homologue Rbm22 has been structurally (Figure 4) and sequence (Figure 5) aligned with Cwc2ΔC to illustrate the conserved RNA binding domains (RRM and ZnF domains)
- Rbm22 has also been found to be implicated in other non-splicing specific activities such as lipid biogenesis, DNA damage response, and cellular senescence.
Figure 4: Crystal Structures of Cwc2ΔC and the homologous, human Rbm22.Their crystal structures are aligned, revealing the sites of the conserved RNA binding domains.
Figure 5: Sequence alignment with “EMBOSS Water pairwise sequence alignment.” The results present a 44.4% similarity between the two sequences, with an overall and impressive score of 97, further confirming the presence of these conserved domains.
