2008 Rustbelt RNA Meeting
The TRAMP complex is critical for quality control and 3’ end processing of nuclear RNA in S. cerevisiae. The TRAMP complex, which consists of the poly(A) polymerase Trf4p, the RNA binding protein Air2p, and the DExH RNA helicase Mtr4p, appends short poly(A) tails to RNAs designated for degradation by the nuclear exosome. Both the 5’ to 3’ polyadenylation activity of Trf4p, and the 3’ to 5’ helicase activity of Mtr4p are required for TRAMP to stimulate RNA degradation by the exosome. However, it is unclear how these two activities, given their opposing directionality, function together in one complex. To illuminate this question we have quantitatively analyzed polyadenylation and RNA unwinding activities for a reconstituted, purified TRAMP complex. Using a newly developed method that allows us to measure rate constants for addition of individual adenylate residues to the RNA, we show that poly(A) addition does not occur in equal steps but in a characteristic pattern where RNA species with 4-10 A residues accumulate. Functional Mtr4p is required for this characteristic pattern; it is not observed for TRAMP with non-functional Mtr4p, or in the absence of Mtr4p. Interestingly, we find that TRAMP requires a minimal number of 4 unpaired nucleotides to unwind RNA duplex substrates, suggesting that the polyadenylation by Trf4p serves to provide a landing site for Mtr4p, which then unwinds RNA secondary structure. We further show dramatic stimulation of the helicase activity of Mtr4p by Trf4p/Air2p, with a preference for duplexes with short overhangs. Collectively, our data indicate strong functional interdependence of Trf4p and Mtr4p during polyadenylation and duplex unwinding by TRAMP. The opposing polarities of polyadenylation and unwinding activities are critical for RNA recognition and ensure that only a minimal number of adenylates are added to RNAs prior to further processing.
Keywords: poly(A) polymerase, helicase