Biology subjects

Nasif, S.

Publications and source records attributed to Nasif, S..

4 recordsLinked to original sources

bioRxiv2025

UPF1 shuttles between nucleus and cytoplasm independently of its RNA binding and ATPase activities

The ATP-dependent RNA helicase Up-frameshift 1 (UPF1) is an essential protein in mammalian cells and a key factor in nonsense-mediated mRNA decay (NMD), a translation-dependent mRNA surveillance process. UPF1 is mainly cytoplasmic at steady state but accumulates in the nucleus after inhibiting CRM1-mediated nuclear export by Leptomycin B (LMB), indicating that UPF1 shuttles between the nucleus and the cytoplasm. Consistent with its dual localization, there is evidence for nuclear functions of UPF1, for instance in DNA replication, DNA damage response, and telomere maintenance. However, whether any of UPF1s biochemical activities are required for its nuclear-cytoplasmic shuttling remains unclear. To investigate this, we examined two UPF1 mutants: the well-described ATPase-deficient UPF1-DE (D636A/E637A) and a newly generated RNA-binding mutant UPF1-NKR (N524A/K547A/R843A). Biochemical assays confirmed that the UPF1-NKR mutant cannot bind RNA or hydrolyze ATP in vitro but retains interaction with UPF2, UPF3B, and SMG6. Overexpression of UPF1-NKR exerted a dominant-negative eaect on endogenous UPF1 and inhibited NMD. Subcellular localization studies revealed that UPF1-DE accumulates in cytoplasmic granules (P-bodies), even in the presence of LMB, whereas UPF1-NKR shuttles normally. This indicates that UPF1s shuttling does not require its RNA-binding or ATPase activities. Notably, the UPF1-DE.NKR double mutant restored nuclear-cytoplasmic shuttling and prevented accumulation in P-bodies, suggesting that the shuttling defect of UPF1-DE arises from its tight binding to RNA. Overall, our findings demonstrate that UPF1s shuttling is independent of its ATPase and RNA-binding activities, with RNA binding itself being a key determinant of its cytoplasmic retention.

molecular biology↗

bioRxiv2023

Inhibition of nonsense-mediated mRNA decay reduces the tumorigenicity of human fibrosarcoma cells

Nonsense-mediated mRNA decay (NMD) is a eukaryotic RNA degradation pathway that targets for degradation faulty mRNAs with premature termination codons as well as many physiological mRNAs encoding full-length proteins. Consequently, NMD functions in both, quality control and post-transcriptional regulation of gene expression, and it has been implicated in the modulation of cancer progression. To investigate the role of NMD in cancer, we knocked out SMG7 in the HT1080 human fibrosarcoma cell line. SMG7 is involved in deadenylation-coupled exonucleolytic mRNA decay, one of the two main degradation pathways in mammalian NMD. Genome-wide proteomic and transcriptomic analyses confirmed that NMD is severely compromised in these SMG7-knockout HT1080 cells. We compared the oncogenic properties between the parental, the SMG7-knockout, and a rescue cell line in which we re-introduced both isoforms of SMG7. In parallel, we tested the effect of a drug inhibiting the NMD factor SMG1 on the HT1080 cells to distinguish NMD-dependent effects from putative NMD-independent functions of SMG7. Using cell-based assays as well as a mouse xenograft tumor model, we show that the oncogenic properties of the parental HT1080 cells are severely compromised when NMD is inhibited. Molecular pathway analysis revealed a strong reduction of the matrix metalloprotease 9 (MMP9) gene expression in NMD-suppressed cells. Since MMP9 expression promotes cancer cell migration and invasion, metastasis and angiogenesis, its downregulation in NMD-suppressed cells explains, at least partially, their reduced tumorigenicity. Collectively, our findings emphasize the therapeutic potential of NMD inhibition for the treatment of certain types of cancer. SignificanceNonsense-mediated mRNA decay (NMD) is a eukaryotic RNA decay pathway with reported roles in regulating cellular stress responses, differentiation, and viral defense. NMD has also emerged as a modulator of cancer progression, however, the available evidence supports both, a tumor suppressor as well as a pro-tumorigenic role for NMD. We discovered that NMD inhibition results in impaired tumorigenicity in the HT1080 human fibrosarcoma cell line and uncovered a direct correlation between NMD activity and the expression levels the pro-tumorigenic gene MMP9. Restoring MMP9 expression in NMD-suppressed cells partially improved their oncogenic properties. These results show that the tumorigenicity of the HT1080 fibrosarcoma cells relies on NMD activity and highlights the potential use of NMD inhibition as a therapeutic approach.

molecular biology↗

bioRxiv2021

Dissecting the import and export pathways of the human RNA helicase UPF1

The RNA helicase UPF1 is best known for its key role in mRNA surveillance but has been implicated in additional cellular processes both in the nucleus and in the cytoplasm. In human cells, the vast majority of UPF1 resides in the cytoplasm and only small amounts can be detected in the nucleus at steady state. It was previously shown that its export from the nucleus to the cytoplasm is Crm1-dependent, yet neither the nuclear export signal (NES) nor the nuclear localization signal (NLS) has been identified. Here, we provide evidence for a noncanonical NLS in UPF1, map the NES to amino acids 89-105 and show that L103 and F105 are essential for UPF1s export to the cytoplasm. Examination of additional UPF1 mutants revealed that a functional helicase domain but not the association with RNA is crucial for the shuttling capacity of UPF1.

cell biology↗

bioRxiv2018

NKX2.1 is critical for melanocortin neuron identity, hypothalamic Pomc expression and body weight

Food intake is tightly regulated by a group of neurons present in the mediobasal hypothalamus which activate satiety by releasing Pomc-encoded melanocortins. Although the relevance of hypothalamic POMC neurons in the regulation of energy balance and body weight is well appreciated, little is known about the transcription factors that establish their cellular fate, terminal differentiation and phenotypic maintenance. Here, we report that the transcription factor Nkx2.1 activates hypothalamic Pomc expression from early development to adulthood by binding to conserved canonical NKX motifs present in the neuronal Pomc enhancers nPE1 and nPE2. Transgenic and mutant mouse studies showed that the NKX motifs present in nPE1 and nPE2 are essential for their transcriptional enhancer activity. Early inactivation of Nkx2.1 in the ventral hypothalamus prevented the onset of Pomc expression and selective Nkx2.1 ablation from POMC neurons impaired Pomc expression and increased body weight and adiposity. These results demonstrate that NKX2.1 is critical in the early establishment of arcuate melanocortin neurons and the regulation of Pomc expression and body weight in adulthood.

genetics↗