mRNA Decay In Cells Research Articles

Fragile X-Related Protein 1 (FXR1) Promotes Trophoblast Migration at Early Pregnancy via Downregulation of GDF-15 Expression

Fragile X-related protein 1 (FXR1) is an RNA-binding protein that can regulate specific mRNA decay in cells. Our previous study showed that FXR1 expression was significantly decreased in trophoblasts from patients with unexplained recurrent spontaneous abortion (RSA); however, the role of FXR1 in trophoblast function during early placenta development has not been fully elucidated. In this study, we found that knockdown of FXR1 using siRNA effectively inhibited the migration of HTR-8 cells and extravillous trophoblast (EVT) outgrowth in an ex vivo extravillous explant culture model. Furthermore, through analysis of a panel of cytokines, we found that the GDF-15 protein was upregulated after knockdown of FXR1 in HTR-8/SVneo cells. This was further confirmed by western blotting and immunofluorescence in HTR-8/SVneo cells and an extravillous explant. Our data also showed that FXR1 expression was downregulated and GDF-15 was upregulated in chorionic villous tissues from RSA patients compared with those from healthy controls (HCs). Further, immunohistochemistry showed a strong expression of GDF-15 in chorionic villous tissue in the RSA group, which was mainly distributed in villous trophoblasts (CTBs) and syncytiotrophoblasts (STBs). Moreover, knockdown of GDF-15 enhanced the migration of HTR-8 cells, while overexpression of GDF-15 using plasmid or treatment with recombinant human GDF-15 protein inhibited trophoblast migration. Importantly, RNA-binding protein immunoprecipitation showed that FXR1 directly bound to the 3′-UTR of GDF-15 mRNA to promote GDF-15 mRNA decay. Together, our data provide new insight into the function of FXR1 in human placenta via regulation of GDF-15 expression in trophoblasts and suggest a possible pathological process involved in RSA.

Amlexanox Enhances Premature Termination Codon Read-Through in COL7A1 and Expression of Full Length Type VII Collagen: Potential Therapy for Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare monogenic blistering disorder caused by the lack of functional type VII collagen, leading to skin fragility and subsequent trauma-induced separation of the epidermis from the underlying dermis. A total of 46% of patients with RDEB harbor at least one premature termination codon (PTC) mutation in COL7A1, and previous studies have shown that aminoglycosides are ableto overcome RDEB PTC mutations by inducing "read-through" and incorporation of an amino acid at the PTCsite. However, aminoglycoside toxicity will likely prevent widespread clinical application. Here the FDA-approved drug amlexanox was tested for its ability to read-through PTC mutations in cells derived frompatients with RDEB. Eight of 12 different PTC alleles responded to treatment and produced full length protein, in some cases more than 50% relative to normal controls. Read-through type VII collagen was readily detectable in cell culture media and also localized to the dermal-epidermal junction in organotypic skin culture. Amlexanox increased COL7A1 transcript and the phosphorylation of UPF-1, an RNA helicase associated with nonsense-mediated mRNA decay, suggesting that amlexanox inhibits nonsense-mediated mRNA decay in cells from patients with RDEB that respond to read-through treatment. This preclinical study demonstrates the potential of repurposing amlexanox for the treatment of patients with RDEB harboring PTC mutation in COL7A1.

Quantitative analysis of CAPN3 transcripts in LGMD2A patients: Involvement of nonsense-mediated mRNA decay

Limb girdle muscular dystrophy type 2A (LGMD2A) is caused by single or small nucleotide changes widespread along the CAPN3 gene, which encodes the muscle-specific proteolytic enzyme calpain-3. About 356 unique allelic variants of CAPN3 have been identified to date. We performed analysis of the CAPN3 gene in LGMD2A patients at both the mRNA level using reverse transcription-PCR, and at the DNA level using PCR and denaturing high performance liquid chromatography. In four patients, we detected homozygous occurence of a missense mutation or an in-frame deletion at the mRNA level although the DNA was heterozygous for this mutation in conjuction with a frame-shift mutation. The relationship observed in 12 patients between the quantity of CAPN3 mRNA, determined using real-time PCR, and the genotype leads us to propose that CAPN3 mRNAs which contain frame-shift mutations are degraded by nonsense-mediated mRNA decay. Our results illustrate the importance of DNA analysis for reliable establishment of mutation status, and provide a new insight into the process of mRNA decay in cells of LGMD2A patients.

The chemotherapeutic agent VP16 increases the stability of HB-EGF mRNA by a mechanism involving the 3′-UTR

VP16 is a chemotherapeutic agent that introduces DNA damage. We demonstrate that cellular stress induced by VP16 in the human cervix cancer cell line HeLa increases the HB-EGF (heparin binding epidermal growth factor like growth factor) mRNA level dose dependently. Maximal induction (10-fold) was observed at 20–40 μM VP16. Increased HB-EGF peptide levels accompanied the increase in HB-EGF mRNA. We investigated the molecular mechanism involved in HB-EGF mRNA induction by VP16. Transcription was only slightly increased (60%) as determined by real-time PCR quantification of transcription from a reporter plasmid containing the HB-EGF promoter in front of the luciferase gene. In contrast, HB-EGF mRNA stability was increased significantly by VP16 as demonstrated by monitoring HB-EGF mRNA decay in cells treated with the transcriptional inhibitor actinomycin D. The 3′-UTR (3′-untranslated region) of HB-EGF was inserted at the 3′-end of LacZ mRNA. VP16 treatment of the cells caused a 5-fold increase in this chimeric mRNA, as compared to LacZ without this 3′-UTR. A 186 nucleotide region of HB-EGF contains five of the six AUUUA sequences found in the 1454 nucleotide 3′-UTR of HB-EGF and we demonstrate that this region caused an approximately 3-fold induction of LacZ mRNA when inserted at the 3′-end, as compared to LacZ without any insertion at the 3′-end, demonstrating that a significant proportion of the effect resides in this region. Induction of HB-EGF by VP16 has important implications as HB-EGF has been reported to prevent cell death, which might lower the efficacy of chemotherapy. We demonstrate that mRNA stability and in particular the HB-EGF 3′-UTR is involved in the HB-EGF mRNA induction.

Bcl-2 Protein Is Required for the Adenine/Uridine-rich Element (ARE)-dependent Degradation of Its Own Messenger

We have shown previously that the decay of human bcl-2 mRNA is mediated by an adenine/uridine-rich element (ARE) located in the 3'-untranslated region. Here, we have utilized a non-radioactive cell-free mRNA decay system to investigate the biochemical and functional mechanisms regulating the ARE-dependent degradation of bcl-2 mRNA. Using RNA substrates, mutants, and competitors, we found that decay is specific and ARE-dependent, although maximized by the ARE-flanking regions. In unfractionated extracts from different cell types and in whole cells, the relative enzymatic activity was related to the amount of Bcl-2 protein expressed by the cells at steady state. The degradation activity was lost upon Bcl-2 depletion and was reconstituted by adding recombinant Bcl-2. Ineffective extracts from cells that constitutively do not express Bcl-2 acquire full degradation activity by adding recombinant Bcl-2 protein. We conclude that Bcl-2 is necessary to activate the degradation complex on the relevant RNA target.

The c-fos transcript is targeted for rapid decay by two distinct mRNA degradation pathways.

Rapid degradation of c-fos proto-oncogene mRNA is crucial for transient c-fos gene expression. Experiments were performed to investigate the cellular mechanisms responsible for the extremely short half-life of human c-fos mRNA in growth-factor-stimulated fibroblasts. These experiments demonstrate the existence of two distinct cellular pathways for rapid c-fos mRNA degradation. Each of these pathways recognizes a different, functionally independent instability determinant within the c-fos transcript. One instability determinant, which is located within the c-fos 3'-untranslated region, is a 75-nucleotide AU-rich segment. Insertion of this element into beta-globin mRNA markedly reduces the half-life of that normally long-lived message. Nevertheless, specific deletion of the AU-rich element from c-fos mRNA has little effect on the transcript's cytoplasmic half-life due to the presence of the other c-fos instability determinant, which is located in the protein-coding segment of the c-fos message. Examination of mRNA decay in cells treated with transcription inhibitors indicates that one c-fos mRNA degradation pathway is dependent on RNA synthesis, whereas the other is not.