Figure 1. Mpp6 stimulates the nuclear RNA exosome and binds RNA.

(A) Mpp6 stimulates Rrp6 activities when degrading 5’ fluorescein-labeled 49 nt polyA in 10- (top) and 11- subunit exosomes (bottom). Relative positions of RNA substrate, Rrp6 products and the Dis3 product are indicated to the right of gels in panels A, B, and C. Quantitation of initial rates shown to the right of representative gels. For Rrp6 initial rates, the median length of Rrp6 products was determined at the earliest time points and used to calculate initial rates using the equation (1 – (median product length/substrate length)/min) to yield a fraction of length degraded per minute. For Dis3, initial rates were determined by calculating the fraction of full-length substrate degraded based on the accumulation of 4–5 nt product. For the lower gel, Dis3 and Rrp6 initial rates were determined at the earliest time points where distributive products of Rrp6 are easily distinguished/separated from the 4–5 nt processive products of Dis3. (B) Dis3 exoribonuclease activity can be stimulated on 5’ fluorescein-labeled 49 nt polyA in 11-subunit exosomes if Rrp6 is present but catalytically inert. (C) Top: Predicted domain structure of S. cerevisiae Mpp6. Calculated with Jpred (Cole et al., 2008). Previously identified conserved regions (Milligan et al., 2008) are marked with red stars, with Mtr4 recruiting and exosome stimulating domains as described in this work labeled. Below: a minimal fragment of Mpp6 (residues 81 to 120) is necessary and sufficient to stimulate Rrp6 activity in Exo11^Dis3/Rrp6. Representative decay assays of Exo11^Dis3/Rrp6 on 5’ fluorescein-labeled polyA 49 nt RNA with different Mpp6 constructs added in 2-fold molar excess. (D) Mpp6 enhances RNA binding of Rrp6-containing exosomes on polyA RNA, and alleviates binding defects caused by Exo9 channel occlusions. Fluorescence polarization of Mpp6 and Exo10^Rrp6exo- with or without PH-like ring occlusions in the lower half of the Exo9 channel, with binding to 5’ fluorescein-labeled polyA 37 nt RNA. Bar graphs and error bars in panels A, B, and D are the result of triplicate experiments with error bars indicating plus or minus one standard deviation.

DOI: http://dx.doi.org/10.7554/eLife.29062.002

Figure 1—figure supplement 1. Mpp6 is a RNA-binding protein cofactor of the nuclear RNA exosome that stimulates exosome exoribonuclease activities.

(A) Mpp6 binds various sequences of single-stranded RNA. Fluorescence polarization of Mpp6 bound to 5’ fluorescein-labeled polyA 37 nt, AU-rich 36 nt, and polyU 36 nt RNA. Results of triplicate experiments shown with error bars representing plus and minus one standard deviation. (B) Mpp6 associates with Exo9 by analytical gel-filtration (Superose 6). Fractions were analyzed by SDS-PAGE and stained with Sypro Ruby. A N-terminal truncation corresponding to residues 59–186 was used to better separate Mpp6 from Exo9 subunits with similar molecular weight as full-length Mpp6. (C) Mpp6 does not stimulate free Dis3 on 5’ fluorescein-labeled 49 nt polyA. Reaction products were analyzed by denaturing PAGE. (D) Mpp6 does not stimulate free Rrp6 on 5’ fluorescein-labeled 49 nt polyA. Reaction products were analyzed by denaturing PAGE. (E) Mpp6 does not stimulate either the exoribonuclease or endoribonucease activities of Dis3 in Exo10^Dis3 on 5’ fluorescein-labeled 49 nt polyA. Reaction products were analyzed by denaturing PAGE. (F) Mpp6 stimulation of Rrp6 exoribonuclease activity on 5’ fluorescein 36 nt AU-rich RNA in Exo10^Rrp6. Initial rates from triplicate experiments, with error bars representing plus and minus one standard deviation. (G) Exo10^Dis3 exoribonuclease activity can be stimulated by Mpp6 on 5’ fluorescein-labeled AU-rich 49 nt RNA. Bar graphs represent (left) initial rates and (right) apparent Kd values of Exo10^Dis3exo- determined by fluorescence polarization on 5’ fluorescein 36 nt AU-rich RNA. (H) Mpp6 exosomes utilize the S1/KH ring to degrade RNA. Mpp6 can partially, but not completely alleviate the inhibitory effects of point mutations within the S1/KH ring on Rrp6 activity in Exo10^Rrp6. Assays performed on 5’ fluorescein-labeled polyA 49 nt RNA, and intermediates analyzed by denaturing PAGE.

DOI: http://dx.doi.org/10.7554/eLife.29062.003