The neuronal DNA-/RNA-binding protein Pur-alpha is a transcription regulator and core factor for mRNA localization. Pur-alpha-deficient mice die after birth with pleiotropic neuronal defects. Here, we report the crystal structure of the DNA-/RNA-binding domain of Pur-alpha in complex with ssDNA. It reveals base-specific recognition and offers a molecular explanation for the effect of point mutations in the 5q31.3 microdeletion syndrome. Consistent with the crystal structure, biochemical and NMR data indicate that Pur-alpha binds DNA and RNA in the same way, suggesting binding modes for tri- and hexanucleotide-repeat RNAs in two neurodegenerative RNAopathies. Additionally, structure-based in vitro experiments resolved the molecular mechanism of Pur-alpha's unwindase activity. Complementing in vivo analyses in Drosophila demonstrated the importance of a highly conserved phenylalanine for Pur-alpha's unwinding and neuroprotective function. By uncovering the molecular mechanisms of nucleic-acid binding, this study contributes to understanding the cellular role of Pur-alpha and its implications in neurodegenerative diseases.
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.