Characterizing the RNA-binding activity of MEX-5

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Research Strategy – Approach

Specific Aim 1: How does the primary sequence of MEX-5 determine its RNA-binding specificity?

The CCCH-type TZF proteins recognize their RNA targets though a template of H-bonds between the amide and carbonyl groups of the protein backbone and the Watson-Crick edges of the bases (ref Hudson, Teplova). This observation suggests that in CCCH-type TZF proteins any variation of the primary sequence that results in a change of the backbone conformation alters the pattern of interactions between the protein and the RNA molecules and therefore the ability to bind specific sequences of RNA.

TIS11d is a CCCH-type TZF protein that shares 40% of sequence identity with MEX-5. It has been previously demonstrated that TIS11d binds with high affinity (Kd=8±2 nM) to the sequence UUAUUUAUU, while alterations of any nucleotide, particularly the two adenosines, is poorly tolerate. MEX-5 instead binds with similar affinities (Kd=10-100 nM) to a heterogeneous set of uridine-rich sequences of at least 10 nucleotides (ref Pagano). The comparison of the sequences of MEX-5 and TIS11d reveals a different scheme in the spacing between the CCCH zinc coordinating residues in each ZF. In MEX-5 this scheme is CX9CX5CX3H for ZF1 and CX10CX5CX3H for ZF2. For TIS11d, both ZFs have the same spacing, with the first two cysteines closer than in MEX-5: CX8CX5CX3H (Figure 1). In addition, the region between the two ZFs (linker) consists of 23 residues in MEX-5 and 18 in TIS11d (Figure 1). These differences in the primary sequence are thought to result in a different architecture of the protein backbone of MEX-5 respect to TIS11d. To verify this hypothesis, variants of the TZF domain of MEX-5, in which the spacing of the CCCH residues and the linker length are changed, will be constructed using site-directed mutagenesis. The characterization of the RNA-binding activity of the MEX-5 variants will help to identify the elements of the primary sequence that allow MEX-5 to be more promiscuous than TIS11d in RNA binding.

1.1 Is the spacing between CCCH residues or the length of the linker between the ZFs affecting the RNA-binding specificity of MEX-5?

Strategy: The CCCH-type TZF domain of MEX-5 (residues 268-346) will be expressed and purified to perform RNA-binding studies. Residues Gly282 (in ZF1) and Thr328-Gly329 (in ZF2) will be deleted in turn from the MEX-5 sequence to generate the variants MEX-5ZF1, MEX-5ZF2 and MEX-5ZF1,ZF2 in which the original spacing between the CCCH residues is altered in order to resemble the scheme CX8CX5CX3H of TIS11d.

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