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Tethered function assay

Project leader: Jeff Coller (jmc71@case.edu)

Tethered function assays. Once putative RNA-binding proteins are identified, we will determine their role in the transport of mRNA. This is particularly challenging because the mRNA substrate may be unknown. We will first determine which isolated protein(s) have functional relevance in the process of controlling mRNA trafficking before searching for their target mRNAs. The function of putative RNA binding proteins will be determined using a novel assay called the “tethered function” assay (Coller et al., 1998; Coller and Wickens 2002 & 2007).

This approach allows us to directly demonstrate a role of candidate proteins in mRNA trafficking without prior knowledge of the mRNA target. The rationale for the tethered function assay is based on the observation that proteins and protein complexes that regulate mRNA metabolism often possess two activities. They bind mRNA, and then they elicit some function, that is regulate mRNA transport, translation, localization, and/or stability. These two activities can often reside in different proteins in a complex, or in different regions of a single polypeptide. Much can be learned about the function of the protein or complex once it is stripped of the constraints imposed by RNA binding. With this in mind, we developed a “tethered function” assay, in which the mRNA regulatory protein is brought to a mRNA reporter through a heterologous RNA-protein interaction (Coller et al., 1998). In this manner, the functional activity of the protein can be studied independent of its intrinsic ability to recognize and bind RNA. This simple assay has proven powerful in dissecting numerous proteins identified as being involved in post-transcriptional regulation (Coller and Wickens, 2002 & 2007). This is especially true in instances where the natural mRNA target for a given protein is unknown. The design of the tethered function assay is straightforward. To determine the effects of a putative RNA regulator, the protein is expressed as a chimera with a tethering polypeptide.

The tethering protein is an RNA-binding protein that recognizes an RNA tag sequence with high specificity and affinity. The effect of the fusion protein on mRNA metabolism is determined by co-expressing the chimera with an mRNA reporter into which a tag RNA sequence has been embedded. Usually, the tethering site lies in the 3’ UTR of the mRNA; this region is relatively unconstrained evolutionarily and the natural site of action of many mRNA regulators. The fusion protein’s effects on mRNA metabolism are assayed by conventional means. For more information link to Coller and Wickens, 2007.