Identification of target mRNAs for the clock-controlled RNA-binding protein Chlamy 1 from Chlamydomonas reinhardtii

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Abstract
An endogenous clock regulates the temporal expression of genes/mRNAs that are involved in the circadian output pathway. In the green alga Chlamydomonas reinhardtii, a clock-controlled RNA-binding protein (Chlamy 1) was identified recently, which represents an analog of the circadian trans-acting factor CCTR from the phylogenetically diverse alga Gonyaulax polyedra. In order to identify in C. reinhardtii target mRNAs that can be recognized by Chlamy 1, gel mobility-shift assays and UV-crosslinking experiments were carried out, and revealed that this protein interacts specifically with the 3′ untranslated regions of several mRNAs and recognizes them all via a common cis-acting element, composed of at least seven UG repeats. By using competition assays, it was found that the affinity of Chlamy 1 is highest for mRNAs whose products are key components of nitrogen and CO2 metabolism. Since the activities of enzymes involved in nitrogen metabolism vary in a temporal pattern that is opposite in phase to that of Chlamy 1 binding activity, the protein may repress the translation of the cognate mRNAs.

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