STAU1-mediated mRNA Decay Research Articles

Ghrelin alleviates 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y cells.

Ghrelin is a neuropeptide that has various physiological functions and has been demonstrated to be neuroprotective in a number of neurological disease models. However, the underlying mechanisms of ghrelin in Parkinson's disease remain largely unexplored. The current study aimed to study the effects of ghrelin in a 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease model and evaluate the potential underlying mechanisms. In the present study, we treated an SH-SY5Y cell model with 6-OHDA, and observed that pretreatment with different concentrations of ghrelin (1, 10, and 100 nM) for 30 minutes relieved the neurotoxic effects of 6-OHDA, as revealed by Cell Counting Kit-8 and Annexin V/propidium iodide (PI) apoptosis assays. Reverse transcription quantitative polymerase chain reaction and western blot assay results demonstrated that 6-OHDA treatment upregulated α-synuclein and lincRNA-p21 and downregulated TG-interacting factor 1 (TGIF1), which was predicted as a potential transcription regulator of the gene encoding α-synuclein (SNCA). Ghrelin pretreatment was able to reverse the trends caused by 6-OHDA. The Annexin V/PI apoptosis assay results revealed that inhibiting either α-synuclein or lincRNA-p21 expression with small interfering RNA (siRNA) relieved 6-OHDA-induced cell apoptosis. Furthermore, inhibiting lincRNA-p21 also partially upregulated TGIF1. By retrieving information from a bioinformatics database and performing both double luciferase and RNA immunoprecipitation assays, we found that lincRNA-p21 and TGIF1 were able to form a double-stranded RNA-binding protein Staufen homolog 1 (STAU1) binding site and further activate the STAU1-mediated mRNA decay pathway. In addition, TGIF1 was able to transcriptionally regulate α-synuclein expression by binding to the promoter of SNCA. The Annexin V/PI apoptosis assay results showed that either knockdown of TGIF1 or overexpression of lincRNA-p21 notably abolished the neuroprotective effects of ghrelin against 6-OHDA-induced neurotoxicity. Collectively, these findings suggest that ghrelin exerts neuroprotective effects against 6-OHDA-induced neurotoxicity via the lincRNA-p21/TGIF1/α-synuclein pathway.

IGF2BP2 stabilized FBXL19-AS1 regulates the blood-tumour barrier permeability by negatively regulating ZNF765 by STAU1-mediated mRNA decay

ABSTRACT Blood-tumour barrier (BTB) has been known to significantly attenuate the efficacy of chemotherapy for glioma. In this report, we identified that insulin-like grown factor 2 mRNA-binding protein 2 (IGF2BP2) was over-expressed in glioma microvessel and glioma endothelial cells (GECs). Knockdown of IGF2BP2 decreased the expression of lncRNA FBXL19-AS1 and tight junction-related proteins, thereby promoting BTB permeability. FBXL19-AS1 was over-expressed and more enriched in the cytoplasm of GECs. In addition, FBXL19-AS1 could bind to 3ʹ-UTR of ZNF765 mRNA and down-regulate ZNF765 mRNA expression through STAU1-mediated mRNA decay (SMD). The low expression of ZNF765 was discovered in GECs and verified to increase BTB permeability by inhibiting the promoter activities of tight junction-related proteins. Meanwhile, ZNF765 also inhibited the transcriptional activity of IGF2BP2, thereby forming a feedback loop in regulating the BTB permeability. Single or combined application of silenced IGF2BP2 and FBXL19-AS1 improved the delivery and antitumor efficiency of doxorubicin (DOX). In general, our study revealed the regulation mechanism of IGF2BP2/FBXL19-AS1/ZNF765 axis on BTB permeability, which may provide valuable insight into treatment strategy for glioma.

The PABPC5/HCG15/ZNF331 Feedback Loop Regulates Vasculogenic Mimicry of Glioma via STAU1-Mediated mRNA Decay

Glioma is the most common primary malignancy in the brain, and vasculogenic mimicry (VM) is one of the blood supply methods. Here we investigated the possibility that lncRNAs regulate the stability of transcription factors through the SMD pathway, which affects proliferation, migration, invasion, and the ability to form VMs in glioma. Expression of PABPC5, HCG15, and ZNF331 was detected by real-time qPCR or western blot in glioma. Cell Counting Kit-8, Transwell assays, and in vitro VM tube formation were used to investigate PABPC5, HCG15, and ZNF331 function in cell proliferation, migration, invasion, and VM, respectively. ChIP assays were used to ascertain the interaction betweenZNF331 and LAMC2 or PABPC5. PABPC5 and HCG15 were highly expressed in glioma cells. ZNF331 was lowly expressed. PABPC5 bound HCG15 to increase its stability. Knockdown HCG15 reduced the degradation of ZNF331 mRNA by the SMD pathway. ZNF331 inhibited transcription through binding to the promoter region of LAMC2 and PABPC5 and inhibited the ability to form VMs in glioma cells. The PABPC5/HCG15/ZNF331 feedback loop plays an important role in regulating VM formation in glioma and provides new targets for glioma treatment.

Staufen1 reads out structure and sequence features in ARF1 dsRNA for target recognition.

Staufen1 (STAU1) is a dsRNA binding protein mediating mRNA transport and localization, translational control and STAU1-mediated mRNA decay (SMD). The STAU1 binding site (SBS) within human ADP-ribosylation factor1 (ARF1) 3′UTR binds STAU1 and this downregulates ARF1 cytoplasmic mRNA levels by SMD. However, how STAU1 recognizes specific mRNA targets is still under debate. Our structure of the ARF1 SBS–STAU1 complex uncovers target recognition by STAU1. STAU1 dsRNA binding domain (dsRBD) 4 interacts with two pyrimidines and one purine from the minor groove side via helix α1, the β1–β2 loop anchors the dsRBD at the end of the dsRNA and lysines in helix α2 bind to the phosphodiester backbone from the major groove side. STAU1 dsRBD3 displays the same binding mode with specific recognition of one guanine base. Mutants disrupting minor groove recognition of ARF1 SBS affect in vitro binding and reduce SMD in vivo. Our data thus reveal how STAU1 recognizes minor groove features in dsRNA relevant for target selection.

Role of ANKHD1/LINC00346/ZNF655 Feedback Loop in Regulating the Glioma Angiogenesis via Staufen1-Mediated mRNA Decay

Accumulating evidence shows that long non-coding RNAs (lncRNAs) dysregulation plays a critical role in tumor angiogenesis. Glioma is characterized by angiogenesis. Herein, we investigated the expression and function of LINC00346 in the regulation of glioma angiogenesis. The present study first demonstrated that ANKHD1 was significantly increased in glioma-associated endothelial cells (GECs) and facilitated angiogenesis of GECs. LncRNA microarray analysis was conducted to identify the differentially expressed LINC00346 in GECs treated with sh-ANKHD1. We verified that LINC00346 was increased in GECs and then the mRNA microarray data indicated the differentially expressed ZNF655 in GECs treated with sh-LINC00346. Further we demonstrated that ZNF655 was reduced in GECs. Meanwhile, LINC00346 inhibition or ZNF655 overexpression impeded angiogenesis of GECs. Moreover, ANKHD1 targeted LINC00346 and enhanced LINC00346's stability. In addition, LINC00346 bound to ZNF655 mRNA through their Alu elements so that LINC00346 facilitated the degradation of ZNF655 mRNA via STAU1-mediated mRNA decay (SMD) mechanism. Futhermore, ZNF655 targeted the promoter region of ANKHD1 and formed an ANKHD1/LINC00346/ZNF655 feedback loop that regulated glioma angiogenesis. Finally, knockdown of ANKHD1 and LINC00346 combined with overexpression of ZNF655 resulted a significant decrease in new vessels and hemoglobin content in vivo. The results identified an ANKHD1/LINC00346/ZNF655 feedback loop in the regulation of glioma angiogenesis that may provide new targets and strategies for targeted therapy against glioma. Funding Statement: This work is supported by grants from the Natural Science Foundation of China (81672511 and 81573010), Liaoning Science and Technology Plan Project (No. 2017225020, 2015225007), Project of Key Laboratory of Neuro-oncology in Liaoning Province (112-2400017005), special developmental project guided by central government of Liaoning Province (No. 2017011553-301) and outstanding scientific fund of Shengjing hospital (No. 201304). Declaration of Interests: We declare that all the authors have no conflict of interest in relation to the work described. Ethics Approval Statement: Not required.

Kruppel-like factor 4-dependent Staufen1-mediated mRNA decay regulates cortical neurogenesis

Kruppel-like factor 4 (Klf4) is a zinc-finger-containing protein that plays a critical role in diverse cellular physiology. While most of these functions attribute to its role as a transcription factor, it is postulated that Klf4 may play a role other than transcriptional regulation. Here we demonstrate that Klf4 loss in neural progenitor cells (NPCs) leads to increased neurogenesis and reduced self-renewal in mice. In addition, Klf4 interacts with RNA-binding protein Staufen1 (Stau1) and RNA helicase Ddx5/17. They function together as a complex to maintain NPC self-renewal. We report that Klf4 promotes Stau1 recruitment to the 3′-untranslated region of neurogenesis-associated mRNAs, increasing Stau1-mediated mRNA decay (SMD) of these transcripts. Stau1 depletion abrogated SMD of target mRNAs and rescued neurogenesis defects in Klf4-overexpressing NPCs. Furthermore, Ddx5/17 knockdown significantly blocked Klf4-mediated mRNA degradation. Our results highlight a novel molecular mechanism underlying stability of neurogenesis-associated mRNAs controlled by the Klf4/Ddx5/17/Stau1 axis during mammalian corticogenesis.

Staufen1-mediated mRNA decay induces Requiem mRNA decay through binding of Staufen1 to the Requiem 3'UTR.

Requiem (REQ/DPF2) was originally identified as an apoptosis-inducing protein in mouse myeloid cells and belongs to the novel Krüppel-type zinc finger d4-protein family of proteins, which includes neuro-d4 (DPF1) and cer-d4 (DPF3). Interestingly, when a portion of the REQ messenger ribonucleic acid (mRNA) 3′ untranslated region (3′UTR), referred to as G8, was overexpressed in K562 cells, β-globin expression was induced, suggesting that the 3′UTR of REQ mRNA plays a physiological role. Here, we present evidence that the REQ mRNA 3′UTR, along with its trans-acting factor, Staufen1 (STAU1), is able to reduce the level of REQ mRNA via STAU1-mediated mRNA decay (SMD). By screening a complementary deoxyribonucleic acid (cDNA) expression library with an RNA–ligand binding assay, we identified STAU1 as an interactor of the REQ mRNA 3′UTR. Specifically, we provide evidence that STAU1 binds to putative 30-nucleotide stem–loop-structured RNA sequences within the G8 region, which we term the protein binding site core; this binding triggers the degradation of REQ mRNA and thus regulates translation. Furthermore, we demonstrate that siRNA-mediated silencing of either STAU1 or UPF1 increases the abundance of cellular REQ mRNA and, consequently, the REQ protein, indicating that REQ mRNA is a target of SMD.

MRNA–mRNA duplexes that autoelicit Staufen1-mediated mRNA decay

We report a new mechanism by which human mRNAs crosstalk: an Alu element in the 3'-untranslated region (3' UTR) of one mRNA can base-pair with a partially complementary Alu element in the 3' UTR of a different mRNA thereby creating a Staufen1 (STAU1)-binding site (SBS). STAU1 binding to a 3' UTR SBS was previously shown to trigger STAU1-mediated mRNA decay (SMD) by directly recruiting the ATP-dependent RNA helicase UPF1, which is also a key factor in the mechanistically related nonsense-mediated mRNA decay (NMD) pathway. In the case of a 3' UTR SBS created via mRNA–mRNA base-pairing, we show that SMD targets both mRNAs in the duplex provided that both mRNAs are translated. If only one mRNA is translated, then it alone is targeted for SMD. We demonstrate the importance of mRNA–mRNA-triggered SMD to the processes of cell migration and invasion.

Non-structural protein 1 of influenza viruses inhibits rapid mRNA degradation mediated by double-stranded RNA-binding protein, staufen1

Although non-structural protein 1 (NS1) of influenza viruses is not essential for virulence, this protein is involved in host–virus interactions, viral replication, and translation. In particular, NS1 is known to interact with the host protein, staufen1 (Stau1). This interaction is important for efficient viral replication. However, the underlying molecular mechanism by which NS1 influences the viral life cycle remains obscure. Here, we show using immunoprecipitation and artificial tethering that the N-terminus of NS1, NS1(1-73), interacts with Stau1, blocks the Stau1–Upf1 interaction, and consequently inhibits the efficiency of Stau1-mediated mRNA decay (SMD), but not nonsense-mediatedmRNA decay (NMD). The regulation of SMD efficiency by NS1 may contribute to building a more favorable cellular environment for viral replication. Structured summary of protein interactionsSTAU1-55physically interacts with UPF1 by anti tag-coimmunoprecipitation (View interaction)NS1physically interacts with STAU1-55 by anti tag-coimmunoprecipitation (View interaction)

Staufen1-Mediated mRNA Decay Functions in Adipogenesis

The double-stranded RNA binding protein Staufen1 (Stau1) is involved in diverse gene expression pathways. For Stau1-mediated mRNA decay (SMD) in mammals, Stau1 binds to the 3' untranslated region of target mRNA and recruits Upf1 to elicit rapid mRNA degradation. However, the events downstream of Upf1 recruitment and the biological importance of SMD remain unclear. Here we show that SMD involves PNRC2, decapping activity, and 5'-to-3' exonucleolytic activity. In particular, Upf1 serves as an adaptor protein for the association of PNRC2 and Stau1. During adipogenesis, Stau1 and PNRC2 increase in abundance, Upf1 becomes hyperphosphorylated, and consequently SMD efficiency is enhanced. Intriguingly, downregulation of SMD components attenuates adipogenesis in a way that is rescued by downregulation of an antiadipogenic factor, Krüppel-like factor 2 (KLF2), the mRNA of which is identified as a substrate of SMD. Our data thus identify a biological role for SMD in adipogenesis.

LncRNAs transactivate STAU1-mediated mRNA decay by duplexing with 3′ UTRs via Alu elements

Staufen1 (STAU1)-mediated mRNA decay (SMD) degrades translationally active mRNAs that bind the double-stranded (ds)RNA binding protein STAU1 within their 3'-untranslated regions (3'UTRs)1,2. Earlier studies defined the STAU1 binding site (SBS) within ADP ribosylation factor 1 (ARF1) mRNA as a 19-base-pair stem with a 100-nucleotide apex2. However, we were unable to identify comparable structures within the 3'UTRs of other SMD targets. Here we report that SBSs can be formed by imperfect base-pairing between an Alu element within the 3'UTR of an SMD target and another Alu element within a cytoplasmic and polyadenylated long noncoding RNA (lncRNA). Individual lncRNAs can downregulate a subset of SMD targets, and distinct lncRNAs can downregulate the same SMD target. These are previously unappreciated functions for ncRNAs and Alu elements3–5. Not all mRNAs that contain a 3'UTR Alu element are targeted for SMD despite the presence of a complementary lncRNA that targets other mRNAs for SMD. Most known trans-acting RNA effectors consist of fewer than 200 nucleotides and include snoRNAs and microRNAs. Our finding that STAU1 binding to mRNAs can be transactivated by lncRNAs uncovers an unexpected strategy used by cells to recruit proteins to mRNAs and mediate their decay. We name these lncRNAs “half(½)-sbsRNAs”.

Stau1 negatively regulates myogenic differentiation in C2C12 cells

Sequential expression of myogenic regulatory factors (MRFs) such as MyoD and myogenin drives myogenic differentiation. Besides transcriptional activation of MRFs, this process is also coordinated by post-transcriptional regulation; MyoD and myogenin mRNAs are stabilized by RNA-binding protein HuR. Stau1 is known to regulate mRNA stability in a complex with Upf1, which is termed Stau1-mediated mRNA decay (SMD). We describe here that Stau1 is involved in the regulation of myogenesis. We found that knockdown of Stau1 promotes myogenesis including the expression of a muscle-specific marker protein, myoglobin, in C2C12 myoblasts. MyoD induces myogenin expression in response to induction of myogenesis, which is a key step to start myogenesis. The level of MyoD protein was not affected when Stau1 was depleted by siRNA, whereas the levels of myogenin mRNA and protein were increased in Stau1-knockdown cells. Interestingly, myogenin promoter activity was also increased in Stau1-knockdown cells in the absence of induction of myogenesis. Furthermore, Stau1-knockdown cells spontaneously progressed myogenesis including the expression of muscle-specific protein. Although Stau1 is involved in mRNA decay together with Upf1, Upf1-knockdown did not affect progression of myogenesis. Our results suggest that Stau1 negatively regulates myogenesis in C2C12 myoblasts through a mechanism that is different from SMD.

Staufen1 regulates diverse classes of mammalian transcripts

It is currently unknown how extensively the double-stranded RNA-binding protein Staufen (Stau)1 is utilized by mammalian cells to regulate gene expression. To date, Stau1 binding to the 3'-untranslated region (3'-UTR) of ADP ribosylation factor (ARF)1 mRNA has been shown to target ARF1 mRNA for Stau1-mediated mRNA decay (SMD). ARF1 SMD depends on translation and recruitment of the nonsense-mediated mRNA decay factor Upf1 to the ARF1 3'-UTR by Stau1. Here, we demonstrate that Stau1 binds to a complex structure within the ARF1 3'-UTR. We also use microarrays to show that 1.1 and 1.0% of the 11 569 HeLa-cell transcripts that were analyzed are upregulated and downregulated, respectively, at least two-fold upon Stau1 depletion in three independently performed experiments. We localize the Stau1 binding site to the 3'-UTR of four mRNAs that we define as natural SMD targets. Additionally, we provide evidence that the efficiency of SMD increases during the differentiation of C2C12 myoblasts to myotubes. We propose that Stau1 influences the expression of a wide variety of physiologic transcripts and metabolic pathways.