MLN51 stimulates the RNA-helicase activity of eIF4AIII

Abstract

The core of the exon-junction complex consists of Y14, Magoh, MLN51 and eIF4AIII, a DEAD-box RNA helicase. MLN51 stimulates the ATPase activity of eIF4AIII, whilst the Y14-Magoh complex inhibits it. We show that the MLN51-dependent stimulation increases both the affinity of eIF4AIII for ATP and the rate of enzyme turnover; the K(M) is decreased by an order of magnitude and k(cat) increases 30 fold. Y14-Magoh do inhibit the MLN51-stimulated ATPase activity, but not back to background levels. The ATP-bound form of the eIF4AIII-MLN51 complex has a 100-fold higher affinity for RNA than the unbound form and ATP hydrolysis reduces this affinity. MLN51 stimulates the RNA-helicase activity of eIF4AIII, suggesting that this activity may be functionally important.

Figure 1

ATPase activity of eIF4AIII. (A) ATPase rates for 400 nM eIF4AIII in the presence or absence of 800 nM MLN51 SELOR, 800 nM Y14-Magoh and 200 µg.ml⁻¹ yeast RNA. Increasing the Y14-Magoh concentration (lane 7; 3.2 µM) did not further inhibit the ATPase activity. RNA-dependent rates were measured at increasing ATP concentrations for 1 µM eIF4AIII alone (B) or for 400 nM eIF4AIII with 800 nM MLN51 SELOR (C) and the data were fitted to the Michaelis-Menten equation to determine values for K _M and k _cat.

Figure 2

RNA binding by the core of the exon-junction complex. Binding to a biotinylated 35-mer RNA oligonucleotide was measured using surface plasmon resonance. 160 nM protein samples were injected across the chip surface and the interactions measured. The injections started at time zero and finished at 200 s. The sensorgrams are indicated in the key.

Figure 3

RNA-helicase activity of eIF4AIII. Helicase activity was assayed for 0.25 to 4 µM eIF4AIII with 10 nM ³²P-labelled duplex RNA. Experiments were performed for (A) eIF4AIII alone, (B) with 5 µM MLN51 SELOR in lanes 2-8 or (C, D) with 5 µM MLN51 SELOR and Y14-Magoh in lanes 2-8. D is the same as C except the assays were terminated with a higher SDS concentration to disrupt the core EJC, affecting the resolution of the gel. The duplex was heated to 95°C (lane 1) as a marker for the migration of single-stranded RNA (ssRNA) and the input duplex double-stranded RNA (dsRNA), in the absence of additional protein, is shown in lane 2 top panel. A control assay in the absence of ATP was also performed (lane 8). A schematic illustration of the RNA substrate is shown at the top of the Figure.