Identification of cis and trans Elements Involved in the Cell Cycle Regulation of Multiple Genes in Crithidia fasciculata

Abstract

Transcripts of several DNA replication genes, including the RPA1 and TOP2 genes, encoding the large subunit of nuclear replication protein A and the kinetoplast topoisomerase II, accumulate periodically during the cell cycle in the trypanosomatid Crithidia fasciculata. An octamer consensus sequence, CAUAGAAG, present in the 5′ untranslated regions (UTR) of these mRNAs is required for periodic accumulation of the TOP2 and RPA1 transcripts and also for binding of a nuclear factor(s) to the 5′ UTR RNAs of these genes. We show here that insertion of multiple (six) copies of this octamer sequence (6× octamer) into the 5′ UTR of a reporter gene confers periodic accumulation on its transcript. Competition experiments and UV cross-linking studies show that the 6× octamer RNA and TOP2 5′ UTR RNA bind to the same nuclear factor(s). Single-nucleotide substitutions in the 6× octamer that abolish the RNA gel shift also prevent cyclic accumulation of the reporter gene transcript. A protein termed cycling element binding protein, purified by affinity chromatography using 6× octamer RNA as a ligand, binds to RNAs containing wild-type octamers and not to those with mutant octamers. These results define a small sequence element in C. fasciculata mRNAs required for their cell cycle regulation and report the identification and purification of a putative regulatory protein that binds specifically to these elements.

ACKNOWLEDGMENTS

We thank Lisa Brown for performing the experiment with a single octamer cloned in pΔ10Not.

This research was supported by NIH grant GM53254.