Using C. elegans you have devised a screen to identify mutants in the RNA interf

Question

Using C. elegans you have devised a screen to identify mutants in the RNA interference pathway. A functional RNAi pathway will inhibit reporter GFP expression. You have discovered many mutants that express GFP. You identify one mutant lies within a gene encoding a protein with two domains. The first domain is a leucine zipper while the other domain has a high number of acidic amino acids. You hypothesize that the gene product regulates expression of some of the key players in RNA interference and therefore you name the gene TRI for Transcriptional Regulator of RNA Interference.


From the information above, why do you think TRI encodes a transcriptional regulator?

Do you think TRI is an activator or repressor and why?


You hypothesize that TRI regulates either Drosha or Dicer gene expression.
What assay can you run in vitro to determine if TRI binds to potential regulatory DNA sequences upstream of Dicer or Drosha?

Explanation / Answer

From the information above, we know that TRI encodes a transcriptional regulator as it possesses a leucine zipper domain. Leucine zipper domains are usually found as a part of a dimerization domain in transcription factors. In addition, the leucine zipper contains leucine residues which are necessary for binding to DNA. -------------------------------------------- TRI is a repressor as the mutant shows reporter GFP expression. The normal gene would repress the transcription of a gene. When such a repressor is mutated, it can no longer produce the repressor and transcription of the reporter GFP occurs. --------------------------------------------- To check whether TRI regulates Drosha or Dicer genes, we can isolate C. elegans which is normal and C. elegans which contains a TRI-mutant. We then wait to see if the genes are expressed in the RNAi interference pathway. The gene product of Dicer is needed for the processing of pre-miRNA to mature miRNA. The gene product of Drosha is responsible for initiation of the processing of microRNA. In the TRI-mutant,  the processing of miRNA does not occur.  This can help us determine whether the TRI binds to regulatory sequences of DNA.

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