PQBP1 is a rather unpopular (reflected by 87 hits at PubMed for the search term PQBP1 as of 23 May 2019 [875]) polyglutamine-binding protein that was first identified in 1998 [876]. It is localized in the nucleus and the cytoplasm, with dominant nuclear localization and mutations within the gene have been linked to neurodegenerative disorders. In human tissues, Pqbp1 is ubiquitously expressed at largely homogenous expression levels which are higher in ovaries and lower in the pancreas [877]. PQBP1 is a disordered or denatured protein with a unique domain
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structure. Its N-terminal WW domain binds proline-rich motifs [878]. The subsequent disordered polar amino-acid-rich domain consists of five hepta- and an imperfect stretch of di-amino acid repeats, which are required for interaction with polyglutamine tracts [878], [879]. The third largely disordered C-terminal domain is unique with no homologous sequences found in other proteins [879], [880].
PQBP1 functions in mRNA metabolism by regulating transcription, splicing and translation of its targets. For instance, it can bind to the C-terminal domain of RNA Pol II, which leads to reduced transcription [881]. The binding is enhanced by RNA Pol II phosphorylation suggesting that PQBP1 interacts with actively transcribing RNA Pol II [881]. The interaction is further enhanced by mutant ATXN1 that causes spinocerebellar ataxia type-1 (SCA1), leading to an even greater decrease in transcription, reduced phosphorylation of RNA Pol II and eventually cell death [881]. As PQBP1 can also interact with the polyglutamine tract of HTT with increased affinity to mutant HTT carrying expanded polyglutamine stretches, it is suspected that a similar mechanism of impaired transcription might play a role in the pathology of Huntington’s Disease [878], [880]. Moreover, transgenic mouse expressing the human Pqbp1 gene showed a late-onset, gradually progressing motor neuron disease-like phenotype with reduced amounts of on neurons in the spinal anterior horn and fewer Purkinje and granule cells in the cerebellum [882]. Anterior horn tissue of the spinal cord displayed an upregulated transcription of mitochondrial genes prior to cell death, indicating that PQBP1 induced mitochondrial stress, which is a common pathological defect among human neurodegenerative disorders [883].
Moreover, PQBP1 seems to function at the interface of transcription and splicing as it is not only binding RNA Pol II but also splicing factors like SF3B1, WBP11 and U5-15kD [884]–
[887]. In complex with U5-15kD and U5-52K, PQBP1 might even be integrated in the early spliceosome prior to its activation [888], [889]. In HeLa cells PQBP1 was found to bind several members of the SF3B protein complex and in mouse embryonic cortical neurons it was shown to regulate about 1,400 alternative splicing events of 457 target mRNAs [887]. Targets were enriched for GO terms of neuron projection development and morphogenesis, dendrite development and axogenesis and PQBP1 deletion lead to reduced dendritic outgrowth [887]. In a conditional knockout mouse model, Pqbp1 was depleted from neural stem progenitor cells, which resulted in an increased cell cycle length, especially affecting the M phase [890]. The mice displayed microcephaly and a decreased stem cell pool during development [890]. Changes in transcription and splicing patterns were detected in a group of cell cycle genes that control the M phase [890]. Interestingly, Pqbp1 itself was deregulated, showing more pronounced aberrant splicing than transcription [890]. Further, the phenotype could be rescued by in utero gene therapy [890]. In another neuron-specific conditional knockout mouse model, Pqbp1 KD resulted in
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reduced dendritic spine formation and cognitive decline due to aberrant splicing of genes with synapse-related functions [891]. Remarkably, changes in splicing patterns of PQBP1 conditional KD mice were similar to splicing patterns of Alzheimer’s disease model mice and 40 % of exon skipping and inclusion events in the Alzheimer’s disease model could be restored by PQBP1 overexpression [891]. It should be noted that PQBP1 stability is impaired in Alzheimer’s disease due to SRRM2 which gets phosphorylated and shifts to the cytoplasm at early stages of the disease [891].
However, PQBP1 is also located in the cytoplasm where it is associated with a function in granule formation and translational regulation. In neuronal U373MG cells, PQBP1 was shown to interact with KHSRP, SFPQ/PSF, DDX1, Caprin-1 and two subunits of the intracellular transport-related dynactin complex [892]. This interaction was also found in primary neurons, where PQBP1 and its interaction partners formed RNA-dependent granules at the perikaryon, dendrites and axons, but not at synapses [892]. PQBP1 relocates to stress granules upon stress, where it also interacts with FMR1. This suggests that PQBP1 together with its partner might be involved in cytoplasmic mRNA metabolism including transport, localization and local translation [892]. In accordance with that, the fly homolog of PQBP1 was suggested to play a role in photoreceptor cells, where it might regulate mRNA translation through interaction with dFMR1 [893].
Apart from directing intellectual ability in neuronal cells, PQBP1 was identified to be one out of seven key drivers of a regulatory gene network related to atherosclerosis and coronary artery disease [894]. Silencing of PQBP1 in macrophages activated genes from the network and decreased the cholesterol-ester accumulation in foam cells, indicating that PQBP1 is also playing a role in vascular tissues [894]. Furthermore, PQBP1 is involved in the IRF3-dependent innate immune response to HIV-1 infection [895]. By directly binding reverse-transcribed HIV-1 DNA, associating with and stimulating cGAS activity to initiate signaling in dendritic cells, PQBP1 is acting as an immune regulator [895].
Our analyses revealed that Pqbp1 can be cap-independently translated in vitro and that it mediates internal translation initiation with comparable efficiency as EMCV IRES in bicistronic reporter assays in vivo. Potential internal translation initiation was confirmed by RNAi-based validation, however Pqbp1-driven internal translation was not stimulated during cap-dependent translation inhibition in vivo. Further, Pqbp1 enhanced cap-dependent translation when located downstream of the ORF, which suggests a CITE-like function. Cap-independent translation was as efficient as cap-dependent translation and could be stimulated by inhibition of cap-dependent translation in vitro, although Pqbp1 was overall inefficiently translated in the Drosophila in vitro system. Thus, validation in a mammalian in vitro system or preferably RNA reporter transfection
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is recommended. Overall it is unclear whether Pqbp1 acts as a bona fide IRES, but it was shown that Pqbp1 has a low cap-dependency and that Pqbp1 remains to be efficiently translated during cap-dependent translation inhibition by 4E-PB1 in vivo. Such resistance to cap-dependent translation inhibition could play a role during stress conditions like for example HIV-1 infection or mitochondrial oxidative stress in neurodegenerative or cardiovascular disease [896], [897]. As PQBP1 was shown to mediate innate immune response by binding reverse transcribed HIV-1 DNA, persistent translation of Pqbp1 mRNA during virus infection might be required for effective immune response [895]. Indeed, infection by HIV-1 was shown to activate GCN2 kinase resulting in phosphorylation of eIF2α [898], [899]. This leads to a decrease of global protein synthesis through sequestration of eIF2α by eIF2B and abrogation of ternary complex formation before GCN2 is eventually cleaved by HIV-1 protease [898], [899]. But also other translation initiation factors like eIF4G, PABP and eIF3d were reported to be cleaved during HIV-1 infection [900]. To test if Pqbp1 continues to be translated during HIV-1 induced cap-dependent translation inhibition, in vitro translation assays with purified HIV-1 protease or reporter assays in co-transfected/infected cells could be performed.
In the case of disease-related mitochondrial oxidative stress, sustained translational control of Pqbp1 might be rather harmful. In both, neurodegenerative and cardiovascular, disease contexts PQBP1 overexpression is suggested to promote disease progression [883], [894]. Hence, persistent Pqbp1 translation during oxidative stress might reinforce the devil’s circle and further intensify mitochondrial dysfunction in neurodegeneration or accumulation of cholesteryl esters in atherosclerosis causing more severe outcomes. To test whether Pqbp1 translation is maintained during oxidative stress, reporter assays could be performed in cells treated with arsenite or hydrogen peroxide.
Interestingly, both PQBP1 overexpression and under-expression were associated with pathophysiologic conditions [880]. Thus, it seems that tight regulation of Pqbp1 translation is critical at least for specific Pqbp1 functions. For instance, dPQBP1 mutant flies exhibit learning disability and a shortened lifespan [901]. Both can be reversed in a dose-dependent manner but while excessive dPQBP1 expression recovers learning disability, neither insufficient nor excessive dPQBP1 expression is recovering lifespan [901]. Hence, relaxed cap-dependency of Pqbp1 is likely well controlled, potentially involving one or more interaction partners or specific translation initiation factors, whose identity would be of interest in order to uncover Pqbp1s mechanism of cap-independent translation. Analysis of Pqbp1’s 5’UTR for binding sites of those factors or for RNA modification sites like m6A methylation could help to identify factors enabling cap-independent translation. In case no motifs can be found, deletion assays could help to narrow
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down sequence stretches that are required for cap-independent translation. EMSA or RNA pull-down assays could further help to identify proteins binding to Pqbp1 mRNA.
Forward-thinking, after elucidating the mechanism of action of cap-independent translation of Pqbp1, the next step would be to figure out whether interference with this mechanism could help to prevent or slow down disease progression in neurodegenerative disorders or atherosclerosis. As Pqbp1 was identified as a key driver in coronary artery disease, specific interference with cap-independent translation of Pqbp1 might have potential to decrease mitochondrial stress fueling atherosclerosis. However, atherosclerosis as well as neurodegenerative diseases are chronic conditions, wherefore potential medication would be administered over long periods of time. It would be challenging to find a way to interfere with the translational machinery (providing the mechanism would rely on standard eIFs) in the long term without causing substantial side effects. Nevertheless, strategies to interfere with mRNA translation in neuronal cells could be reconsidered in treating neurodegenerative diseases given the lack of efficient treatment options to date.
Summarizing, Pqbp1 has the potential to regulate a wide range of target mRNAs through transcriptional, alternative splicing or translational control. Over- and under-expression of Pqbp1 has previously been linked to disease conditions like mental retardation syndromes or atherosclerosis. Tight translational control of Pqbp1 therefore seems warranted. The reported ability of Pqbp1 to undergo cap-independent translation initiation could play a role in Pqbp1 dysfunction in disease contexts and might contribute to development and progression of disease outcomes. Hence, further investigation of Pqbp1 translational regulation will provide insights into pathological mechanisms and might provide new approaches to advance the development of corresponding treatments.