Question

5. ZBP1 binds mRNAs. How would you determine whether there is a specific sequence in the mRNA that is required for ZBP1 binding? (Hint: they discuss relevant techniques at the bottom of page 1899 in the article, but make sure you use your own words in your answer). (5 points)

Answer 1

Introduction to ZBP1 protein:

ZBP1 or Zipcode binding protein 1 is an oncofetal RNA-binding protein, essential for development of nervous system. It mediates the transport and local translation of b-actin by the KH3-KH4 double domain. Reduced ZBP1 expression results in impaired neural development and a smaller cerebral cortex in the embryo.

ZBP binds to mRNA by a KH3-KH4 di-domain complex, where KH4 recognizes a non-canonical GGA sequence via an enlarged and dynamic hydrophobic groove. mRNA looping drives the ZBP1-mRNA binding and the ZBP1 concentration regulates the binding.

Determination of binding sequence.

ZBP1 contains 6 mRNA binding domains. To determine whether there is a specific sequence in the mRNA that is required for ZBP1 binding, we can use RNA oligos with KH3-KH4 recognition motifs present in it. The KH3-KH4 has to mRNA binding groove on opposite side and are separated by a spacer of about 14 nucleotide base long. The 5' to 3' order of the KH4-KH3 target sequence can be swapped to create a recognition unit, where the RNA spacer binds the sequence either in 5' to 3' or 3' to 5' direction. This binding can be studied by NMR spectroscopy to determine the specific mRNA sequence that has bind the ZBP1 protein.