Depletion of the RNA binding protein HNRNPD impairs homologous recombination by inhibiting DNA-end resection and inducing R-loop accumulation.

DNA double strand break (DSB) repair through homologous recombination (HR) is crucial to maintain genome stability. DSB resection generates a single strand DNA intermediate, which is crucial for the HR process. We used a synthetic DNA structure, mimicking a resection intermediate, as a bait to identify proteins involved in this process. Among these, LC/MS analysis identified the RNA binding protein, HNRNPD. We found that HNRNPD binds chromatin, although this binding occurred independently of DNA damage. However, upon damage, HNRNPD re-localized to γH2Ax foci and its silencing impaired CHK1 S345 phosphorylation and the DNA end resection process. Indeed, HNRNPD silencing reduced: the ssDNA fraction upon camptothecin treatment; AsiSI-induced DSB resection; and RPA32 S4/8 phosphorylation. CRISPR/Cas9-mediated HNRNPD knockout impaired in vitro DNA resection and sensitized cells to camptothecin and olaparib treatment. We found that HNRNPD interacts with the heterogeneous nuclear ribonucleoprotein SAF-A previously associated with DNA damage repair. HNRNPD depletion resulted in an increased amount of RNA:DNA hybrids upon DNA damage. Both the expression of RNase H1 and RNA pol II inhibition recovered the ability to phosphorylate RPA32 S4/8 in HNRNPD knockout cells upon DNA damage, suggesting that RNA:DNA hybrid resolution likely rescues the defective DNA damage response of HNRNPD-depleted cells.

Posttranscriptional regulation of cyclin D1 by ARE-binding proteins AUF1 and HuR in cycling myoblasts.

RNA binding proteins (RBPs) can regulate the stability and/or translatability of messengerRNAs (mRNAs) through interactions with their 30-untranslated regions. However, individual mRNAs may be regulated simultaneously or successively by more than one RBP, as well as by Argonaute (AGO)-bound miRNAs; the coordination of these various influences on an individual mRNA is therefore complex and not well studied. In this report we examine the roles of two RBPs that bind to AU-rich elements (ARE) - AUF1 and HuR - in the stability and translation of cyclin D1 (Ccnd1) mRNA in rat myoblasts transiting the G phase of the cell cycle, and their interactions with miRNAs. Knockdown (KD) of AUF1 resulted in (1) transient upregulation of the mRNA level as well as an advancement of translation onset time (TOT) from 6 to 5 h post-serum addition, (2) loss of miRNA loading on AGO1 and AGO2 and (3) reduction in the level of AGO-1 and AGO-2 bound mRNA. In contrast, KD of HuR had no effect on the mRNA level, or on the AGO-mRNA complexes, but delayed TOT by 1 h independent of miRNA let-7. Thus the dynamics of RBP-mRNA binding and -RBP-AGO-miRNA interactions are coordinated to fine tune the expression of Ccnd1 in the G1 phase.

Post-transcriptional regulation of CD83 expression by AUF1 proteins.

Mature dendritic cells (DC), activated lymphocytes, mononuclear cells and neutrophils express CD83, a surface protein apparently necessary for effective DC-mediated activation of naïve T-cells and T-helper cells, thymic T-cell maturation and the regulation of B-cell activation and homeostasis. Although a defined ligand of CD83 remains elusive, the multiple cellular subsets expressing CD83, as well as its numerous potential implications in immunological processes suggest that CD83 plays an important regulatory role in the mammalian immune system. Lately, nucleocytoplasmic translocation of CD83 mRNA was shown to be mediated by direct interaction between the shuttle protein HuR and a novel post-transcriptional regulatory element (PRE) located in the CD83 transcript's coding region. Interestingly, this interaction commits the CD83 mRNA to efficient nuclear export through the CRM1 protein translocation pathway. More recently, the cellular phosphoprotein and HuR ligand ANP32B (APRIL) was demonstrated to be directly involved in this intracellular transport process by linking the CD83 mRNA:HuR ribonucleoprotein (RNP) complex with the CRM1 export receptor. Casein kinase II regulates this process by phosphorylating ANP32B. Here, we identify another RNA binding protein, AUF1 (hnRNP D) that directly interacts with CD83 PRE. Unlike HuR:PRE binding, this interaction has no impact on intracellular trafficking of CD83 mRNA-containing complexes; but it does regulate translation of CD83 mRNA. Thus, our data shed more light on the complex process of post-transcriptional regulation of CD83 expression. Interfering with this process may provide a novel strategy for inhibiting CD83, and thereby cellular immune activation.

Differential effects of hnRNP D/AUF1 isoforms on HIV-1 gene expression.

Control of RNA processing plays a major role in HIV-1 gene expression. To explore the role of several hnRNP proteins in this process, we carried out a siRNA screen to examine the effect of depletion of hnRNPs A1, A2, D, H, I and K on HIV-1 gene expression. While loss of hnRNPs H, I or K had little effect, depletion of A1 and A2 increased expression of viral structural proteins. In contrast, reduced hnRNP D expression decreased synthesis of HIV-1 Gag and Env. Loss of hnRNP D induced no changes in viral RNA abundance but reduced the accumulation of HIV-1 unspliced and singly spliced RNAs in the cytoplasm. Subsequent analyses determined that hnRNP D underwent relocalization to the cytoplasm upon HIV-1 infection and was associated with Gag protein. Screening of the four isoforms of hnRNP D determined that, upon overexpression, they had differential effects on HIV-1 Gag expression, p45 and p42 isoforms increased viral Gag synthesis while p40 and p37 suppressed it. The differential effect of hnRNP D isoforms on HIV-1 expression suggests that their relative abundance could contribute to the permissiveness of cell types to replicate the virus, a hypothesis subsequently confirmed by selective depletion of p45 and p42.

AUF-1 and YB-1 are critical determinants of ß-globin mRNA expression in erythroid cells.

The normal accumulation of ß-globin protein in terminally differentiating erythroid cells is critically dependent on the high stability of its encoding mRNA. The molecular basis for this property, though, is incompletely understood. Factors that regulate ß-globin mRNA within the nucleus of early erythroid progenitors are unlikely to account for the constitutively high half-life of ß-globin mRNA in the cytoplasm of their anucleate erythroid progeny. We conducted in vitro protein-RNA binding analyses that identified a cytoplasm-restricted ß-globin messenger ribonucleoprotein (mRNP) complex in both cultured K562 cells and erythroid-differentiated human CD34(+) cells. This novel mRNP targets a specific guanine-rich pentanucleotide in a region of the ß-globin 3'untranslated region that has recently been implicated as a determinant of ß-globin mRNA stability. Subsequent affinity-enrichment analyses identified AUF-1 and YB-1, 2 cytoplasmic proteins with well-established roles in RNA biology, as trans-acting components of the mRNP. Factor-depletion studies conducted in vivo demonstrated the importance of the mRNP to normal steady-state levels of ß-globin mRNA in erythroid precursors. These data define a previously unrecognized mechanism for the posttranscriptional regulation of ß-globin mRNA during normal erythropoiesis, providing new therapeutic targets for disorders of ß-globin gene expression.

RNA-binding protein AUF1 regulates lipopolysaccharide-induced IL10 expression by activating IkappaB kinase complex in monocytes.

Exposure of monocytes and macrophages to endotoxin/lipopolysaccharide (LPS) from Gram-negative bacteria activates the NF-κB signaling pathway. At early times, this leads to their production of proinflammatory cytokines, but subsequently, they produce anti-inflammatory interleukin-10 (IL-10) to quell the immune response. LPS-mediated induction of IL10 gene expression requires the p40 isoform of the RNA-binding protein AUF1. As LPS exerts modest effects upon IL10 mRNA stability, we hypothesized that AUF1 controls the expression of signaling proteins. Indeed, knockdown of AUF1 impairs LPS-mediated p38 mitogen-activated protein kinase (MAPK) and NF-κB signaling, and the expression of an RNA interference-refractory p40(AUF1) cDNA restores both signaling pathways. To define the molecular mechanisms by which p40(AUF1) controls IL10 expression, we focused on the NF-κB pathway in search of AUF1-regulated targets. Here, we show that p40(AUF1) serves to maintain proper levels of the kinase TAK1 (transforming growth factor-ß-activated kinase), which phosphorylates the IKKß subunit within the IκB kinase complex to activate NF-κB-regulated genes. However, p40(AUF1) does not control the TAK1 mRNA levels but instead promotes the translation of the mRNA. Thus, p40(AUF1) regulates a critical node within the NF-κB signaling pathway to permit IL10 induction for the anti-inflammatory arm of an innate immune response.

Polyamines regulate the stability of JunD mRNA by modulating the competitive binding of its 3' untranslated region to HuR and AUF1.

Polyamines critically regulate all mammalian cell growth and proliferation by mechanisms such as the repression of growth-inhibitory proteins, including JunD. Decreasing the levels of cellular polyamines stabilizes JunD mRNA without affecting its transcription, but the exact mechanism whereby polyamines regulate JunD mRNA degradation has not been elucidated. RNA-binding proteins HuR and AUF1 associate with labile mRNAs bearing AU-rich elements located in the 3' untranslated regions (3'-UTRs) and modulate their stability. Here, we show that JunD mRNA is a target of HuR and AUF1 and that polyamines modulate JunD mRNA degradation by altering the competitive binding of HuR and AUF1 to the JunD 3'-UTR. The depletion of cellular polyamines enhanced HuR binding to JunD mRNA and decreased the levels of JunD transcript associated with AUF1, thus stabilizing JunD mRNA. The silencing of HuR increased AUF1 binding to the JunD mRNA, decreased the abundance of HuR-JunD mRNA complexes, rendered the JunD mRNA unstable, and prevented increases in JunD mRNA and protein in polyamine-deficient cells. Conversely, increasing the cellular polyamines repressed JunD mRNA interaction with HuR and enhanced its association with AUF1, resulting in an inhibition of JunD expression. These results indicate that polyamines modulate the stability of JunD mRNA in intestinal epithelial cells through HuR and AUF1 and provide new insight into the molecular functions of cellular polyamines.

AUF1 p42 isoform selectively controls both steady-state and PGE2-induced FGF9 mRNA decay.

Fibroblast growth factor 9 (FGF9) is an autocrine/paracrine growth factor that plays vital roles in many physiologic processes including embryonic development. Aberrant expression of FGF9 causes human diseases and thus it highlights the importance of controlling FGF9 expression; however, the mechanism responsible for regulation of FGF9 expression is largely unknown. Here, we show the crucial role of an AU-rich element (ARE) in FGF9 3'-untranslated region (UTR) on controlling FGF9 expression. Our data demonstrated that AUF1 binds to this ARE to regulate FGF9 mRNA stability. Overexpression of each isoform of AUF1 (p37, p40, p42 and p45) showed that only the p42 isoform reduced the steady-state FGF9 mRNA. Also, knockdown of p42(AUF1) prolonged the half-life of FGF9 mRNA. The induction of FGF9 mRNA in prostaglandin (PG) E(2)-treated human endometrial stromal cells was accompanied with declined cytoplasmic AUF1. Nevertheless, ablation of AUF1 led to sustained elevation of FGF9 expression in these cells. Our study demonstrated that p42(AUF1) regulates both steady-state and PGE(2)-induced FGF9 mRNA stability through ARE-mediated mRNA degradation. Since almost half of the FGF family members are ARE-containing genes, our findings also suggest that ARE-mediated mRNA decay is a common pathway to control FGFs expression, and it represents a novel RNA regulon to coordinate FGFs homeostasis in various physiological conditions.

The role of AUF1 in thyroid carcinoma progression.

AUF1/heterogeneous nuclear ribonucleoprotein D is an adenylate-uridylate-rich elements (AREs) -binding protein, which regulates the mRNA stability of many genes related to growth regulation, such as proto-oncogenes, growth factors, cytokines, and cell cycle-regulatory genes. Several studies demonstrated AUF1 involvement in the processes of apoptosis, tumorigenesis, and development by its interactions with ARE-bearing mRNAs. We report here that AUF1 may be involved in thyroid carcinoma progression. Investigations on thyroid tissues revealed that cytoplasmic expression of AUF1 in malignant tissues was increased when compared with benign thyroid tissues. In thyroid carcinoma cell lines, AUF1 was mostly detectable in the nucleus; however, in dividing cells, its increased production was also observed in the cytoplasm. We found AUF1 in complexes with ARE-bearing mRNAs, previously described to be crucial for proliferation and cell cycle of thyroid carcinoma. Total or exon-selective knockdown of AUF1 led to growth inhibition accompanied by induction of cell cycle inhibitors and decreased levels of cell cycle promoters. Our data demonstrate the existence of a complex network between AUF1 and mRNAs encoding proteins related to cell proliferation. AUF1 may control the balance between stabilizing and destabilizing effects, both of which are exerted on cell cycle machinery in thyroid carcinoma. Although we cannot exclude participation of other factors, thyroid carcinoma may recruit cytoplasmic AUF1 to disturb the stability of mRNAs encoding cyclin-dependent kinase inhibitors, leading to uncontrolled growth and progression of tumor cells. Thus, AUF1 may be considered as a new, additional marker for thyroid carcinoma.

Differential stability of thymidylate synthase 3'-untranslated region polymorphic variants regulated by AUF1.

A 6-nucleotide insertion (I)/deletion (D) polymorphism in the 3'-untranslated region of the thymidylate synthase gene was shown to influence mRNA stability, but the molecular basis of this effect has not been elucidated. Here, studies of both endogenous and ectopically expressed thymidylate synthase alleles revealed that the mRNA-binding, decay-promoting protein AUF1 has higher affinity for allele D mRNA. AUF1 overexpression preferentially suppressed D allele mRNA levels, whereas AUF1 silencing selectively elevated D allele mRNA levels. Our results illustrate the functional consequences of ribonucleoprotein associations involving a polymorphic RNA sequence and uncover a novel mechanism of action for non-coding RNA polymorphisms.

Roles of AUF1 isoforms, HuR and BRF1 in ARE-dependent mRNA turnover studied by RNA interference.

HT1080 cells stably expressing green fluorescent protein (GFP) linked to a 3' terminal AU-rich element (ARE) proved to be a convenient system to study the dynamics of mRNA stability, as changes in mRNA levels are reflected in increased or decreased fluorescence intensity. This study examined whether mRNA stability can be regulated by small interfering RNAs (siRNAs) targeted to AU-binding proteins (AUBPs), which in turn should reveal their intrinsic role as stabilizers or destabilizers of ARE-mRNAs. Indeed, siRNAs targeting HuR or BRF1 decreased or increased fluorescence, respectively. This effect was abolished if cells were treated with both siRNAs, thus indicating antagonistic control of ARE-mRNA stability. Unexpectedly, downregulation of all four AUF1 isoforms by targeting common exons did not affect fluorescence whereas selective downregulation of p40AUF1/p45AUF1 strongly increased fluorescence by stabilizing the GFP-ARE reporter mRNA. This observation was fully confirmed by the finding that only selective reduction of p40AUF1/p45AUF1 induced the production of GM-CSF, an endogenous target of AUF1. These data suggest that the relative levels of individual isoforms, rather than the absolute amount of AUF1, determine the net mRNA stability of ARE-containing transcripts, consistent with the differing ARE-binding capacities of the isoforms.