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Αlpha-synuclein (SNCA) has been associated with the central nervous system, is localized mainly in the presynaptic terminals of neurons and is thought to be important for synaptic plasticity, vesicular packaging and trafficking. Although its function has not been fully elucidated, it is thought to regulate the amount of synaptic vesicles that bind to the synapse during neurotransmitter release. Abnormal expression of SNCA is a fundamental feature in several diseases including Parkinson's disease (PD), Lewy body dementia and Multiple system atrophy, with a critical role in the initiation and exacerbation of neurodegeneration. Understanding the mechanisms underlying SNCA expression is essential for the development of potential therapeutic interventions. Research activity to date has focused on the processes of misfolding and protein accumulation with little attention paid to the regulatory networks that shape the fate of the α-synuclein messenger RNA (mRNA). Studies have reported SNPs in ...
Αlpha-synuclein (SNCA) has been associated with the central nervous system, is localized mainly in the presynaptic terminals of neurons and is thought to be important for synaptic plasticity, vesicular packaging and trafficking. Although its function has not been fully elucidated, it is thought to regulate the amount of synaptic vesicles that bind to the synapse during neurotransmitter release. Abnormal expression of SNCA is a fundamental feature in several diseases including Parkinson's disease (PD), Lewy body dementia and Multiple system atrophy, with a critical role in the initiation and exacerbation of neurodegeneration. Understanding the mechanisms underlying SNCA expression is essential for the development of potential therapeutic interventions. Research activity to date has focused on the processes of misfolding and protein accumulation with little attention paid to the regulatory networks that shape the fate of the α-synuclein messenger RNA (mRNA). Studies have reported SNPs in the 3' untranslated region (3'UTR) of α-synuclein mRNA and changes in its length distribution in PD patients compared to healthy individuals. The 3'UTR of α-synuclein, through the mechanism of alternative polyadenylation, can produce mRNAs of many different lengths. In particular, 3'UTRs with lengths of 290, 480, 560, 1070 and 2520 nuleotides have been identified. RNA-binding proteins (RBPs) contribute to a plethora of functions in the mRNA life cycle from splicing and alternative polyadenylation to nucleocytoplasmic shuttling and further to subcellular localization, stability, granule stress dynamics and even translation levels. The aim of this study was to map the points in the life cycle of α-synuclein mRNA at which regulatory control is exerted by two such proteins, AUF1 (AU-binding Factor 1) and hnRNPA1 (heterogeneous ribonucleoprotein A1).Heterogeneous nuclear ribonucleoprotein D (hNRNPD), also known as AU-binding factor 1 (AUF1), comprises four protein isoforms (p37, p40, p42 and p45) capable of moving between the nucleus and the cytoplasm. AUF1 binds U-, GU- and UG-rich sequences and is involved in many processes such as transcriptional activation and alternative splicing and either promotes degradation or enhances the stability and translation of target mRNAs by mechanisms that are yet poorly understood. AUF1 protein was precipitated using biotinylated SNCA mRNA segments (5'UTR, coding region, proximal 3'UTR, distal 3'UTR) where it was found bound to the 3'UTR segments and absent from the other functional regions of the mRNA. Upregulated AUF1 was able to reduce SNCA mRNA levels leaving the levels of the long 3'UTR isoform consisting of 2520 bases (3'UTR2520) unaffected. The decrease in total mRNA levels coincided with destabilization of the short mRNA isoforms and a shortening of their average poly(A) tail length. The length of the poly(A) tail is a determinant of the lifetime of an mRNA molecule, with its shortening signaling destruction processes. Further biotinylated RNA precipitation and co-immunoprecipitation experiments showed that AUF1 recruits the CNOT1-CNOT7 deadenylation complex to the α-synuclein mRNA. Finally, it prevents SNCA mRNA-ribosome binding, leading to an overall 35% reduction in α-synuclein protein levels. On the other hand, reduction of AUF1 levels resulted in a 35% decrease in α-synuclein levels, a surprising fact as it elevated the total α-synuclein mRNA levels, as well as the 3'UTR2520 isoform. Additionally, it increased the stability of the short mRNA of α-synuclein and increased the average length of the poly(A) tail. A possible explanation for the inhibition of α-synuclein protein synthesis in the absence of AUF1 is the strong retention of both short and long mRNA isoforms of α-synuclein in the nucleus. Given the strong nuclear presence of AUF1 it seems important for the 'healthy' exit of α-synuclein mRNA from the nucleus. Finally, given the literature-known interaction of AUF1 with miRNAs, the interaction of AUF1 with the strong negative regulators of α-synuclein mRNA miRNA-7 and miRNA-153 was tested. Manipulation of AUF1 levels left the levels of miRNAs unaffected and did not appear to participate in feedback loops in inhibiting α-synuclein expression. Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) is an evolutionarily conserved and highly expressed protein involved in a variety of RNA processing events, including transcription, telomere maintenance, miRNA splicing, mRNA stability and translation. It has been associated with neurodegenerative diseases such as Amyotrophic Lateral Sclerosis and Multisystem proteinopathy. Similarly to AUF1, hnRNPA1 binds to both the proximal and distal 3'UTR without showing affinity to the 5'UTR and the SNCA mRNA coding region, indicating that it can exert control over all 3'UTR isoforms. The knowledge that hnRNPA1 is involved in the exit of certain target mRNAs from the nucleus led us to investigate its influence on the subcellular localization of α-synuclein mRNA, where it led to a prolonged persistence of the 3'UTR2520 isoform in the nucleus (with hnRNPA1 silencing leading to increased mRNA exit from the nucleus). Furthermore, in a catecholaminergic cell model, it was found to be able to alter the cytoplasmic localization of mRNA favoring the persistence of more elongated isoforms in the soma relative to the outgrowth, an observation which was paralleled in protein levels. Given the toxic effect of excessive α-synuclein on neurons, the function of hnRNPA1 as a regulator of mRNA and α-synuclein protein localization suggests its potential importance in α-synuclein-mediated neurodegeneration. The hnRNPA1 appeared to be a regulator of alternative polyadenylation (APA), with its overexpression leading to a 45% increase in the long isoform-to-total ratio. Three possible mechanisms underlying the APA of α-synuclein were examined, interference with the 3' end processing factors, cooperation with the U1 spliceosome component and finally enhancement of transcription elongation factors by promoting the binding of phosphorylated-TEFb to RNA polymerase II (Pol II), which seemed to be the most likely mechanism. Differential control of α-synuclein mRNA isoforms by hnRNPA1 is also identified in the case of mRNA stability with hnRNPA1 leading to destabilization of short isoforms, with a mechanism so far unknown. It appears independent of regulation via miRNA-7 and miRNA-153, presumably occurring with involvement of the CNOT1-CNOT7 complex as hnRNPA1 induced deadenylation but no direct interaction with the complex has been found. hnRNPA1 has been identified as a regulator of protein degradation and recycling processes. Therefore, its effect on these processes was studied in the SK-N-SH neuroblastoma cell model, where it was found to enhance proteasome activity, catabolism of ubiquitinylated proteins and positively affect autophagic flux with its silencing being associated with a slowing down of protein degradation mechanisms. The direct reduction of SNCA mRNA-ribosome affinity was examined and shown not to be a point of regulation. The sum of the above components was a decrease in protein α-synuclein levels in the presence of hnRNPA1 and an increase in the absence of hnRNPA1.In conclusion, the investigation of the mechanisms involved in the control of SNCA through its 3'UTR by AUF1/hnRNPA1 revealed two important pre-translational regulators of SNCA and highlighted the importance of alternative polyadenylation in the orderly production and presence of α-synuclein. Mapping the interactions of RBPs with their target mRNAs is important for understanding the mechanisms underlying transcript regulation, health/pathogenesis balance and thus adding to the human 'toolbox' for the development of novel therapeutic approaches.
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