Figure 5. Channel occlusion inhibits RNA binding and RNase activities of Rrp44 and Rrp6.

Bar graphs depicting initial rates calculated for Rrp6 and Rrp44 RNase on A) AU-rich and B) polyA RNA. Occluding the central channel of Exo11^44/6 (Exo11^{44/6/channel-}) with loop insertions (Rrp41-M/Rrp45-M) inhibits both Rrp44 activities while Exo11^{44exo-/6/channel-} diminishes Rrp6 exoribonuclease activities and alleviates Rrp6 inhibition by Rrp44^exo-. Rrp44 endonuclease activities are inhibited by channel occlusion in Exo10^44exo- and Exo11^44exo-/6exo-. Assays performed in triplicate and initial rates calculated from data obtained in the linear range (Figures S6A and S6C). Bar graphs on the right depict apparent K_d values for various exosome complexes with RNA. Error bars are ± 1 standard deviation. C) Channel occlusion in Exo9 leads to diminished cross-linking (left). Panel to the right shows that channel occlusion in Exo10^44exo- leads to loss of most RNA-protein adducts to core subunits. Next panel shows that channel occlusion in Exo10^6exo- slightly weakens cross-linking to the S1/KH cap proteins and Rrp6. On the right, channel occlusion in Exo11^44exo-/6exo- shifts the cross-linking pattern from one involving Rrp44, the PH-like and S1/KH rings to one involving Rrp6 and the S1/KH ring. Products separated by SDS-PAGE and imaged by detecting the fluorescence of the 5’ labeled AU-rich RNA. Major cross-linked species are labeled and indicated by arrows.