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Molecular characterization of the neuronal nucleic acid-binding protein Pur-α

2015-07-08 00:01:39

Ludwig Maximilian University of Munich; 2015 July; Doctoral Thesis


Janine Weber



Summary



Pur-α (purine-rich element binding protein A) is a multifunctional protein binding to ss/dsDNA and RNA. It is involved in replication, transcription, mRNA transport and translation in neurons. Homozygous Pur-α mutant mice die within 4 weeks after birth, suffering from severe neurological defects. Pur-α unwinds dsDNA in an ATP-independent manner, thereby providing access for replication and transcriptional regulators. Still, Pur-α's role in cellular functions is not well understood.



Pur-α (purine-rich element binding protein A) is a multifunctional protein binding to ss/dsDNA and RNA. It is involved in replication, transcription, mRNA transport and translation in neurons. Homozygous Pur-α mutant mice die within 4 weeks after birth, suffering from severe neurological defects. Pur-α unwinds dsDNA in an ATP-independent manner, thereby providing access for replication and transcriptional regulators. Still, Pur-α's role in cellular functions is not well understood. has also been implicated in the pathomechanism of heritable, neurodegenerative diseases like ALS/FTLD (amyotrophic lateral sclerosis / frontotemporal lobar degeneration) and FXTAS (fragile X-associated tremor/ataxia syndrome). FXTAS is caused by premutation expansions (55-200 CGG repeats) in the 5'UTR of the fmr1 gene. ALS/FTLD can be triggered by hexanucleotide (G4C2) repeat expansions in the first intron of the C9orf72 gene. The pathological hallmark for both diseases is the formation of neuronal, intranuclear and cytoplasmic inclusions. It is thought that these repeat-RNA containing inclusions sequester RNA-binding proteins, leading to altered transcription, RNA processing and trafficking. Pur-α binds to both types of RNA repeats and accumulates in these pathogenic inclusions.



The first goal of this study was to gain insights into the molecular principles of Pur-α’s binding to nucleic acids and its cellular functions. For this, structural analysis were combined
with various biochemical in vitro and cellular studies. Here, I present the crystal structure of Pur-α/ssDNA co-complex from Drosophila melanogaster at 2.0 A resolution. The structure explains Pur-α’s dsDNA-binding and –unwinding, and its ssDNA stabilizing activity. The protein disrupts the base stacking of DNA by intercalation of a highly conserved phenylalanine. The importance of this structural feature was confirmed by in vitro unwinding assays. NMR titration experiments and EMSAs suggest that short RNA and DNA oligomers interact with Pur-α in identical ways. Filter-binding assays confirmed that the main nucleic acid binding domain of Pur-α binds two molecules of nucleic acid, as suggested by the crystal structure.



The second aim of this study was to investigate Pur-α’s role in neurodegenerative diseases. For this, I generated inducible, mammalian expression vectors coding for the fmr1 5’UTR with normal and disease-related CGG-repeats. These vectors have been tested in COS7 and HeLa cells and can now be used for establishment of a stable cellular FXTAS model.



INTRODUCTION



1.1. Purine-rich element binding protein family



Pur (purine-rich element binding) proteins are nucleic acid-binding proteins that can be found from bacteria to mammals. They bind to purine-rich elements conserved in origins of replication and gene flanking regions. The Pur family consists of 4 members, encoded by genes at three different loci. These four members are Pur-α at chromosome 5q31, Pur-Β at 7p13 and two isoforms of Pur-γ at 8p11. Different transcription termination sites generate the two isoforms of Pur-γ: Pur-γ A and B. Except for Pur-γ B, all Pur proteins are expressed as a single, intronless coding sequence. Transcription of Pur-γ B runs through the Pur-γ A termination signal, resulting in a very long transcript of which a 30 kb intron becomes spliced out. This splicing event results in a loss of the stop codon and a different C-terminus for the protein isoform B.



Human Pur-α, Pur-Β and Pur-γ (both isoforms) possess an N-terminal glycine-rich domain and, except for Pur-γ, a C-terminal gluatmine/glutamate-rich region. A so-called “Psycho” motif at the C-terminus describing the consensus motif of proline, serine, tyrosine and cysteine can be found in all Pur proteins, except for the isoform B of for Pur-γ. All vertebrate Pur proteins contain three strongly conserved repeats of approximately 80 amino acids and are expressed at different time points during development. While Pur-γ is highly expressed at early stages of mouse embryo development (embryonic age 14), Pur-α expression is nearly undetectable at these early stages. Later Pur-γ protein levels decrease drastically whereas Pur-α expression reaches a peak at 18-25 days after birth, together with Pur-Β. These observations implied that Pur-γ is an important factor for embryonic or fetal development that becomes replaced by Pur-α and Pur-Β at a later developmental stage.



Pur-α plays multiple roles in cellular regulation including replication, transcription, mRNA transport and translation. Pur-α’s various functions are further described in section 1.1.2. Pur-Β has been implicated in transcriptional repression of genes encoding for muscle-specific isoforms of actin and myosin in heart, skeletal muscle and vascular smooth muscle. Both Pur-α and Pur-Β have been shown to be present in the same mRNPs (messenger ribonucleoprotein particle) that is transported in dendrites along microtubules by a kinesin motor. Interaction of Pur-α and Pur-Β with nucleic acids results in the formation of multimeric complexes. Still, direct interaction between Pur-α and Pur-Β has not been shown so far.



Abberations in all three Pur genes have been implicated in multiple tumor types and cell proliferation disorders, including myelodysplastic syndrome, myelogenous leukemia and 5q31.3 microdeletion syndrome (Pur-α), brain tumors and glioblastoma (Pur-Β), myeloproliferative syndrome (Pur-γ).



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