Structure-to-function studies on the neuronal transcription and RNA-transport factor Pur-alpha in complex with DNA and RNA.
Final Report Abstract
The protein Pur-α is a member of the purine-rich element (PUR) binding protein family serving as essential neuronal factor that binds to DNA- and RNA. Its protein sequence is strongly conserved throughout metazoan. Pur-α has been mechanistically implicated in different neuronal diseases, such as the fragile X-associated tremor/ataxia syndrome (FXTAS) and a familial form of ALS/FTLD. In both diseases abnormal RNA-repeat expansions cause the formation of aggregates, in which Pur-α is found. Mutational analysis of Pur-α in mice confirmed that it plays an essential role in postnatal brain development. More recently mutations in PURA, the gene encoding the Pur-α protein, have been shown to cause the socalled PURA syndrome. This disease usually occurs by spontaneous mutations and patients display neurodevelopmental delay and learning disability as well as neonatal hypotonia, early feeding difficulties and seizures, or 'seizure-like' movements. Pur-α is ubiquitously expressed and localizes in the nucleus as well as the cytoplasm. By interacting with DNA in the nucleus, Pur-α regulates the transcription of several genes. Via its cytoplasmic mRNA binding Pur-α contributes to their synaptic transport. A number of highaffinity DNA-targets are known for Pur-α. However, only one RNA sequence motif (r(GGN)n) has been identified to bind to Pur-α so far. Our previous work has led to the identification of three so-called PUR repeats in the protein sequence. PUR repeats I and II interact to form a stable PUR domain. PUR repeat III interacts with the corresponding repeat III of another molecule and thereby dimerizes Pur-α. For the PUR domain of repeats I-II we had previously determined the crystals structure. The aim of this funded project was to understand how Pur-α binds to nucleic acids and whether there are differences in RNA and DNA binding. First we solved the crystal structure of the DNA-/RNA-binding domain (repeats I-II) of Pur-α in complex with single-stranded DNA at 2.0 Å resolution (Rfree = 21.5 %). It reveals base-specific recognition of consecutive guanines in the single-stranded DNA. Consistent with insights from the crystal structure, biochemical and NMR data show that Pur-α binds DNA and RNA in the same way and with comparable affinities. This observation suggests that the subcellular localization of Pur-α can be modulated by the expression and thus presence of certain target mRNAs in the cytoplasm. Since the sequence-specific recognition of guanines is compatible with binding to diseasecausing tri- and hexanucleotide-repeat RNAs in FXTAS and ALS/FTLD, our findings are also suited to explain how Pur-α recognizes these sequences. Additionally, structure-based in vitro experiments resolved the molecular mechanism of Pur-α’s unwindase activity. This activity of separating short regions of double-stranded DNA regions seems to mainly depend on a phenylalanine, which undergoes aromatic stacking with DNA bases. Complementing analyses in the fruitfly Drosophila demonstrated the importance of this highly conserved phenylalanine for Pur-α’s in vivo function. Finally, we used the structural information from this project to rationalize the negative impact of PURA-syndrome causing mutations in Pur-α. In summary, by uncovering the molecular mechanisms of nucleic-acid binding, this DFG-funded project makes an important contribution towards the understanding of Pur-α’s cellular role and its implications in neurodegenerative diseases.
Publications
- Ein Baustein des Fragilen X-assoziierten Tremor/Ataxie- Syndroms. Biospektrum Oct. 635-637 (2010)
Graebsch, A, Roche, S, Niessing, D
- Drosophila Pur-α binds to trinucleotide-repeat containing cellular RNAs and translocates to the early oocyte. RNA Biol. 9(5):633-43 (2012)
Aumiller V, Graebsch A, Kremmer E, Niessing D, Förstemann K
(See online at https://doi.org/10.4161/rna.19760) - Structural basis of nucleic-acid recognition and double-strand unwinding by the essential neuronal protein Pur-α. Elife 5. pii e11297 (2016)
Weber J, Bao H, Hartlmüller C, Wang Z, Windhager A, Janowski R, Madl T, Jin P, Niessing D
(See online at https://doi.org/10.7554/eLife.11297)
