Depletion of the RNA-binding protein PURA triggers changes in posttranscriptional gene regulation and loss of P-bodies

Abstract

The RNA-binding protein PURA has been implicated in the rare, monogenetic, neurodevelopmental disorder PURA Syndrome. PURA binds both DNA and RNA and has been associated with various cellular functions. Only little is known about its main cellular roles and the molecular pathways affected upon PURA depletion. Here, we show that PURA is predominantly located in the cytoplasm, where it binds to thousands of mRNAs. Many of these transcripts change abundance in response to PURA depletion. The encoded proteins suggest a role for PURA in immune responses, mitochondrial function, autophagy and processing (P)-body activity. Intriguingly, reduced PURA levels decrease the expression of the integral P-body components LSM14A and DDX6 and strongly affect P-body formation in human cells. Furthermore, PURA knockdown results in stabilization of P-body-enriched transcripts, whereas other mRNAs are not affected. Hence, reduced PURA levels, as reported in patients with PURA Syndrome, influence the formation and composition of this phase-separated RNA processing machinery. Our study proposes PURA Syndrome as a new model to study the tight connection between P-body-associated RNA regulation and neurodevelopmental disorders.

PURA Syndrome-causing mutations impair PUR-domain integrity and affect P-body association

“…Consistent with previous reports (Daigle et al, 2016;Markmiller et al, 2018), we observed that hsPURA co-localizes to stress granules in Very recently, hsPURA was reported to localize to P-bodies (Molitor et al, 2023). In a recent study, it was shown that a hsPURA knock-down in HeLa and in fibroblast cells resulted in down-regulation of the essential P-body proteins LSM14a and DDX6 and, most likely as a direct consequence, in strongly impaired P-body formation (Molitor et al, 2023). In light of this report, our finding that patient-derived mutations impair the P-body association of hsPURA (Fig…”

“…In order to assess the localization of hs PURA to cytoplasmic granules in unstressed conditions, we first recapitulated its recently reported localization to P-bodies (Molitor et al , 2023). Co-staining with the marker DCP1A indeed confirmed a co-localization of overexpressed hs PURA in P-bodies of HeLa cells ( Fig 2A and 2B ).…”

“…Very recently, hs PURA was reported to localize to P-bodies and that a PURA knockdown leads to a significant reduction in HeLa cells (Molitor et al ., 2023). While we recapitulated co-localization of hs PURA to P-bodies ( Fig 2 ), hs PURA K97E and F233del mutants failed to fully localize to P-bodies.…”

“…In a recent study, it was shown that a hs PURA knock-down in HeLa and in fibroblast cells resulted in down-regulation of the essential P-body proteins LSM14a and DDX6 and, most likely as a direct consequence, in strongly impaired P-body formation (Molitor et al ., 2023). In light of this report, our finding that patient-derived mutations impair the P-body association of hs PURA ( Fig 2 ) provide further evidence for a potential role of P-body localization of hs PURA for the etiology of PURA syndrome.…”

“…In order to assess the localization of hsPURA to cytoplasmic granules in unstressed conditions, we first recapitulated its recently reported localization to P-bodies (Molitor et al, 2023). Co- shown to be required for P-body formation in HeLa cells and fibroblasts (Molitor et al, 2023), PURA-dependent liquid phase separation could potentially also directly contribute to the formation of these granules. Furthermore, a loss of such a function could potential contribute to the etiology of PURA syndrome.…”