The next step should logically be to concentrate on the ZnF and connector element domains that bind RNA in the Cwc2 protein because:
- Previously mentioned experiments led to the realisation that the RRM domain alone is insufficient for Cwc2 splicing function
- Although the RNPs motifs are exposed and able to bind RNA, single-point mutations in amino acids Y138 and F183 (both of which are located in the RRM - RNP2 and RNP1 motifs of Cwc2, respectively) are not lethal (McGrail et al, 2009)
· First Y89 in the ZnF region was mutated to alanine because in RNA–protein cocrystals of MBNL1 (RNA splicing factor muscleblind-like) and TIS11d (tandem zinc finger (TZF) domain), there are respectively one and two aromatic amino acids in the ZnF structures that stack against nucleotide bases of the RNA (Hudson et al, 2004; Teplova and Patel, 2008). In Cwc2 one of the corresponding aromatic residues is replaced with a Leucine (L83) and the other is conserved (Y89).
· This Y89A mutant Cwc2 was added to Cwc2 depleted extract, which partially restored RNA binding but only ~34% compared to wildtype, showing the importance of Y89 and thus the ZnF for Cwc2 function.
· As for the connector element, multiple single point mutations were generated, including one where the cross linking residue Y120 is replaced with Alanine – this completely prevents Cwc2 from restoring splicing in a depleted sample. The replacement of K132 and F130 with alanine also leads to a large loss in splicing activity. Therefore, the connector element is important for the function of Cwc2 in splicing in vitro.
Although the assay does not provide direct evidence that the reduction in splicing activity is due to inhibition of an RNA–Cwc2 interaction by the point mutations, this is likely to be the case.
Boosting this theory is the fact that; aromatic residues of other ZnF domains corresponding to Y89 in Cwc2 are known to take part in RNA binding (Hudson et al, 2004; Teplova an Patel, 2008). Furthermore, Y120 of the connector element belongs to the Y120–R121 region that was crosslinked to U6 snRNA and its mutation together with Y138A reduced RNA binding as shown by the EMSA experiments (See below).
Boosting this theory is the fact that; aromatic residues of other ZnF domains corresponding to Y89 in Cwc2 are known to take part in RNA binding (Hudson et al, 2004; Teplova an Patel, 2008). Furthermore, Y120 of the connector element belongs to the Y120–R121 region that was crosslinked to U6 snRNA and its mutation together with Y138A reduced RNA binding as shown by the EMSA experiments (See below).
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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. |
References:
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
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
