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. 2007 Aug;8(8):756-62.
doi: 10.1038/sj.embor.7401031. Epub 2007 Jul 13.

Structural and functional analysis of RNA and TAP binding to SF2/ASF

Aura M Tintaru  1 , Guillaume M Hautbergue, Andrea M Hounslow, Ming-Lung Hung, Lu-Yun Lian, C Jeremy Craven, Stuart A Wilson
Affiliations

Structural and functional analysis of RNA and TAP binding to SF2/ASF

Aura M Tintaru et al. EMBO Rep. 2007 Aug.

Abstract

The serine/arginine-rich (SR) protein splicing factor 2/alternative splicing factor (SF2/ASF) has a role in splicing, stability, export and translation of messenger RNA. Here, we present the structure of the RNA recognition motif (RRM) 2 from SF2/ASF, which has an RRM fold with a considerably extended loop 5 region, containing a two-stranded beta-sheet. The loop 5 extension places the previously identified SR protein kinase 1 docking sequence largely within the RRM fold. We show that RRM2 binds to RNA in a new way, by using a tryptophan within a conserved SWQLKD motif that resides on helix alpha1, together with amino acids from strand beta2 and a histidine on loop 5. The linker connecting RRM1 and RRM2 contains arginine residues, which provide a binding site for the mRNA export factor TAP, and when TAP binds to this region it displaces RNA bound to RRM2.

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Figures

Figure 1
Figure 1
Structure of SF2/ASF RNA-recognition motif 2. (A) Superposition of the 20 lowest energy structures of SF2/ASF RRM2 (amino acids 107–215). (B) Ribbon representation of SF2/ASF RRM2 shown in two orientations. (C) SRPK1 docking motif (blue side chains) highlighted on the RRM2 structure. (D) Space-filled model of RRM2 showing the SRPK1 docking motif in blue. RRM2, RNA-recognition motif 2; SF2/ASF, splicing factor 2/alternative splicing factor; SRPK1, serine/arginine-rich protein kinase 1.
Figure 2
Figure 2
Interaction of SF2/ASF RNA-recognition motif 2 with RNA. (A) Chemical shift perturbation following the addition of 5′-ACGA RNA to 15N-labelled RRM2. The heteronuclear single quantum correlation (HSQC) spectrum of free protein (black) and after the addition of 1.75 molar equivalents of RNA (red) are shown. Amino acids showing marked changes are indicated. (B) Magnitude of weighted amide chemical shift perturbations (wΔδ) following the addition of 5′-ACGA RNA to 15N-labelled RRM2. Mapping of these perturbations to secondary structural elements are shown above the SF2/ASF sequence alignment with related proteins. Residues with chemical shift changes of more than 0.1 p.p.m. are shown by black bars. The side chain Hɛ resonance of W134 also shows a sizeable shift of more than 0.1 p.p.m., and the side chain Hɛ of Q135 is unobservable, following the first addition of ligand, which indicates exchange broadening owing to a large chemical shift change. Filled circles below the secondary structural elements represent amino acids directly involved in RNA binding identified by mutagenesis, and open circles represent amino acids in which mutagenesis does not affect RNA binding. Residues that form the core of the RRM fold are denoted by the letter C above the sequences. Within the sequence alignments, residues highlighted in yellow are strongly conserved and residues in green have conservative substitutions. RRM, RNA recognition motif; SF2/ASF, splicing factor 2/alternative splicing factor.
Figure 3
Figure 3
Mutagenesis defines SF2/ASF residues required for RNA binding. (A) UV crosslinking with RNA 5′-CACACGA and SF2/ASF RRM2 (aa 107–215) and point mutants. The top panel shows a phosphorimage of the Coomassie blue-stained gel shown in the bottom panel. (B) Side chains of amino acids required for RNA binding are shown in blue (left). The ribbon representation of the structure is also coloured according to the magnitude of chemical shifts seen on the addition of RNA 5′-ACGA (red=large). The amino acids required for RNA binding (blue) map to a localized patch on the space-filled model of the structure (right). (C) W134A mutation in RRM2 reduces translational activation in vivo. The pLCS-EDA reporter carries an SF2-binding site sequence and is translationally activated by overexpression of SF2/ASF. The pLCS-EDAmt carries a mutated SF2-binding site, which shows poor activation by SF2/ASF (Sanford et al, 2004, 2005b). pLCS-EDA (EDA) or pLCS-EDAmt (MT) plasmids were co-transfected with Flag-SF2/ASF (aa 11–196) or the W134A mutant into 293T cells and luciferase activity was measured after 24 h. Transfection efficiencies were normalized by co-transfection of a cytomegalovirus (CMV)-driven β-galactosidase expression vector; results represent averages from transfections carried out in triplicate. Flag-tagged SF2/ASF proteins were detected by western blotting (lower panel). (D) W134A mutation in RRM2 reduces the interaction with pLCS-EDA messenger RNA in vivo. 293T cells were transfected with the indicated plasmids, immunoprecipitated with anti-Flag–agarose, RNA extracted and assayed by RT–PCR. The top panel shows the results of RT–PCR analysis from immunoprecipitates, the middle panel results from total extracts before immunoprecipitation and the bottom panel a western blot of immunoprecipitates using Flag antibodies. RRM, RNA recognition motif; RT–PCR, reverse transcriptase–PCR; SF2/ASF, splicing factor 2/alternative splicing factor.
Figure 4
Figure 4
TAP binds to the SF2/ASF linker between RRM1 and RRM2. (A) Schematic of SF2/ASF and linker region sequence. Mutated arginine residues are underlined. (B) Pull-down assays with GST–TAP-p15, and 35S-SF2/ASF truncations and point mutants. (C) Pull-down assays with GST–SF2/ASF linker and 35S-TAP. (D) Co-immunoprecipitation of SF2/ASF mutants with TAP. SF2/ASF has Flag and Myc tags; TAP is Myc tagged. Total extracts (left) and Flag immunoprecipitates (right) were probed with Myc antibody. (E) Schematic of tethered messenger RNA export assay. (F) Western blot detection of Myc-tagged MS2 fusions as indicated by using Myc antibodies. Specific bands are highlighted with asterisks. Chim pLCS-EDAmt (MT) is the same construct carrying R90,93,117,118A mutations. (G) Normalized luciferase activities for MS2 fusions indicated in the tethered export assay. (H) UV crosslinking assay with RNA, GST–TAP-p15 and SF2/ASF (107–196). The right panel shows a phosphorimage of the Coomassie-stained gel (left panel). For all lanes, 32P-labelled RNA was added to the samples at the start together with SF2/ASF (107–196). Lanes 1+2 contain free SF2/ASF; lanes 3–6 show GST pull-downs subsequently eluted from beads and then UV crosslinked as indicated. Chim, REF-RRM-SF2/ASF linker peptide chimaera; ENV, HIV-1 envelope gene; GFP, green fluorescent protein; GST, glutathione-S-transferase; MS2, bacteriophage MS2 coat protein; PCMV, cytomegalovirus promoter; RRM, RNA recognition motif; SA, splice acceptor; SD, splice donor; SF2/ASF, splicing factor 2/alternative splicing factor; TAP, TIP associated protein.
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