Characterization of the Crithidia fasciculata mRNA cycling sequence binding proteins

The Crithidia fasciculata cycling sequence binding protein (CSBP) binds wit h high specificity to sequence elements in several mRNAs that accumulate pe riodically during the cell cycle. Mutations in these sequence elements abol ish both cycling of the mRNA and binding of CSBP. Two genes, CSBPA and CSBP B, encoding putative subunits of CSBP have been cloned and were found to be present in tandem on the same DNA molecule and to be closely related. CSBP A and CSBPB are predicted to encode proteins with sizes of 35.6 and 42.0 kD a, respectively. Both CSBPA and CSBPB proteins have a predicted coiled-coil domain near the N terminus and a novel histidine and cysteine motif near t he C terminus. The latter motif is conserved in other trypanosomatid specie s. Gel sieving chromatography and glycerol gradient sedimentation results i ndicate that CSBP has a molecular mass in excess of 200 kDa and an extended structure. Recombinant CSBPA and CSBPB also bind specifically to the cycli ng sequence and together can be reconstituted to give an RNA gel shift simi lar to that of purified CSBP. Proteins in cell extracts bind to an RNA prob e containing six copies of the cycling sequence. The RNA-protein complexes contain both CSBPA and CSBPB, and the binding activity cycles in near synch rony with target mRNA levels. CSBPA and CSBPB mRNA and protein levels show little variation throughout the cell cycle, suggesting that additional fact ors are involved in the cyclic binding to the cycling sequence elements.