- The spliceosome requires integrated protein factors to aid in its spatial arrangement when performing its catalytic function. These are known as NTC-associated factors. Cwc2 is the only NTC-associated protein that has RNA binding domains. The protein structure could only be crystallised through removal of the C-terminal domain, using limited proteolysis.
- Crystallisation, to 2.4Å resolution, revealed the structure of the two RNA binding domains (RRM and ZnF) which form a single folding unit.
- The canonical RRM domain contains two conserved subdomains with essential amino acid residues required for stabilisation. The ZnF domain adopts the common CCCH circular conformation, with conserved cysteine and histidine residues co-ordinating a single zinc atom. This domain is similarly stabilised through conserved amino acid residues.
- The torus domain surrounds the zinc finger domain and scaffolds it in a manner that improves the catalytic efficiency of cwc2. The RNA recognition motif was shown to bind RNA faster and with higher affinity and this was modeled with the Fox1 protein. This conserved torus fold is seen as an evolution from the zinc finger domain.
- The connector element that links the torus domain and RRM is shown to be potentially involved in RNA binding and therefore improving cwc2 function by guiding RNA to the binding pocket. It is shown to bind certain snRNAs in the spliceosome complex. It shows high conformational flexibility and is similar to the CBC20 N-terminus.
- 7 residues from four different domains in Cwc2 were found to be crosslinked to RNA. Some were found to not be essential for RNA binding, but others were found to be very important in RNA binding, thus Cwc2 is a multipartite RNA binding protein.
- Single point mutations showed that the ZnF domain is important for Cwc2 function and the connector element is important for the function of Cwc2 in splicing in vitro.
- What’s next?
o This information can be used to investigate the human homologue of Cwc2, RBM22.
o I.e. if the structure, function, crosslinked residues, essential/non-essential domains for RNA binding are the same.
o It can also be a framework for understanding the regulatory function of the NTC in RNA splicing
o And be used to understand how exactly Cwc2 links the NTC to spliceosome during pre-mRNA splicing.
References
Maris, C., Dominguez, C., & Allain, F. (2005). The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression. The FEBS Journal , 272, 2118–2131.
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McGrail, J., Krause, A., & O’Keefe, R. (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 Research , 37
(13), 4205–4217.
Lodish, H., Berk, A., Kaiser, C.A., Krieger, M., Bretscher, A., Ploegh, H., Scott, M.P., and Amon, A. (2012). Molecular Cell Biology, 7 edn (W.H. Freeman & Company).
Lu, P., Lu, G., Yan, C., Wang, L., Li, W., and Yin, P. (2012). Structure of the mRNA splicing complex component Cwc2: insights into RNA recognition. Biochemical Journal 441, 591-597.
Rasche, N., Dybkov, O., Schmitzova, J., Akyildiz, B., Fabrizio, P., and Luehrmann, R. (2012). Cwc2 and its human homologue RBM22 promote an active conformation of the spliceosome catalytic centre. Embo Journal 31, 1591-1604.
Schmitzova, J., Rasche, N., Dybkov, O., Kramer, K., Fabrizio, P., Urlaub, H., Luehrmann, R., and Pena, V. (2012). Crystal structure of Cwc2 reveals a novel architecture of a multipartite RNA-binding protein. Embo Journal31, 2222-2234.
Wahl, M.C., Will, C.L., and Luehrmann, R. (2009). The Spliceosome: Design Principles of a Dynamic RNP Machine. Cell 136, 701-718.
Hudson BP, Martinez-Yamout MA, Dyson HJ, Wright PE (2004) Recognition of the mRNA AU-rich element by the zinc finger domain of TIS11d. Nat Struct Mol Biol 11: 257–264
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
Teplova M, Patel DJ (2008) Structural insights into RNA recognition by the alternative-splicing regulator muscleblind-like MBNL1. Nat Struct Mol Biol 15: 1343–1351
Pictures used in techniques page were taken from the following websites:
Size exclusion chromatography
http://www.chemistry.adelaide.edu.au/external/soc-rel/content/size-exc.htm
Mutagenesis experiment
http://www.chem-agilent.com/contents.php?id=300131
Crystallisation
http://www.bio.davidson.edu/courses/molbio/molstudents/spring2003/kogoy/protein.html
Gel shift assay
http://meddic.jp/gel_mobility_shift
