The human U5-200kD DEXH-box protein unwinds U4/U6 RNA duplices in vitro

Abstract

Splicing of nuclear precursors of mRNA (pre-mRNA) involves dynamic interactions between the RNA constituents of the spliceosome. The rearrangement of RNA-RNA interactions, such as the unwinding of the U4/U6 duplex, is believed to be driven by ATP-dependent RNA helicases. We recently have shown that spliceosomal U5 small nuclear ribonucleoproteins (snRNPs) from HeLa cells contain two proteins, U5-200kD and U5-100kD, which share homology with the DEAD/DEXH-box families of RNA helicases. Here we demonstrate that purified U5 snRNPs exhibit ATP-dependent unwinding of U4/U6 RNA duplices in vitro. To identify the protein responsible for this activity, U5 snRNPs were depleted of a subset of proteins under high salt concentrations and assayed for RNA unwinding. The activity was retained in U5 snRNPs that contain the U5-200kD protein but lack U5-100kD, suggesting that the U5-200kD protein could mediate U4/U6 duplex unwinding. Finally, U5-200kD was purified to homogeneity by glycerol gradient centrifugation of U5 snRNP proteins in the presence of sodium thiocyanate, followed by ion exchange chromatography. The RNA unwinding activity was found to reside exclusively with the U5-200kD DEXH-box protein. Our data raise the interesting possibility that this RNA helicase catalyzes unwinding of the U4/U6 RNA duplex in the spliceosome.

Figure 1

ATP-dependent RNA unwinding activity of MonoQ-purified mammalian 20S U5 snRNPs. The unwinding of a [³²P]-U4/U6 snRNA duplex (10 fmol) by 20S U5 snRNPs was assayed in the presence of ATP (lanes 2–5), without ATP (lane 7), or with the nonhydrolyzable analog γS-ATP (lane 8). Unwinding of the U4/U6 RNA duplex also was monitored with U5 snRNA (lane 9), 10S core U5 snRNPs (lane 10), and 12S U1 snRNPs (lane 6), in the presence of ATP. The concentrations of snRNPs or U5 snRNA used are indicated. The [³²P]-U4/U6 snRNA duplex in the absence of protein is shown in lane 1. The positions of duplex and single-stranded U4 RNA are indicated. Details concerning the analysis of RNA unwinding are described in Materials and Methods.

Figure 2

Fractionation of 20S U5 and [U4/U6.U5] snRNPs under high salt conditions yields a Δ100-kDa U5 snRNP active in RNA unwinding. (a) The composition of proteins (Upper; for clarity, only high molecular weight proteins are shown) and snRNAs (Lower) after centrifugation in high salt (0.7 M KCl) glycerol gradients. (b) Immunodetection of the putative RNA helicases U5–200kD (Upper) and U5–100kD (Lower) from fractions displayed in a. (c) RNA unwinding activity of gradient fractions. An equivalent volume of each gradient fraction was assayed for RNA unwinding activity. Lane L, a mixture of 20S U5 and [U4/U6.U5] snRNPs before dissociation in 0.7 M KCl. The positions of proteins and snRNAs are indicated to the left.

Figure 3

Isolation of the U5–200kD protein from purified 20S U5 snRNPs. (a) U5 snRNP proteins fractionated by glycerol gradient centrifugation in the presence of 0.4 M NaSCN. The positions of proteins are indicated to the left. The most prominent U5 snRNP proteins U5–100kD, U5–200kD, U5–220kD, and U5–116kD are indicated on the gel by arrows. The 10S core U5 snRNP is indicated by the presence of the Sm proteins B/B′ (additional Sm proteins are not shown in this figure). The 100- to 110-kDa band contains in addition to U5–100kD the U5-specific 102-kDa and 110-kDa proteins. The U5-specific 40-kDa protein sedimenting across the gradient is a novel WD40-repeat protein and does not encompass an RNA helicase-like domain (T.A. and R.L., unpublished data). (b) RNA unwinding activity of individual fractions from the gradient shown in a. For U4/U6 RNA unwinding analysis, equivalent volumes were taken from the gradient fractions and dialyzed against G-buffer containing 5% glycerol. The amount of U5–200kD protein provided in this assay was ca. 100 ng for a peak fraction (fractions 11–16).

Figure 4

Highly purified U5–200kD protein successively fractionated on NaSCN-glycerol gradients and by MonoQ chromatography is still active in U4/U6 RNA duplex unwinding. (a) Elution profile from the MonoQ column. The absorbance of the eluted fractions is measured at 280 nm (thick line) and 260 nm (thin line). The content of the peak eluates is indicated. The NaSCN concentrations are shown to the right, and the step gradient for elution is displayed by a broken line. (b) Coomassie-staining of the proteins present in the eluted fractions. M, molecular mass standards of 200, 116, 97, 66, 45, and 34 kDa. TP, total proteins from a mixture of spliceosomal snRNPs. (c) RNA unwinding activity of the eluted fractions. After dialysis against G-buffer with 5% glycerol, equivalent volumes of the eluted fractions were assayed for U4/U6 RNA unwinding in the presence of 200 μM ATP.

Figure 5

Substrate specificity of the ATP-dependent RNA unwinding activity. The reactions were incubated with ≈10 fmol of the corresponding duplex in the presence or absence of 200 μM ATP, as indicated. The position of the duplices and radiolabeled single-stranded RNA are indicated. The secondary structures of each substrate are represented schematically at the bottom. (a) RNA unwinding activity of 20S U5 snRNPs or isolated U5–200kD protein with U4/U6 RNA (Left) or the nonspliceosomal C4/MO Ia RNA (Right). The unwinding activity was investigated with 100 fmol U5 snRNPs or 1 pmol U5–200kD. (b) A 37-bp DNA duplex was incubated with ≈100 fmol 20S U5 snRNPs or 20 ng of hexameric large T antigen (denoted T-A.).