Some proteins perform several different tasks inside cells. This is the case for a protein called Pur-alpha, which is essential for neurons to work correctly. For example, Pur-alpha can bind to DNA to regulate gene activity. It also binds to RNA molecules, which are copies of a gene, and helps to distribute them within the neuron. In humans, there are several neurodegenerative diseases in which Pur-alpha is involved. One example is the Fragile X-associated Tremor/Ataxia Syndrome (FXTAS), which causes memory and movement problems.

Experiments with isolated proteins and double-stranded DNA show that Pur-alpha is able to separate the two DNA strands. But it was not clear how this DNA unwinding occurs, and the biological significance of this activity was unknown. Other questions also remained unanswered: how does Pur-alpha recognize DNA and RNA? Does the loss of Pur-alpha’s binding to DNA and RNA contribute to neurodegenerative diseases?

To address these questions, Weber et al. obtained Pur-alpha from the fruit fly and crystallized the protein bound to DNA. A technique called X-ray crystallography was then used to determine the three-dimensional structure of the Pur-alpha/DNA complex in fine enough detail to work out the position of individual atoms.

Based on this structure, Weber et al. could introduce mutations that alter the DNA- and RNA-binding region of the protein to investigate the binding mechanism. The crystal structure and experiments with normal and mutant Pur-alpha protein revealed how it unwinds double-stranded DNA: binding of Pur-alpha to DNA causes a strong twist of the DNA molecule, which contributes to separating the strands. Further experiments in fruit flies revealed that both the DNA-unwinding activity and the ability of Pur-alpha to bind DNA/RNA are needed for the protein to work correctly in neurons.

Because Pur-alpha is involved in a range of different processes inside cells, a future goal is to identify the DNA and RNA sequences it specifically binds to. This information, together with the insights gained from Weber et al.’s study, should advance our understanding of why Pur-alpha is essential for maintaining neurons.

Purine-rich element-binding protein A (Pur-alpha) plays a crucial role in postnatal brain development. Pur-alpha-deficient mice appear normal at birth but develop severe neurological abnormalities after 2 weeks and die shortly after birth (Hokkanen et al., 2012; Khalili et al., 2003). These mice show fewer cells in the brain cortex, hippocampus, and cerebellum as a consequence of decreased proliferation of the precursor cells. Further studies indicated that Pur-alpha co-localizes with Staufen and FMRP and that Pur-alpha ^(-/-) mice display dendritic mislocalization of both proteins (Johnson et al., 2006). In support of its important neuronal function, point mutations in the human Pur-alpha gene have been found to cause the so-called 5q31.3 microdeletion syndrome, which is characterized by neonatal hypotonia, encephalopathy, and severe developmental delay (Lalani et al., 2014; Hunt et al., 2014; Tanaka et al., 2015).

Pur-alpha is an ubiquitously expressed, multifunctional protein that binds to both DNA and RNA and is known to regulate replication, transcription, and translation (Johnson et al., 2013). It has been shown that Pur-alpha binds to single- and double-stranded nucleic acids that contain GGN motifs. Such regions are found at origins of DNA replication and enhancers of TATA-box lacking genes, such as c-myc or the myelin-basic protein, which Pur-alpha regulates. Pur-alpha has also been routinely purified from cytoplasmic kinesin-containing ribonucleoprotein particles (RNPs) (Kanai et al., 2004; Ohashi et al., 2000), further supporting its role in mRNA localization and showing that Pur-alpha is a core factor in localizing mRNPs.

Besides its ability to bind RNA and DNA, Pur-alpha possesses dsDNA-destabilizing activity in an ATP-independent fashion (Darbinian et al., 2001). This function has been suggested as important for DNA replication and transcription regulation. It was postulated that Pur-alpha, being a transcription activator, contacts the purine-rich strand of promoter regions and displaces the pyrimidine-rich strand, which would allow the binding of other proteins and activation of transcription (Darbinian et al., 2001; Wortman et al., 2005). The role of Pur-alpha-dependent unwinding activity in RNA localization and in RNA-based neuropathological disorders is currently unknown.

One particularly interesting interaction partner of Pur-alpha is the RNA helicase Rm62, the Drosophila ortholog of p68. It is implicated in transcriptional regulation, pre-mRNA splicing, RNA interference, and nucleo-cytoplasmic shuttling (Qurashi et al., 2011). Thus, their joint function could be the initial unwinding of short dsRNA regions by Pur-alpha followed by helicase-dependent melting of larger regions for the regulation of RNA processing, translational control, and transport.

Nucleic acid-binding of Pur-alpha is mediated by three central PUR repeats (Graebsch et al., 2010; Graebsch et al., 2009), which are N-terminally flanked by unstructured, glycine-rich sequences and C-terminally by glutamine- and glutamate-rich regions (Figure 1A; Johnson et al., 2013). In the recently published crystal structure of Pur-alpha each of both PUR repeats I and II consist of a four-stranded antiparallel beta-sheet, followed by a single alpha-helix (Graebsch et al., 2009). Repeat I and II fold into an intramolecular dimer that serves as a DNA-/RNA-binding domain. The third repeat leads to intermolecular dimerization (Figure 1A; Graebsch et al., 2009). Despite these insights, it remains unclear how Pur-alpha interacts with its nucleic-acid targets to mediate its cellular functions. Furthermore, the mechanistic basis and physiological importance of its unwinding activity remains unresolved.

Figure 1 with 2 supplements see all

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Pur-alpha has been implicated in two so-called RNA repeat-expansion diseases, which have been the focus of a number of recent studies. The first one contains expansions in the well-studied fmr1 gene. Individuals with 55 to 200 CGG repeats, termed pre-mutation, develop the neurodegenerative Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) (Hagerman et al., 2001), whereas healthy individuals have less than 54 trinucleotide CGG repeats in their 5’-UTR region (Oostra and Willemsen, 2009). It is generally accepted that expression of FMR1 mRNA with abnormal trinucleotide-repeat expansions are the main cause of FXTAS. The second Pur-alpha related disease is caused by repeat expansions of G₄C₂-hexanucleotides in the first intron of the c9orf72 transcript. These repeat expansions are considered as the most common genetic abnormality in amyotrophic lateral sclerosis (ALS) and familial frontotemporal lobal degeneration (FTLD) (Stepto et al., 2014). The diseases associated with both types of repeat expansions are accompanied by the formation of repeat RNA-containing protein inclusions (Sareen et al., 2013; Stepto et al., 2014,; Xu et al., 2013), suggesting sequestration of proteins as potential mechanism of pathology. Pur-alpha is incorporated into the inclusions of both types of disease and associates directly with the repeat RNAs (Jin et al., 2007,; Xu et al., 2013; Rossi et al., 2015). In fly and mouse models, the overexpression of Pur-alpha can overcome repeat-dependent neurodegeneration of both diseases (Jin et al., 2007,; Xu et al., 2013), suggesting a direct contribution of Pur-alpha to neuropathology.

Expression of 95 CGG repeats in human neuroblastoma-derived SK-N-MC cells not only induced the formation of nuclear inclusions but also impairs the architecture of the nuclear laminar and activates DNA repair-associated histone variants (Hoem et al., 2011). The expression of G₄C₂-repeat expansions cause nuclear trafficking defects, which contribute to neurotoxicity in ALS/FTLD (Freibaum et al., 2015; Jovicic et al., 2015; Zhang et al., 2015). Recent studies also showed that repeat-associated non-AUG (RAN) translation occurs from CGG- as well as from G₄C₂-repeat RNAs and that the resulting proteins can form cytoplasmic aggregates, potentially contributing to pathology (Mori et al., 2013; Todd et al., 2013). It is likely that a combination of RNA toxicity and RAN-derived protein aggregates contribute to the full manifestation of FXTAS.

Here, we used NMR chemical shift titrations together with in vitro-binding assays to demonstrate that the nucleic acid-binding domain of Pur-alpha binds RNA and DNA in the same manner. We present the co-crystal structure of Pur-alpha with a CGG trinucleotide-repeat DNA, providing a detailed structural explanation for nucleotide recognition. Pur-alpha interacts with this single-stranded DNA fragment in a sequence-specific manner with guanines and additional contacts to the phosphordiester backbone. The observed binding mode of Pur-alpha also explains its interaction with G₄C₂-hexanucleotide repeats. Mutational analyses as well as determination of the complex stoichiometry confirm that the DNA-/RNA-binding domain of Pur-alpha has two nucleic acid-binding sites. The structure also revealed that a highly conserved phenylalanine causes disruption of the normal base stacking and leads to a strong torsion of the DNA strand, which plays a central role in Pur-alpha’s dsDNA-unwinding activity. In vivo analyses of mutant proteins reveal that nucleic-acid binding and unwinding studied in vitro are both essential for Pur-alpha’s function in vivo. This information together with the crystal structure of its C-terminal dimerization domain allows us to propose a mechanism of how full-length Pur-alpha binds and unwinds dsDNA regions.

Pur-alpha repeat I-II shows strong and specific binding to its physiological target MF0677 DNA located upstream of the c-myc gene (Bergemann et al., 1992), but much weaker binding to CGG-repeat RNA (Graebsch et al., 2009). For this reason, it has been suggested that the binding of Pur-alpha to DNA is stronger than to RNA and, as a consequence, that there might be differences in the binding modes to both nucleic-acid targets. In this study, we directly compared Pur-alpha binding to RNA and DNA oligonucleotides of the same sequence and found no major differences (Figure 1B–D). This suggests that the higher affinity for MF0677 (K_D ~200 nM; Figure 1B) over CGG repeats (K_D ~2 µM; Figure 1C) is due to differences in sequence and not the absence of the 2’ OH group in the DNA. This interpretation found further support from NMR titrations with ¹⁵N-labeled Pur-alpha repeat I-II and oligonucleotides. The spectra showed similar chemical shift perturbations, regardless of whether it was DNA or RNA, indicating that both nucleic acids are bound in the same way (Figure 1E). Finally, the crystal structure of the Pur-alpha/DNA co-complex showed that a hydroxyl-group on the 2’ position of the pentose ring of the RNA sugar backbone would not cause steric clashes (Figure 2A,C-F). Together, our biochemical, NMR, and X-ray crystallographic insights indicate that Pur-alpha binds DNA and RNA in the same way and thus will interact equally with both types of nucleic acids in the cell. It is also consistent with the previously suggested Pur-alpha-dependent gene regulation by competitive RNA binding (Tretiakova et al., 1998).

Previous findings implied that the positively charged β-sheets mediate DNA/RNAbinding, whereas the amphipathic helices might contribute to protein-protein interactions (Graebsch et al., 2009). The crystal structure of the protein-DNA co-complex confirms that the β-sheets, together with their short linkers, are involved in DNA binding, in contrast to the α-helices that show no interaction (Figure 2A). A comparison of the Pur-alpha repeat I-II apo-structure (PDB ID 3K44) with the co-structure presented here revealed no significant conformational changes (Figure 2—figure supplement 1B).

In the crystal structure, Pur-alpha interacts with nucleic acids by clamping them between its two repeats, mostly by interacting with the guanine bases (Figure 2A, B). Only R142 interacts with the cytosine base C2. K54 and K138 additionally stabilize the DNA binding by interacting with the sugar phosphate backbone of guanine G4 and cytosine C5, respectively (Figure 2B–D). Binding therefore occurs sequence specifically and confirms the GGN-binding motif postulated before (Bergemann and Johnson, 1992).

Mutation of the interacting residues resulted in a decreased binding affinity (Figure 3G, H) and therefore confirmed the interaction sites seen in the crystal structure. Also mutation of the corresponding KNR motif on PUR repeat I (KNR I) caused a decrease in affinity (Figure 3G, H). However, in fluorescence-polarization experiments, the mutation of KNR I had a less severe effect on DNA binding (K_D = 1.3 µM) than mutation of KNR II (K_D = 4.1 µM; Figure 3H). This is consistent with the observation that in the crystal structure all three residues of KNR II make contacts with DNA 1 (Figure 2B, C), whereas in KNR I only a single amino acid binds to DNA 2 (Figure 2—figure supplement 3). Also, the F I mutation in repeat I had a less severe effect on MF0677 ssDNA binding than the F II mutation (Figure 3G, H). In summary, these observations suggest that the MF0677 ssDNA is bound asymmetrically by PUR repeats I-II.

In FXTAS patients, Pur-alpha binds to CGG-repeat expansions that cause the formation of nuclear inclusions and neurodegeneration (Oostra and Willemsen, 2003). Pur-alpha is also incorporated into inclusion triggered by G₄C₂-repeat RNA of patients with ALS and FTLD. The nucleic-acid binding of Pur-alpha observed in the crystal structure can explain both binding events, as it makes sequence-specific interactions with a GGC motif found in both repeat RNAs.

The structural model of Pur-alpha repeat I-II forming a PUR domain has two nucleic-acid-binding surfaces. PUR repeats I and II share the identical binding motif (KNR), and adopt the same fold, despite moderate sequence identity of about ~30% (Figure 2—figure supplement 4; Graebsch et al., 2010; Graebsch et al., 2009). Consistent with this finding, we observed a stoichiometric ratio of 1:2 for the PUR domain with ssDNA in filter-binding assays (Figure 2G). Both binding events appear at overlapping but non-identical surface regions (Figure 2A, B), which might prefer different GGN-motifs (GGA, GGG, GGC, GGT) as has been previously suggested (Aumiller et al., 2012). This might also explain why CGG repeats bind less strongly to Pur-alpha than the MF0677 sequence, which mostly consists of GGA and GGT motifs.

Pur-alpha has been previously reported to unwind dsDNA in an ATP-independent manner (Darbinian et al., 2001; Wortman et al., 2005). However, so far, it has not been shown how unwinding is achieved on a molecular level and that this function is physiologically relevant. The crystal structure of our Pur-alpha/DNA co-complex offers a mechanistic explanation: phenylalanine in position 145 of PUR repeat II undertakes base stacking with the guanine G4 and thereby blocks the space for the neighboring cytosine C5 (Figure 2E). Thereupon, the cytosine flips out and the 3’-end of the DNA strand becomes distorted. The interaction of K54 and K138 with the phosphate backbone upstream of the cytosine C5 enforces this strong turn (Figure 2B–D). F145 is highly conserved throughout different species (Figure 2—figure supplement 4A) and its mutation (F II) abolishes unwinding of dsDNA (Figure 3F, G).

Phenylalanine 145 has its structural counterpart in PUR repeat I in position F68. Although F68 is also highly conserved, in the crystal structure the guanine base stacking is not mediated by this residue. Instead, the conserved Y57 in repeat I stacks with G7 (Figure 2B, F). As mentioned before, the two binding sites of Pur-alpha seen in the crystal structure are asymmetric and might account for sequence-specific binding to nucleic acids with different GGN motifs.

To assess the physiological importance of the interactions observed in the crystal structure and validated in vitro, we used the previously reported FXTAS fly model (Jin et al., 2007; Jin et al., 2003). Expression of pre-mutation CGG-repeat RNA in Drosophila induces neurodegeneration, which is easily detectable in abnormalities in the facet eye (compare Figure 5A with 5B). While we observed that overexpression of wild-type Pur-alpha rescues the eye phenotype (Figure 5C), the RNA-binding mutant QSK I - KNR II failed to do so (Figure 5D). Surprisingly, a second, previously published RNA-binding mutant (Pur-alpha R I – R II), which showed strongly reduced MF0677 ssDNA binding (Figure 3H), was able to fully rescue the eye phenotype (Figure 5E). This observation indicates that arginine 80 and 158 are not required for the binding to nucleic acids important for neuroprotection. While the neuroprotection by the R I – R II mutant indicates flexibility in nucleic-acid recognition, the loss of rescue by the QSK I - KNR II mutant formally establishes the requirement of nucleic-acid binding for Pur-alpha-dependent neuroprotection. Additionally, the F II mutation of Pur-alpha, which abolishes its dsDNA-unwinding activity, also impairs the neuroprotective function in the fly model (Figure 5F). These findings indicate that unwinding is important for neuroprotection by Pur-alpha.

Recently, de novo mutations in Pur-alpha have been found to cause the so-called 5q31.3 microdeletion syndrome. This disease is characterized by neonatal hypotonia, encephalopathy, and severe developmental delay (Lalani et al., 2014; Hunt et al., 2014; Tanaka et al., 2015). Of the reported mutations (Figure 6—source data 1), two missense mutations (A89P, K97E) are of particular interest from a structure-to-function point of view (Lalani et al., 2014). Sequence alignment of Pur-alpha from different species shows that the residues A89 and K97 of the human Pur-alpha protein correspond to the residues A72 and R80 of the Drosophila protein, respectively. These residues are highly conserved (Figure 2—figure supplement 4A). In the crystal structure of the protein/DNA co-complex, A72 does not directly interact with the DNA molecule. Instead it forms backbone hydrogen bonds between the β-strands of PUR repeat I to stabilize the nucleic-acid binding β-sheet (Figure 6A, top) (this study and Graebsch et al., 2009). When A72 and its disease-causing counterpart A98 in the human protein (Figure 6A, middle) are substituted by a proline, the backbone interactions that stabilize the β-sheet very likely become disrupted (Figure 6A, bottom) and thus the protein misfolds.

Figure 6

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Figure 6—source data 1

Mutations in the gene encoding for human Pur-alpha that result in the 5q31.3 microdeletion syndrome.

Amino acids in brackets indicate the corresponding positions in Drosophila Pur-alpha, based on the protein-sequence alignment shown in Figure 2—figure supplement 4. “p.” indicates a point mutation, “*” a stop codon, “fs” a frame shift followed by the number of residues produced until the next stop codon, “del” a deletion, and “delins” a combination of deletion and insertion.

https://doi.org/10.7554/eLife.11297.020

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The Drosophila equivalent R80 of the disease-associated human K97 directly binds to the guanine base G7 (Figure 2B, F and 6B, top) and its mutation results in reduced nucleic-acid binding (Graebsch et al., 2009). It is therefore conceivable that a mutation of K97 to glutamate impairs nucleic-acid interaction because of repulsive forces and causes dysfunction of Pur-alpha (Figure 6B, middle, bottom). Although in our fly model the double mutant R80A/R158A (R I – R II) was still able to rescue neurodegeneration (Figure 5E), the reported effect of the K97E mutation in the microdeletion syndrome indicates that nucleic-acid binding by this residue is important at least in humans. Additional interesting disease-causing point mutations in human Pur-alpha are I188T and I206F (Figure 6—source data 1), which likely impair the intramolecular dimerization of PUR repeats I and II (Hunt et al., 2014;, Tanaka et al., 2015). Taken together, the crystal structure of the Pur-alpha/DNA co-complex presented in this study provides a molecular explanation for the effects of missense mutations in the 5q31.3 microdeletion syndrome.

Wild-type Pur-alpha binds to origins of replication and promoter regions (Bergemann and Johnson, 1992,; Bergemann et al., 1992) and regulates the transcription of more than 20 genes (White et al., 2009). Pur-alpha’s ability to unwind dsDNA might therefore play an important role in the initiation of replication and transcription. One recently reported interaction partner of Pur-alpha that might play a role in this context is the RNA helicase Rm62 (Qurashi et al., 2011). In the light of the dsDNA-unwinding activity an intriguing speculation is that Pur-alpha also unwinds dsCGG-repeat RNA. This initial unwinding by Pur-alpha could allow interacting helicases to subsequently regulate RNA processing, transport, and translation. Therefore, it will be important to assess Pur-alpha’s role in unwinding of dsRNA and its interaction with Rm62.

Considering that Pur-alpha repeat I-II has two nucleic-acid-binding sites, it is conceivable that each PUR repeat binds to one of the strands of a duplex DNA molecule thereby unwinding short stretches of dsDNA (Figure 7A–C, top). The insertion of Pur-alpha between both DNA strands might be achieved through spontaneous breathing of the dsDNA helix (Peyrard et al., 2009,; Jose et al., 2012). Intercalating residues (phenylalanine, tyrosine) might cause further separation of the two DNA strands via base stacking with the guanines and thereby causing the strong twist of the DNA strands. The partly melted duplex DNA could then be further unwound by DNA helicases, which are required for initiation of transcription and replication. In the crystal structure, base pairing is observed between the 5’-G1-C2 bases of two symmetry-related DNA molecules (Figure 2—figure supplement 2), indicating, that a PUR domain would unwind a short stretch of approximately four to six bases.

Figure 7

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We also solved the crystal structure of PUR repeat III (Figure 4A; Figure 4—figure supplement 1) and found that it binds only weakly to DNA/RNA (Figure 3G, H) and unwinds dsDNA only slightly (Figure 4C). Since in the crystal structure the C-terminal end of PUR repeat I-II is located on the opposite side of its nucleic-acid-binding surface (Figure 7A, B, bottom), it is unlikely that PUR repeat III causes steric clashes interfering with the nucleic-acid binding by PUR repeat I-II. Hence, PUR repeat III might only facilitate dimerization, thereby guiding a second DNA-/RNA-binding domain (PUR repeat I-II) to another GGN motif further upstream or downstream on the dsDNA, where additional DNA-unwinding events could take place (Figure 7C, bottom). How this effect of dimeric Pur-alpha is achieved on a molecular level and if unwinding of longer dsDNA fragments requires its joint action with helicases are main questions to be addressed in future.

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Author details

1. Janine Weber

   Institute of Structural Biology, Helmholtz Zentrum München - German Research Center for Environmental Health, Neuherberg, Germany

   Contribution

   JW, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

2. Han Bao

   Department of Human Genetics, Emory University, Atlanta, United States

   Contribution

   HB, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

3. Christoph Hartlmüller

   Center for Integrated Protein Science Munich, Department of Chemistry, Technische Universität München, Munich, Germany

   Contribution

   CH, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

4. Zhiqin Wang

   Department of Human Genetics, Emory University, Atlanta, United States

   Contribution

   ZW, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

5. Almut Windhager

   Institute of Structural Biology, Helmholtz Zentrum München - German Research Center for Environmental Health, Neuherberg, Germany

   Contribution

   AW, Conception and design, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

6. Robert Janowski

   Institute of Structural Biology, Helmholtz Zentrum München - German Research Center for Environmental Health, Neuherberg, Germany

   Contribution

   RJ, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

7. Tobias Madl

   1. Institute of Structural Biology, Helmholtz Zentrum München - German Research Center for Environmental Health, Neuherberg, Germany
   2. Center for Integrated Protein Science Munich, Department of Chemistry, Technische Universität München, Munich, Germany
   3. Institute of Molecular Biology & Biochemistry, Center of Molecular Medicine, Medical University of Graz, Graz, Austria
   4. Omics Center Graz, BioTechMed Graz, Graz, Austria

   Contribution

   TM, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

8. Peng Jin

   Department of Human Genetics, Emory University, Atlanta, United States

   Contribution

   PJ, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

9. Dierk Niessing

   1. Institute of Structural Biology, Helmholtz Zentrum München - German Research Center for Environmental Health, Neuherberg, Germany
   2. Department Cell Biology, Biomedical Center of the Ludwig-Maximilians-University München, Planegg-Martinsried, Germany

   Contribution

   DN, Conception and design, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   For correspondence

   dierk.niessing@med.uni-muenchen.de

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-5589-369X

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