PURA, the gene encoding Pur-alpha, member of an ancient nucleic acid-binding protein family with mammalian neurological functions

Abstract

The PURA gene encodes Pur-alpha, a 322 amino acid protein with repeated nucleic acid binding domains that are highly conserved from bacteria through humans. PUR genes with a single copy of this domain have been detected so far in spirochetes and bacteroides. Lower eukaryotes possess one copy of the PUR gene, whereas chordates possess 1 to 4 PUR family members. Human PUR genes encode Pur-alpha (Pura), Pur-beta (Purb) and two forms of Pur-gamma (Purg). Pur-alpha is a protein that binds specific DNA and RNA sequence elements. Human PURA, located at chromosome band 5q31, is under complex control of three promoters. The entire protein coding sequence of PURA is contiguous within a single exon. Several studies have found that overexpression or microinjection of Pura inhibits anchorage-independent growth of oncogenically transformed cells and blocks proliferation at either G1-S or G2-M checkpoints. Effects on the cell cycle may be mediated by interaction of Pura with cellular proteins including Cyclin/Cdk complexes and the Rb tumor suppressor protein. PURA knockout mice die shortly after birth with effects on brain and hematopoietic development. In humans environmentally induced heterozygous deletions of PURA have been implicated in forms of myelodysplastic syndrome and progression to acute myelogenous leukemia. Pura plays a role in AIDS through association with the HIV-1 protein, Tat. In the brain Tat and Pura association in glial cells activates transcription and replication of JC polyomavirus, the agent causing the demyelination disease, progressive multifocal leukoencephalopathy. Tat and Pura also act to stimulate replication of the HIV-1 RNA genome. In neurons Pura accompanies mRNA transcripts to sites of translation in dendrites. Microdeletions in the PURA locus have been implicated in several neurological disorders. De novo PURA mutations have been related to a spectrum of phenotypes indicating a potential PURA syndrome. The nucleic acid, G-rich Pura binding element is amplified as expanded polynucleotide repeats in several brain diseases including fragile X syndrome and a familial form of amyotrophic lateral sclerosis/fronto-temporal dementia. Throughout evolution the Pura protein plays a critical role in survival, based on conservation of its nucleic acid binding properties. These Pura properties have been adapted in higher organisms to the as yet unfathomable development of the human brain.

Keywords: AIDS; ALS; AML; Acute myelogenous leukemia; Amyotrophic lateral sclerosis; C9ORF72; Dementia; FMR1; FXS; Fragile X syndrome; JCV; Myelodysplastic syndrome; PML; Polyomavirus JC; Progressive multifocal leukoencephalopathy; Pur-beta; Pur-gamma-A and Pur-gamma-B; Pura (Pur-alpha); Purb; Purg.

Figure 1. Comparison of the signature Pur domain in similar proteins of diverse species from spirochetes to humans

The sequences shown were aligned to genomic databases using the CDD/SPARCLE protocol with a 2-bit color identity (Marchler-Bauer et al., 2017) The sequences presented are from eight of the most diverse species containing the Pur domain. The species as numbered at left are: 1. Caenorhabditis elegans; 2. Drosophila melanogaster; 3. Arabadopsis thaliana; 5. Caenorhabditis elegans, repeat 2; 5. Homo sapiens Pura, repeat 3; 6. Schistosoma mansoni; 7. Treponema denticola; 8. Treponema pallidum. Note that all the Pur domains shown from these species align with human repeat 3 (Line 5). C. elegans, D. melanogaster and A. thaliana also have three repeats. The bacterial sequences shown have only one Pur domain. Several amino acids evolutionarily identically conserved in these domains are indicated by arrows at bottom.

Figure 2. Sequence comparison of human Pur proteins, Pura, Purb and two forms of Purg

All four of the human protein Pur family members are aligned using the Clustal Omega algorithm. Pura, Purb and Purg-A are each derived from a distinct gene locus. Their entire sequences are aligned at top. Stars indicate amino acid identities; one dot indicates a substitution; two dots indicate a conservative substitution. Purg-B is transcribed from the Purg-A gene locus. Transcription of Purg-B, however, passes through the Purg-A stop point producing a 38 kb intron that is spliced out to make Purg-B mRNA. It is only the C-termini that differ between Purg-A and Purg-B. Sequences of these are presented at the bottom. Vertical lines indicate identities, and 2 dots indicate conservative substitutions. The Psycho motif is lightly underlined in both top and bottom alignments. In Pura it binds the Rb protein, among others (Ma et al., 1994; Johnson et al., 1995), and it is conserved in evolution. It is eliminated in Purg-B. Class 1 (Cl1) and Class2 (Cl2) segments of Pura repeats (R) 1, 2 and 3 are as indicated and heavily underlined.

Figure 3. Diagram of reported Pura nucleic acid-binding and protein-binding regions in relation to signature Pur domains and to structural features from crystallographic studies. A. Box diagram map of nucleic acid and protein binding regions

Red indicates the region containing long tracts of poly-Gly. Structure: not determined. Green indicates the psycho motif, which is present in Repeat III of eukaryotic organisms and in the only repeat of bacterial Pura (Figure 1). It is present in several other DNA replicative proteins (Ma et al., 1994). Structure: alpha helix (a). Black indicates a Gln-rich region in mammalian Pura proteins. Structure: not determined. Horizontal lines in the boxes denote the Class 1 (lower lines) and Class 2 (upper lines) portions of each Pur repeat. Note that sequences of variable length separate each repeat and the portions of Repeats I and II. X-ray crystallographic analyses of Drosophila Pura reveal that the N-terminal sequence of each repeat, comprising all of Class 1 and a part of Class 2, is structured as four beta-strands comprising a beta sheet, reported to be primarily involved in nucleic acid binding (Graebsch et al., 2009; Weber et al., 2016). The C-terminal 14–15 amino acids of each repeat are in an alpha helical structure (a) primarily involved in protein binding. Horizontal lines with vertical stops below the map denote indicated Pura regions. Positions of reported protein binding are from (White et al., 2009). B. Human Pura amphipathic helix. The wheel is a 2-dimensional depiction looking down the axis of the Repeat III 14 amino acid helix. Aromatic residues are present on one side of the helix and basic residues on the other. For comparison, the homologous region of Purb is included on the wheel. Purb has a very similar amphipathic helix, as shown by the residues in parentheses. C. Alignment comparison of human vs. Drosophila Repeat III. Alignment is with the Clustal Omega algorithm. The human Pura sequence is from Genbank M96684.1. Drosophila melanogaster is from Genbank AF021259.1. The entire gene coding sequence of human Pura is shown compared to that of Drosophila in Supplementary Fig. 1S. In Fig. 3C, stars indicate amino acid identities; one dot indicates a substitution; two dots indicate a conservative substitution. Beta sheet and helical structural regions are conserved. The helical regions in the psycho motifs differ. The human Rb binding site is altered in Drosophila. Drosophila lacks a double-sided amphipathic helix, but residues on the aromatic side of the helix are strongly conserved.(Figure 3B from Bergemann et al., 1992, Mol Cell Biol, 12, 5673–5682 used with permission.)

Figure 4. Pura visualized by laser confocal microscopy with cytoplasmic proteins in rat hippocampal neuronal dendrites

Pura localizations with proteins exemplifying aspects of dendritic mRNA transport are shown. A. Pura (red) colocalized with microtubule-associated protein 2 (Map2, green). Colocalization (yellow) is seen primarily in long neuronal processes. Map2 is a protein associated with microtubules, along which Pura- and RNA-containing granules are transported via a kinesin motor. B. Pura colocalized with Staufen in a hippocampal neuronal dendrite. Colocalized Pura (yellow) is seen at dendritic branch junctions. These junctions are reportedly sites of dendritic mRNA translation. C. Pura localized specifically in rat hippocampal neuronal dendrites, not in axons. Ankyrin G (green) is a protein marker specifically located in axons. A single green axon protrudes from each hippocampal neuron. Pura (red) is visualized in multiple protruding dendrites. Pura is not significantly present with ankyrin G in the axon. D. Granular nature of structures containing Pura (red) in a rat hippocampal neuronal dendrite. Pura is detected in such structures together with multiple proteins, including Purb, FMRP and Staufen together with long non-coding RNA BC1 (BC200 in humans).(Portions of Figures 5 & 6 from Johnson et al., 2006, J Neurosci Res, 83:6, 929–943 used with license from Journal of Neuroscience Research.)

Figure 5. Map of PURA and nearby genes located on Chromosome 5, band 31

Location of the PURA gene in relation to other genes and markers that have been mapped to band region 5q31. The order of the markers shown is adopted from bacmaps at the LBNL Human Genome Center. Other genes mapped to chromosome 5 band region q31 encode proteins that include: interferon regulatory factor 1, IRF1; interleukin-9, IL-9; a CDC2 phosphatase, CDC25C; early growth response-1 zinc-finger protein, EGR-1; myelomonocytic differentiation antigen, CD14; and a histone deacetylase, HDAC3. (Figure from Lezon-Geyda et al., 2001, Leukemia, 15:6, 954–962 used with permission.)