Structural basis for high-order complex of SARNP and DDX39B to facilitate mRNP assembly

Abstract

mRNA in eukaryotic cells is packaged into highly compacted ribonucleoprotein particles (mRNPs) in the nucleus and exported to the cytoplasm for translation. mRNP packaging and export require the evolutionarily conserved transcription-export (TREX) complex. TREX facilitates loading of various RNA-binding proteins on mRNA through the action of its DDX39B subunit. SARNP (Tho1 [transcriptional defect of Hpr1 by overexpression 1] in yeast) is shown to interact with DDX39B and affect mRNA export. The molecular mechanism of how SARNP recognizes DDX39B and functions in mRNP assembly is unclear. Here, we determine the crystal structure of a Tho1/DDX39B/RNA complex, revealing a multivalent interaction mediated by tandem DDX39B interacting motifs in SARNP/Tho1. The high-order complex of SARNP and DDX39B is evolutionarily conserved, and human SARNP can engage with five DDX39B molecules. RNA sequencing (RNA-seq) from SARNP knockdown cells shows the most affected RNAs in export are GC rich. Our work suggests the role of the high-order SARNP/DDX39B/RNA complex in mRNP assembly and export.

Keywords: CP: Molecular biology; DDX39B; DEAD-box ATPase; SARNP; Sub2; TREX complex; Tho1; UAP56; mRNA nuclear export; mRNP assembly.

Figure 1.. SARNP and DEAD-box protein DDX39B assemble on RNA

(A) Domain organization of human DDX39B and SARNP. DDX39B contains two RecA-like domains, DDX39B-NTD and DDX39B-CTD. SARNP contains a SAP domain. (B) DDX39B and SARNP assemble on RNA. EMSA was carried out with poly(U) 15-mer RNA at 100 nM, DDX39B at 0.3 μM, and SARNP at increasing concentrations (1, 3, and 10 μM) as indicated. Data are representative of three technical replicates. (C) Evolutionarily conserved assembly as shown with yeast Sub2 and Tho1. EMSA was carried out with poly(U) 15-mer RNA at 100 nM, Sub2 at 3 μM, and Tho1 at increasing concentrations (0.3, 1, and 3 μM) as indicated. Data are representative of three technical replicates. (D) A chimeric assembly between human DDX39B and yeast Tho1 on RNA. EMSA was carried out with poly(U) 15-mer RNA at 100 nM, DDX39B at 0.3 μM, and Tho1 at 3 μM. Data are representative of three technical replicates. (E) SARNP-C is sufficient to assemble with DDX39B on RNA. EMSA was carried out with poly(U) 15-mer RNA at 100 nM, DDX39B at 0.3 μM, and different SARNP proteins at 3 μM. Data are representative of three technical replicates.

Figure 2.. Crystal structure of a Tho1/DDX39B/RNA complex at 2.5 Å resolution

(A) DDX39B was crystallized with a C-terminal domain of yeast Tho1 in the presence of poly(U) 15-mer RNA and the non-hydrolyzable ATP analog ADP-BeF₃. The model shown in two orientations features a 1:2 assembly of Tho1 and DDX39B. The structure reveals a DDX39B interacting motif (DIM) that recognizes the DDX39B-CTD domain. (B) DDX39B interfaces with DIM-1 (left) and DIM-2 (right) of Tho1. (C) Alignment of the two DIMs in yeast Tho1 and five DIMs in human SARNP. Conserved residues are highlighted in cyan. The two invariant residues (R₅ and F₉) are shown in bold.

Figure 3.. Evolutionarily conserved multivalent assembly between SARNP and DDX39B

(A) Domain organization of SARNP in different organisms. DIM is represented as cyan rectangles. (B) Sequence logo of the DIM motif generated with WebLogo. (C) Comparison of SARNP structures in different organisms featuring 2–6 DIMs. For simplicity, only the DIM-containing C-terminal region is shown. Except for S. cerevisiae protein, which uses the structure reported here, all others use AlphaFold-predicted structures (AF-O74871-F1 for S. pombe, AF-Q9VHC8-F1 for D. melanogaster, AF-P82979-F1 for H. sapiens, and AF-Q9N3G0-F1 for C. elegans).

Figure 4.. Characterization of DIM interaction with Sub2/DDX39B

(A) Mutation of the R₅ and F₉ residues in yeast Tho1 DIMs (Tho1-mut) disrupted binding of DDX39B. EMSA was carried out with poly(U) 15-mer RNA at 100 nM, DDX39B at 0.3 μM, and Tho1 or Tho1-mut (R139A/F143A/R159A/F163A) at 3 μM. Data are representative of three technical replicates. (B) Mutation of the R₅ and F₉ residues in all SARNP DIMs (SARNP-mut1) disrupted binding of DDX39B. EMSA was carried out with poly(U) 15-mer RNA at 100 nM, DDX39B at 0.3 μM, and SARNP or SARNP-mut1 (R106A/F110A/R123A/F127A/R153A/F157A/R177A/F181A/R203A/F207A) at 3 μM. Data are representative of three technical replicates. (C) Microscale thermophoresis (MST) analysis of the SARNP-DDX39B interaction. Measurements of SARNP, SARNP-mut2 (R153A/F157A/R177A/F181A/R203A/F207A, DIM3–5 mutated), or SARNP-mut1 binding to DDX39B-CTD are colored in green, red, and blue, respectively. Measurements of SARNP binding to DDX39B-CTD-D283R are colored in brown. Data were fitted using the Hill equation. EC50 and Hill coefficient (n) are shown. Error bars represent SD from three technical replicates.

Figure 5.. Comparison of SARNP, Yra1/ALYREF, and THO complex binding to DDX39B

(A) Yra1/ALYREF and DIM bind to different domains of DDX39B. The DDX39B/Tho1/RNA structure, colored as in Figure 2A, is overlayed on the Sub2/Yra1/RNA structure (PDB: 5SUP), colored in gray except for Yra1 (orange). (B) THO complex (PDB: 7LUV) and DIM bind to overlapping regions (indicated by an oval) on DDX39B-CTD. (C) Comparison of Tho1/SARNP, Yra1/ALYREF, and THO binding interfaces on DDX39B. DDX39B is shown in surface model. Yra1/ALYREF and THO binding interfaces are analyzed based on their structures in complex with Sub2 shown in (A) and (B); corresponding residues in DDX39B are colored in white and orange, respectively.

Figure 6.. SARNP knockdown inhibits nuclear export of a subset of mRNAs with high GC content

(A–D) A549 cells were transfected with nontargeting siRNAs or with siRNAs that target SARNP. After 48 h, cells were subjected to immunofluorescence microscopy (A) or western blot analysis (B) with the depicted antibodies or were subjected to RNA-FISH to detect the intracellular distribution of poly(A) RNA in control cells and in cells depleted of SARNP (C). The nuclear speckle assembly and splicing factor SON is used as a nuclear speckle marker. The graph in (D) depicts accumulation of poly(A) RNA at nuclear speckles upon SARNP depletion (right) compared to control cells (left). The surface plot tool of Fiji (ImageJ) was used to calculate the fluorescence intensity of poly(A) RNA. (E–M) A549 cells were transfected with non-targeting siRNAs or with siRNAs that target SARNP. After 48 h, RNA was isolated from whole-cell, cytoplasmic, and nuclear fractions. RNA-seq data obtained from these samples in two biological replicates were analyzed to identify RNA features associated with cellular mRNAs that are dependent on SARNP for their nuclear export. Violin plots show the distribution of RNA features between exports not affected (left) and export-inhibited (right) transcripts upon SARNP knockdown. The red line indicates the median, and the green line indicates quartiles. Mann-Whitney U rank test was used to calculate statistical significance. p values are shown on the top of each plot. Number of transcripts in each group (n) is mentioned below the graphs.

Figure 7.. Hypothetical model of high-order mRNP assembly mediated by SARNP, ALYREF, and DDX39B

Multivalent interactions between Tho1/SARNP and the DEAD-box protein Sub2/DDX39B facilitate mRNP assembly and export. In yeast, one Tho1 molecule and one Yra1 molecule can assemble with two Sub2 molecules on RNA. In humans, one SARNP molecule can engage with five DDX39B molecules and up to three ALYREF molecules on RNA.