The CAF1-NOT complex of Trypanosoma brucei, like that of other eukaryotes, contains several NOT proteins (NOT1, NOT3, NOT3/5, NOT10, and NOT11), NOT9/CAF40, and the CAF1 deadenylase, which targets 3' poly(A) tails. Again like other eukaryotes, deadenylation is the first step in the degradation of most trypanosome mRNAs. In animal cells, destruction of unstable mRNAs is accelerated by proteins that bind the RNA in a sequence-specific fashion, and also recruit the CAF1-NOT complex. However, this has not yet been demonstrated for T. brucei. To find interaction partners for the trypanosome NOT complex, we did a genome-wide yeast two-hybrid screen, using a random shotgun protein fragment library, with the subunits CAF40, NOT2, NOT10 and NOT11 as baits. To assess interaction specificity, we compared the results with those from other trypanosome proteins, including the cyclin-F-box protein CFB1. The yeast 2-hybrid screen yielded four putatively interacting proteins for NOT2, eleven for NOT11, but only one for NOT9/CAF40. Both CFB1 and NOT10 had over a hundred potential interactions, indicating a lack of specificity. Nevertheless, a detected interaction between NOT10 and NOT11 is likely to be genuine. We also identified proteins that co-purify with affinity tagged NOT9/CAF40 by mass spectrometry. The co-purifying proteins did not include the 2-hybrid partner, but the results confirmed NOT9/CAF40 association with the CAF1-NOT complex, and suggested interactions with expression-repressing RNA-binding proteins (ZC3H8, ZC3H30, and ZC3H46) and the deadenylase PARN3.
An enzyme-mediated bioorthogonal labeling method for genome-wide mapping of 5-hydroxymethyluracil