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Cited by 4Protein Structure and DynamicsGenomics and Chromatin DynamicsRNA Research and Splicing

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Insight into the Structural Basis for Dual Nucleic Acid—Recognition by the Scaffold Attachment Factor B2 Protein

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Korn, Sophie M., Von Ehr, Julian, Dhamotharan, Karthikeyan, Tants, Jan-Niklas, Abele, Rupert, Schlundt, Andreas·2023·International Journal of Molecular Sciences
DOI 10.3390/ijms24043286PMC PMC9958909

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Abstract

The family of scaffold attachment factor B (SAFB) proteins comprises three members and was first identified as binders of the nuclear matrix/scaffold. Over the past two decades, SAFBs were shown to act in DNA repair, mRNA/(l)ncRNA processing and as part of protein complexes with chromatin-modifying enzymes. SAFB proteins are approximately 100 kDa-sized dual nucleic acid-binding proteins with dedicated domains in an otherwise largely unstructured context, but whether and how they discriminate DNA and RNA binding has remained enigmatic. We here provide the SAFB2 DNA- and RNA-binding SAP and RRM domains in their functional boundaries and use solution NMR spectroscopy to ascribe DNA- and RNA-binding functions. We give insight into their target nucleic acid preferences and map the interfaces with respective nucleic acids on sparse data-derived SAP and RRM domain structures. Further, we provide evidence that the SAP domain exhibits intra-domain dynamics and a potential tendency to dimerize, which may expand its specifically targeted DNA sequence range. Our data provide a first molecular basis of and a starting point towards deciphering DNA- and RNA-binding functions of SAFB2 on the molecular level and serve a basis for understanding its localization to specific regions of chromatin and its involvement in the processing of specific RNA species.

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Figures

Figure 1 Scaffold attachment factor proteins (SAF) type A (hnRNP U) and B (SAFB1, SAFB2 and SLTM). (A) Domain organizations of human SAFA, SAFB1, SAFB2 and SLTM as annotated in Uniprot [30] (IDs: Q00839, Q15424, Q14151, Q9NWH9). (B) Sequence alignment of the SAP domain (based on domain boundaries of the SAFB2 SAP defined in this study) with residues color-coded by conservation amongst the four proteins according to Clustal Omega [31] (black—no conservation, light orange—weak-to-strong conservation, dark orange—full conservation). Above, secondary structure elements derived from the SCS analysis for SAFB2 SAP in Figure 2 are given, with rectangles indicating α-helices. The helix–loop–helix DNA-binding motif spans helices 2 (H2) and 3 (H3). (C) Sequence alignment of the SAFB RRM domains (boundaries are based on those of the SAFB2 RRM domain defined in this study) with residues colored according to the degree of conservation as by Clustal Omega (black—no conservation, light green—weak-to-strong conservation, dark green—full conservation). Above, the two conserved RNA-binding motifs (RNP) and secondary structure elements derived from the SCS analysis for SAFB2 RRM in Figure 3 are given, with rectangles indicating α-helices and arrows indicating β-strands, respectively.
Figure 1 Scaffold attachment factor proteins (SAF) type A (hnRNP U) and B (SAFB1, SAFB2 and SLTM). (A) Domain organizations of human SAFA, SAFB1, SAFB2 and SLTM as annotated in Uniprot [30] (IDs: Q00839, Q15424, Q14151, Q9NWH9). (B) Sequence alignment of the SAP domain (based on domain boundaries of the SAFB2 SAP defined in this study) with residues color-coded by conservation amongst the four proteins according to Clustal Omega [31] (black—no conservation, light orange—weak-to-strong conservation, dark orange—full conservation). Above, secondary structure elements derived from the SCS analysis for SAFB2 SAP in Figure 2 are given, with rectangles indicating α-helices. The helix–loop–helix DNA-binding motif spans helices 2 (H2) and 3 (H3). (C) Sequence alignment of the SAFB RRM domains (boundaries are based on those of the SAFB2 RRM domain defined in this study) with residues colored according to the degree of conservation as by Clustal Omega (black—no conservation, light green—weak-to-strong conservation, dark green—full conservation). Above, the two conserved RNA-binding motifs (RNP) and secondary structure elements derived from the SCS analysis for SAFB2 RRM in Figure 3 are given, with rectangles indicating α-helices and arrows indicating β-strands, respectively.CC BY · PubMed Central
Figure 2 Resonance assignment of the SAFB2 SAP domain. (A) 1H-15N HSQC of the SAP domain amide groups. Straight lines indicate side chain assignments. (B) Combined Cα/Cβ carbon secondary chemical shifts (SCS) (upper panel, as suggested by [32]—rectangles indicating α-helices) and the {1H}15N heteronuclear NOE values (lower panel) of SAFB2 SAP plotted against the residue number. Errors are calculated from the program CCPNMR Analysis.
Figure 2 Resonance assignment of the SAFB2 SAP domain. (A) 1H-15N HSQC of the SAP domain amide groups. Straight lines indicate side chain assignments. (B) Combined Cα/Cβ carbon secondary chemical shifts (SCS) (upper panel, as suggested by [32]—rectangles indicating α-helices) and the {1H}15N heteronuclear NOE values (lower panel) of SAFB2 SAP plotted against the residue number. Errors are calculated from the program CCPNMR Analysis.CC BY · PubMed Central
Figure 3 Resonance assignment of the SAFB2 RRM domain. (A) 1H-15N HSQC of the RRM domain amide groups. Assignments of peaks in the red dashed-lined box are shown in the inset zoom-in. See also Supplementary Figure S1B. (B) Combined Cα/Cβ carbon secondary chemical shifts (SCS) (upper panel, as suggested by [32]—rectangles indicating α helices, arrows indicating β-strands) and the {1H}15N heteronuclear NOE values (lower panel) of the SAFB2 RRM plotted against residue numbers. Errors are calculated from the program CCPNMR Analysis.
Figure 3 Resonance assignment of the SAFB2 RRM domain. (A) 1H-15N HSQC of the RRM domain amide groups. Assignments of peaks in the red dashed-lined box are shown in the inset zoom-in. See also Supplementary Figure S1B. (B) Combined Cα/Cβ carbon secondary chemical shifts (SCS) (upper panel, as suggested by [32]—rectangles indicating α helices, arrows indicating β-strands) and the {1H}15N heteronuclear NOE values (lower panel) of the SAFB2 RRM plotted against residue numbers. Errors are calculated from the program CCPNMR Analysis.CC BY · PubMed Central
Figure 4 RNA-binding preferences of the SAFB2 RRM domain. (A) 1H-15N HSQC zoom-ins showing overlays of 2× molar excess of RNAs as indicated (color code depicted in upper left corner) with the apo RRM, respectively. Assignments for RNP residues involved in RNA interaction are included. Addition of G7 resulted in severe precipitation and was, thus, not evaluated accordingly. See Supplementary Figure S4 for full spectra. (B) CSP plots of individual RNAs over amino acid sequence. Indicated above is the RRM secondary structure with RNP1 and RNP2 highlighted. Dotted grey lines represent the CSP average. (C) Mapping of the ten significantly perturbed CS for residues shown as spheres (N-atoms) to a structural model of the SAFB2 RRM domain, i.e., the lowest energy representative of a CS-Rosetta-derived ensemble (Supplementary Figure S2). Maximum CSPs locate to the two RNP motifs and the C-terminal tail, respectively.
Figure 4 RNA-binding preferences of the SAFB2 RRM domain. (A) 1H-15N HSQC zoom-ins showing overlays of 2× molar excess of RNAs as indicated (color code depicted in upper left corner) with the apo RRM, respectively. Assignments for RNP residues involved in RNA interaction are included. Addition of G7 resulted in severe precipitation and was, thus, not evaluated accordingly. See Supplementary Figure S4 for full spectra. (B) CSP plots of individual RNAs over amino acid sequence. Indicated above is the RRM secondary structure with RNP1 and RNP2 highlighted. Dotted grey lines represent the CSP average. (C) Mapping of the ten significantly perturbed CS for residues shown as spheres (N-atoms) to a structural model of the SAFB2 RRM domain, i.e., the lowest energy representative of a CS-Rosetta-derived ensemble (Supplementary Figure S2). Maximum CSPs locate to the two RNP motifs and the C-terminal tail, respectively.CC BY · PubMed Central
Figure 5 DNA-binding preferences of the SAFB2 SAP domain. (A) EMSAs of SAP domain with ss (left) and ds (right) DNAs, either AT-rich (top) or GC-rich (bottom) (with sense sequences as follows: AT13mer 5′-GCAATAAATACG-3′; GC13mer 5′-GCCCCCGCGCCCG-3′). Increasing SAP protein concentration (0–40 µM) is indicated by black bar above gel pictures. EMSAs shown here are representatives of technical triplicates (Supplementary Figure S6). (B) 1H-15N HSQC of SAP titrated with increasing molar ratios (0.5×, 1×, 1.85×, 4×, 6×) of the dsDNA AT16mer (5′-GCGCACAATATAACGC-3′). The most perturbed resonances are labelled with their assignments. The zoom-in of the boxed region below shows the highly conserved Lys53 (see Figure 1), which is most strongly affected. Note the different contour levels used to visualize this peak relative to the settings of the full spectrum above. (C) CSP plots of 6× molar excess of AT16mer plotted over amino acid sequence. Indicated above is the SAP secondary structure. Dotted grey line represents the CSP average, the solid black line the threshold of 2× standard deviation. (D) Mapping of the four significantly perturbed CS for residues shown as spheres (N-atoms) plotted on a structural model of the SAFB2 SAP domain. The structural model is the lowest energy representative of a CS-Rosetta-derived ensemble.
Figure 5 DNA-binding preferences of the SAFB2 SAP domain. (A) EMSAs of SAP domain with ss (left) and ds (right) DNAs, either AT-rich (top) or GC-rich (bottom) (with sense sequences as follows: AT13mer 5′-GCAATAAATACG-3′; GC13mer 5′-GCCCCCGCGCCCG-3′). Increasing SAP protein concentration (0–40 µM) is indicated by black bar above gel pictures. EMSAs shown here are representatives of technical triplicates (Supplementary Figure S6). (B) 1H-15N HSQC of SAP titrated with increasing molar ratios (0.5×, 1×, 1.85×, 4×, 6×) of the dsDNA AT16mer (5′-GCGCACAATATAACGC-3′). The most perturbed resonances are labelled with their assignments. The zoom-in of the boxed region below shows the highly conserved Lys53 (see Figure 1), which is most strongly affected. Note the different contour levels used to visualize this peak relative to the settings of the full spectrum above. (C) CSP plots of 6× molar excess of AT16mer plotted over amino acid sequence. Indicated above is the SAP secondary structure. Dotted grey line represents the CSP average, the solid black line the threshold of 2× standard deviation. (D) Mapping of the four significantly perturbed CS for residues shown as spheres (N-atoms) plotted on a structural model of the SAFB2 SAP domain. The structural model is the lowest energy representative of a CS-Rosetta-derived ensemble.CC BY · PubMed Central
Figure 6 DNA-binding capacity of SAFB2 SAP domain mutants. 1H-15N HSQC overlay of apo SAP proteins (yellow) with 6- or 8-fold excess (red) of AT9mer (5′-CAATATAAC-3′), as indicated. Top panel: SAPWT (left) and SAPR45A (right); bottom panel: SAPS31A (left) and SAPK53A/S54A (right). Contour levels are scaled individually between proteins, for peaks to be visible. Contour levels for each apo and DNA-bound spectra of one protein are adjusted.
Figure 6 DNA-binding capacity of SAFB2 SAP domain mutants. 1H-15N HSQC overlay of apo SAP proteins (yellow) with 6- or 8-fold excess (red) of AT9mer (5′-CAATATAAC-3′), as indicated. Top panel: SAPWT (left) and SAPR45A (right); bottom panel: SAPS31A (left) and SAPK53A/S54A (right). Contour levels are scaled individually between proteins, for peaks to be visible. Contour levels for each apo and DNA-bound spectra of one protein are adjusted.CC BY · PubMed Central
Figure 7 SAFB2 SAP domain mutants. (A) aSEC of SAP wild-type domain and mutants. (B) Estimation of molecular weights (MW) based on MW standards. Shown is the partition coefficient (Kav) plotted as a function of MW. Colored points (in dotted circle) are the Kav from aSEC over their theoretical MWs (expecting a monomer), respectively. Colored points on the fit line are the MWs determined based on the calibration standards (see also Supplementary Figure S12). (C) SEC-MALS-derived MWs of the SAP wild-type domain and mutants compared to the theoretical MW of a mono-mer.
Figure 7 SAFB2 SAP domain mutants. (A) aSEC of SAP wild-type domain and mutants. (B) Estimation of molecular weights (MW) based on MW standards. Shown is the partition coefficient (Kav) plotted as a function of MW. Colored points (in dotted circle) are the Kav from aSEC over their theoretical MWs (expecting a monomer), respectively. Colored points on the fit line are the MWs determined based on the calibration standards (see also Supplementary Figure S12). (C) SEC-MALS-derived MWs of the SAP wild-type domain and mutants compared to the theoretical MW of a mono-mer.CC BY · PubMed Central
Figure 8 Summary of nucleic acid-binding preferences of SAFB2 SAP and RRM domains as determined in this study. In addition, possible inter- and intra-molecular domain interactions based on current experimental knowledge are depicted. SAFB2 and its domains are not shown in scale.
Figure 8 Summary of nucleic acid-binding preferences of SAFB2 SAP and RRM domains as determined in this study. In addition, possible inter- and intra-molecular domain interactions based on current experimental knowledge are depicted. SAFB2 and its domains are not shown in scale.CC BY · PubMed Central

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Metadata

Authors
Korn, S. M., Von Ehr, J., Dhamotharan, K., Tants, J.-N., Abele, R., & Schlundt, A.
Affiliations
Goethe University Frankfurt (DE)
Corresponding author
Schlundt, Andreas
ORCID
Venue
International Journal of Molecular Sciences
Publisher
MDPI AG
Published
2023-02-07
Volume
24
Issue
4
Pages
3286
ISSN
1422-0067 (ISSN-L)
DOI
10.3390/ijms24043286
PMC
PMC9958909
OpenAlex
W4319600471
Cited by
4
References
90
Citations by year
2026: 1 · 2025: 1 · 2024: 2
FWCI
0.35
Type
journal article
Language
en
Open access
Licence
Locations
  • International Journal of Molecular Sciences (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Publication Server of Goethe University Frankfurt am Main (Goethe University Frankfurt) (submittedVersion, cc-by-nc)
  • Publication Server of Goethe University Frankfurt am Main (Goethe University Frankfurt) (submittedVersion, cc-by)
Funders
  • State of Hesse (SCHL2062/2-1, 2019/AS01)
  • German Research Council (DFG) (SCHL2062/2-1, 2019/AS01)
  • Johanna Quandt Young Academy at Goethe (SCHL2062/2-1, 2019/AS01)
  • Goethe University Excellence Initiative “SCALE” (SCHL2062/2-1, 2019/AS01)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
Keywords
nucleic acid recognition, DNA binding, RNA binding, SAP domain, RRM domain, domain structure, protein dimerization, chromatin, nuclear matrix, RNA processing, DNA repair
MeSH terms
Base Sequence, Binding Sites, Chromatin, Magnetic Resonance Spectroscopy, RNA, RNA, Messenger

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