Staufen1 Represses the FOXA1-Regulated Transcriptome by Destabilizing FOXA1 mRNA in Colorectal Cancer Cells.

Transcription factors play key roles in development and disease by controlling gene expression. Forkhead box A1 (FOXA1), is a pioneer transcription factor essential for mouse development and functions as an oncogene in prostate and breast cancer. In colorectal cancer (CRC), FOXA1 is significantly downregulated and high FOXA1 expression is associated with better prognosis, suggesting potential tumor suppressive functions. We therefore investigated the regulation of FOXA1 expression in CRC, focusing on well-differentiated CRC cells, where FOXA1 is robustly expressed. Genome-wide RNA stability assays identified FOXA1 as an unstable mRNA in CRC cells. We validated FOXA1 mRNA instability in multiple CRC cell lines and in patient-derived CRC organoids, and found that the FOXA1 3'UTR confers instability to the FOXA1 transcript. RNA pulldowns and mass spectrometry identified Staufen1 (STAU1) as a potential regulator of FOXA1 mRNA. Indeed, STAU1 knockdown resulted in increased FOXA1 mRNA and protein expression due to increased FOXA1 mRNA stability. Consistent with these data, RNA-seq following STAU1 knockdown in CRC cells revealed that FOXA1 targets were upregulated upon STAU1 knockdown. Collectively, this study uncovers a molecular mechanism by which FOXA1 is regulated in CRC cells and provides insights into our understanding of the complex mechanisms of gene regulation in cancer.

Benzomorphan and non-benzomorphan agonists differentially alter sigma-1 receptor quaternary structure, as does types of cellular stress.

Sigma-1 receptor (S1R) is a calcium-sensitive, ligand-operated receptor chaperone present on the endoplasmic reticulum (ER) membrane. S1R plays an important role in ER-mitochondrial inter-organelle calcium signaling and cell survival. S1R and its agonists confer resilience against various neurodegenerative diseases; however, the molecular mechanism of S1R is not yet fully understood. At resting state, S1R is either in a monomeric or oligomeric state but the ratio of these concentrations seems to change upon activation of S1R. S1R is activated by either cellular stress, such as ER-calcium depletion, or ligands. While the effect of ligands on S1R quaternary structure remains unclear, the effect of cellular stress has not been studied. In this study we utilize cellular and an in-vivo model to study changes in quaternary structure of S1R upon activation. We incubated cells with cellular stressors (H2O2 and thapsigargin) or exogenous ligands, then quantified monomeric and oligomeric forms. We observed that benzomorphan-based S1R agonists induce monomerization of S1R and decrease oligomerization, which was confirmed in the liver tissue of mice injected with (+)-Pentazocine. Antagonists block this effect but do not induce any changes when used alone. Oxidative stress (H2O2) increases the monomeric/oligomeric S1R ratio whereas ER calcium depletion (thapsigargin) has no effect. We also analyzed the oligomerization ability of various truncated S1R fragments and identified the fragments favorizing oligomerization. In this publication we demonstrate that quaternary structural changes differ according to the mechanism of S1R activation. Therefore, we offer a novel perspective on S1R activation as a nuanced phenomenon dependent on the type of stimulus.

Noncoding RNA circuitry in melanoma onset, plasticity, and therapeutic response.

Melanoma, the cancer of the melanocyte, is the deadliest form of skin cancer with an aggressive nature, propensity to metastasize and tendency to resist therapeutic intervention. Studies have identified that the re-emergence of developmental pathways in melanoma contributes to melanoma onset, plasticity, and therapeutic response. Notably, it is well known that noncoding RNAs play a critical role in the development and stress response of tissues. In this review, we focus on the noncoding RNAs, including microRNAs, long non-coding RNAs, circular RNAs, and other small RNAs, for their functions in developmental mechanisms and plasticity, which drive onset, progression, therapeutic response and resistance in melanoma. Going forward, elucidation of noncoding RNA-mediated mechanisms may provide insights that accelerate development of novel melanoma therapies.

Immunohistochemical study of MMP-2, MMP-9, EGFR and IL-8 in decidual and trophoblastic specimens of recurrent pregnancy loss cases.

BACKGROUND AND AIM: Unexplained recurrent pregnancy loss has been a a challenging research task to experts since there is no explicit pathophysiological mechanism and therefore, the treatment remains elusive. Immunological imbalance and morphological abnormalities are under investigation. This study aims to evaluate the implication of MMP-2, MMP-9, EGFR, and IL-8 in recurrent pregnancy loss cases. MATERIALS & METHODS: The study was carried out through comparison among two groups; the unexplained miscarriage group which consisted of 22 women, and the control group consisted of 18 women, who had electively terminated their pregnancies. Both groups were in the first trimester of gestation. The specimens included the trophoblast, decidua basalis, and decidua parietalis. The study was conducted via immunohistochemical methods. Antibodies were used against MMP-2, MMP-9, EGFR, and IL-8. The results were presented at a contingency table and were statistically analyzed with the Chi-Square Test (X2). RESULTS: There were remarkable disparities in some cases in the comparison of the two groups. MMP-9 was detected significantly high in recurrent pregnancy loss (RPL) cases, both on trophoblastic and decidual specimens (p-value < .00001), MMP-2 displayed no difference among the two groups (mild to moderate detection on trophoblast and almost negative on decidual tissues). EGFR was highly detected in trophoblastic tissue (p-value = .014). IL-8 detection was particularly different in both trophoblast and decidua parietalis of the two groups (p-value < .01). CONCLUSION: The study revealed both morphological and immunological dysregulations that might participate in the RPL pathogenesis.

Matrin3 regulates mitotic spindle dynamics by controlling alternative splicing of CDC14B.

Matrin3 is an RNA-binding protein that regulates diverse RNA-related processes, including mRNA splicing. Although Matrin3 has been intensively studied in neurodegenerative diseases, its function in cancer remains unclear. Here, we report Matrin3-mediated regulation of mitotic spindle dynamics in colorectal cancer (CRC) cells. We comprehensively identified RNAs bound and regulated by Matrin3 in CRC cells and focused on CDC14B, one of the top Matrin3 targets. Matrin3 knockdown results in increased inclusion of an exon containing a premature termination codon in the CDC14B transcript and simultaneous down-regulation of the standard CDC14B transcript. Knockdown of CDC14B phenocopies the defects in mitotic spindle dynamics upon Matrin3 knockdown, and the elongated and misoriented mitotic spindle observed upon Matrin3 knockdown are rescued upon overexpression of CDC14B, suggesting that CDC14B is a key downstream effector of Matrin3. Collectively, these data reveal a role for the Matrin3/CDC14B axis in control of mitotic spindle dynamics.

Context-Dependent Function of Long Noncoding RNA PURPL in Transcriptome Regulation during p53 Activation.

PURPL is a p53-induced lncRNA that suppresses basal p53 levels. Here, we investigated PURPL upon p53 activation in liver cancer cells, where it is expressed at significantly higher levels than other cell types. Using isoform sequencing, we discovered novel PURPL transcripts that have a retained intron and/or previously unannotated exons. To determine PURPL function upon p53 activation, we performed transcriptome sequencing (RNA-Seq) after depleting PURPL using CRISPR interference (CRISPRi), followed by Nutlin treatment to induce p53. Strikingly, although loss of PURPL in untreated cells altered the expression of only 7 genes, loss of PURPL resulted in altered expression of ~800 genes upon p53 activation, revealing a context-dependent function of PURPL. Pathway analysis suggested that PURPL is important for fine-tuning the expression of specific genes required for mitosis. Consistent with these results, we observed a significant decrease in the percentage of mitotic cells upon PURPL depletion. Collectively, these data identify novel transcripts from the PURPL locus and suggest that PURPL delicately moderates the expression of mitotic genes in the context of p53 activation to control cell cycle arrest.

HNRNPH1 destabilizes the G-quadruplex structures formed by G-rich RNA sequences that regulate the alternative splicing of an oncogenic fusion transcript.

In the presence of physiological monovalent cations, thousands of RNA G-rich sequences can form parallel G-quadruplexes (G4s) unless RNA-binding proteins inhibit, destabilize, or resolve the formation of such secondary RNA structures. Here, we have used a disease-relevant model system to investigate the biophysical properties of the RNA-binding protein HNRNPH1's interaction with G-rich sequences. We demonstrate the importance of two EWSR1-exon 8 G-rich regions in mediating the exclusion of this exon from the oncogenic EWS-FLI1 transcripts expressed in a subset of Ewing sarcomas, using complementary analysis of tumor data, long-read sequencing, and minigene studies. We determined that HNRNPH1 binds the EWSR1-exon 8 G-rich sequences with low nM affinities irrespective of whether in a non-G4 or G4 state but exhibits different kinetics depending on RNA structure. Specifically, HNRNPH1 associates and dissociates from G4-folded RNA faster than the identical sequences in a non-G4 state. Importantly, we demonstrate using gel shift and spectroscopic assays that HNRNPH1, particularly the qRRM1-qRRM2 domains, destabilizes the G4s formed by the EWSR1-exon 8 G-rich sequences in a non-catalytic fashion. Our results indicate that HNRNPH1's binding of G-rich sequences favors the accumulation of RNA in a non-G4 state and that this contributes to its regulation of RNA processing.

An Evolutionarily Conserved AU-Rich Element in the 3' Untranslated Region of a Transcript Misannotated as a Long Noncoding RNA Regulates RNA Stability.

One of the primary mechanisms of post-transcriptional gene regulation is the modulation of RNA stability. We recently discovered that LINC00675, a transcript annotated as a long noncoding RNA (lncRNA), is transcriptionally regulated by FOXA1 and encodes a highly conserved small protein that localizes to the endoplasmic reticulum, hence renamed as FORCP (FOXA1-regulated conserved small protein). Here, we show that the endogenous FORCP transcript is rapidly degraded and rendered unstable as a result of 3'UTR-mediated degradation. Surprisingly, although the FORCP transcript is a canonical nonsense-mediated decay (NMD) and microRNA (miRNA) target, we found that it is not degraded by NMD or miRNAs. Targeted deletion of an evolutionarily conserved region in the FORCP 3'UTR using CRISPR/Cas9 significantly increased the stability of the FORCP transcript. Interestingly, this region requires the presence of an immediate downstream 55-nt-long sequence for transcript stability regulation. Functionally, colorectal cancer cells lacking this conserved region expressed from the endogenous FORCP locus displayed decreased proliferation and clonogenicity. These data demonstrate that the FORCP transcript is destabilized via conserved elements within its 3'UTR and emphasize the need to interrogate the function of a given 3'UTR in its native context.

Significance of lncRNA abundance to function.

Long noncoding RNAs (lncRNAs) have emerged as regulators of diverse cellular processes. Although the vast majority of lncRNAs are expressed at lower levels compared to messenger RNAs (mRNAs), many lncRNAs play a central role in the regulation of cellular homeostasis and gene expression. With the advancement of next generation sequencing technologies, recent studies illustrate the diversity of lncRNA function. This diversity can be due to differences in their mechanisms of action, spatio-temporal expression, and/or abundance, all of which can vary depending on the particular cell type or tissue. Here, we discuss how the abundance of lncRNAs is an important feature that is often linked to their functions, and why it is crucial to quantitate lncRNA abundance, its local concentration within a cell or a tissue or the dynamic changes in expression levels during cell cycle progression or upon environmental stimuli, to shed light on their physiological roles.

Inflammatory myoglandular polyp as a cause of recurrent abdominal pain in a patient with quiescent Crohn's disease.

Chronic inflammation of the intestinal mucosa has been associated with the appearance of inflammatory polyps or pseudopolyps. Among the distinct categories of inflammatory polyps are inflammatory myoglandular polyps (IMGP) usually found in the colorectum. Only one case of IMGP in the terminal ileus has been described since their first description. We report the first case of an inflammatory polyp with both hyperplastic and myoglandular histological characteristics, in the terminal ileum of a patient with quiescent Crohn's disease, causing recurrent intussusception.

AUF1 ligand circPCNX reduces cell proliferation by competing with p21 mRNA to increase p21 production.

Mammalian circRNAs can influence different cellular processes by interacting with proteins and other nucleic acids. Here, we used ribonucleoprotein immunoprecipitation (RIP) analysis to identify systematically the circRNAs associated with the cancer-related protein AUF1. Among the circRNAs interacting with AUF1 in HeLa (human cervical carcinoma) cells, we focused on hsa_circ_0032434 (circPCNX), an abundant target of AUF1. Overexpression of circPCNX specifically interfered with the binding of AUF1 to p21 (CDKN1A) mRNA, thereby promoting p21 mRNA stability and elevating the production of p21, a major inhibitor of cell proliferation. Conversely, silencing circPCNX increased AUF1 binding to p21 mRNA, reducing p21 production and promoting cell division. Importantly, eliminating the AUF1-binding region of circPCNX abrogated the rise in p21 levels and rescued proliferation. Therefore, we propose that the interaction of circPCNX with AUF1 selectively prevents AUF1 binding to p21 mRNA, leading to enhanced p21 mRNA stability and p21 protein production, thereby suppressing cell growth.

The p53-induced RNA-binding protein ZMAT3 is a splicing regulator that inhibits the splicing of oncogenic CD44 variants in colorectal carcinoma.

p53 is an intensely studied tumor-suppressive transcription factor. Recent studies suggest that the RNA-binding protein (RBP) ZMAT3 is important in mediating the tumor-suppressive effects of p53. Here, we globally identify ZMAT3-regulated RNAs and their binding sites at nucleotide resolution in intact colorectal cancer (CRC) cells. ZMAT3 binds to thousands of mRNA precursors, mainly at intronic uridine-rich sequences and affects their splicing. The strongest alternatively spliced ZMAT3 target was CD44, a cell adhesion gene and stem cell marker that controls tumorigenesis. Silencing ZMAT3 increased inclusion of CD44 variant exons, resulting in significant up-regulation of oncogenic CD44 isoforms (CD44v) and increased CRC cell growth that was rescued by concurrent knockdown of CD44v Silencing p53 phenocopied the loss of ZMAT3 with respect to CD44 alternative splicing, suggesting that ZMAT3-mediated regulation of CD44 splicing is vital for p53 function. Collectively, our findings uncover a p53-ZMAT3-CD44 axis in growth suppression in CRC cells.

A small protein encoded by a putative lncRNA regulates apoptosis and tumorigenicity in human colorectal cancer cells.

Long noncoding RNAs (lncRNAs) are often associated with polysomes, indicating coding potential. However, only a handful of endogenous proteins encoded by putative lncRNAs have been identified and assigned a function. Here, we report the discovery of a putative gastrointestinal-tract-specific lncRNA (LINC00675) that is regulated by the pioneer transcription factor FOXA1 and encodes a conserved small protein of 79 amino acids which we termed FORCP (FOXA1-Regulated Conserved Small Protein). FORCP transcript is undetectable in most cell types but is abundant in well-differentiated colorectal cancer (CRC) cells where it functions to inhibit proliferation, clonogenicity, and tumorigenesis. The epitope-tagged and endogenous FORCP protein predominantly localizes to the endoplasmic reticulum (ER). In response to ER stress, FORCP depletion results in decreased apoptosis. Our findings on the initial characterization of FORCP demonstrate that FORCP is a novel, conserved small protein encoded by a mis-annotated lncRNA that regulates apoptosis and tumorigenicity in well-differentiated CRC cells.

Genome-Wide Analysis of the FOXA1 Transcriptional Network Identifies Novel Protein-Coding and Long Noncoding RNA Targets in Colorectal Cancer Cells.

Differentiation status of tumors is correlated with metastatic potential and malignancy. FOXA1 (forkhead box A1) is a transcription factor known to regulate differentiation in certain tissues. Here, we investigate FOXA1 function in human colorectal cancer (CRC). We found that FOXA1 is robustly expressed in the normal human colon but significantly downregulated in colon adenocarcinoma. Applying FOXA1 chromatin immunoprecipitation coupled with deep sequencing and transcriptome analysis upon FOXA1 knockdown in well-differentiated CRC cells and FOXA1 overexpression in poorly differentiated CRC cells, we identified novel protein-coding and lncRNA genes regulated by FOXA1. Among the numerous novel FOXA1 targets we identified, we focused on CEACAM5, a tumor marker and facilitator of cell adhesion. We show that FOXA1 binds to a distal enhancer downstream of CEACAM5 and strongly activates its expression. Consistent with these data, we show that FOXA1 inhibits anoikis in CRC cells. Collectively, our results uncover novel protein-coding and noncoding targets of FOXA1 and suggest a vital role of FOXA1 in enhancing CEACAM5 expression and anoikis resistance in CRC cells.

A Circular RNA from the MDM2 Locus Controls Cell Cycle Progression by Suppressing p53 Levels.

Circular RNAs (circRNAs) are a class of noncoding RNAs produced by a noncanonical form of alternative splicing called back-splicing. To investigate a potential role of circRNAs in the p53 pathway, we analyzed RNA sequencing (RNA-seq) data from colorectal cancer cell lines (HCT116, RKO, and SW48) that were untreated or treated with a DNA-damaging agent. Surprisingly, unlike the strong p53-dependent induction of hundreds of p53-induced mRNAs upon DNA damage, only a few circRNAs were upregulated from p53-induced genes. circ-MDM2, an annotated circRNA from the MDM2 locus, was one of the handful of circRNAs that originated from a p53-induced gene. Given the central role of MDM2 in suppressing p53 protein levels and p53 activity, we investigated the function of circ-MDM2 Knocking down circ-MDM2 with small interfering RNAs (siRNAs) that targeted circ-MDM2 did not alter MDM2 mRNA or MDM2 protein levels but resulted in increased basal p53 levels and growth defects in vitro and in vivo Consistent with these results, transcriptome profiling showed increased expression of several direct p53 targets, reduced retinoblastoma protein (Rb) phosphorylation, and defects in G1-S progression upon silencing circ-MDM2 Our results on the initial characterization of circ-MDM2 identify a new player from the MDM2 locus that suppresses p53 levels and cell cycle progression.

Senolytic therapy alleviates Aß-associated oligodendrocyte progenitor cell senescence and cognitive deficits in an Alzheimer's disease model.

Neuritic plaques, a pathological hallmark in Alzheimer's disease (AD) brains, comprise extracellular aggregates of amyloid-beta (Aß) peptide and degenerating neurites that accumulate autolysosomes. We found that, in the brains of patients with AD and in AD mouse models, Aß plaque-associated Olig2- and NG2-expressing oligodendrocyte progenitor cells (OPCs), but not astrocytes, microglia, or oligodendrocytes, exhibit a senescence-like phenotype characterized by the upregulation of p21/CDKN1A, p16/INK4/CDKN2A proteins, and senescence-associated ß-galactosidase activity. Molecular interrogation of the Aß plaque environment revealed elevated levels of transcripts encoding proteins involved in OPC function, replicative senescence, and inflammation. Direct exposure of cultured OPCs to aggregating Aß triggered cell senescence. Senolytic treatment of AD mice selectively removed senescent cells from the plaque environment, reduced neuroinflammation, lessened Aß load, and ameliorated cognitive deficits. Our findings suggest a role for Aß-induced OPC cell senescence in neuroinflammation and cognitive deficits in AD, and a potential therapeutic benefit of senolytic treatments.

Loss of miR-451a enhances SPARC production during myogenesis.

MicroRNAs (miRNAs) are small noncoding RNAs that critically regulate gene expression. Their abundance and function have been linked to a range of physiologic and pathologic processes. In aged monkey muscle, miR-451a and miR-144-3p were far more abundant than in young monkey muscle. This observation led us to hypothesize that miR-451a and miR-144-3p may influence muscle homeostasis. To test if these conserved microRNAs were implicated in myogenesis, we investigated their function in the mouse myoblast line C2C12. The levels of both microRNAs declined with myogenesis; however, only overexpression of miR-451a, but not miR-144-3p, robustly impeded C2C12 differentiation, suggesting an inhibitory role for miR-451a in myogenesis. Further investigation of the regulatory influence of miR-451a identified as one of the major targets Sparc mRNA, which encodes a secreted protein acidic and rich in cysteine (SPARC) that functions in wound healing and cellular differentiation. In mouse myoblasts, miR-451a suppressed Sparc mRNA translation. Together, our findings indicate that miR-451a is downregulated in differentiated myoblasts and suggest that it decreases C2C12 differentiation at least in part by suppressing SPARC biosynthesis.

Senescence-Associated MicroRNAs.

Senescent cells arise as a consequence of cellular damage and can have either a detrimental or advantageous impact on tissues and organs depending on the specific cell type and metabolic state. As senescent cells accumulate in tissues with advancing age, they have been implicated in many age-related declines and diseases. The major facets of senescence include two pathways responsible for establishing and maintaining a senescence program, p53/CDKN1A(p21) and CDKN2A(p16)/RB, as well as the senescence-associated secretory phenotype. Numerous MicroRNAs influence senescence by modulating the abundance of key senescence regulatory proteins, generally by lowering the stability and/or translation of mRNAs that encode such factors. Accordingly, understanding the molecular mechanisms by which MicroRNAs influence senescence will enable diagnostic and therapeutic opportunities directed at senescent cells. Here, we review senescence-associated (SA)-MicroRNAs and discuss their implications in senescence-relevant pathologies.

High-purity circular RNA isolation method (RPAD) reveals vast collection of intronic circRNAs.

High-throughput RNA sequencing methods coupled with specialized bioinformatic analyses have recently uncovered tens of thousands of unique circular (circ)RNAs, but their complete sequences, genes of origin and functions are largely unknown. Given that circRNAs lack free ends and are thus relatively stable, their association with microRNAs (miRNAs) and RNA-binding proteins (RBPs) can influence gene expression programs. While exoribonuclease treatment is widely used to degrade linear RNAs and enrich circRNAs in RNA samples, it does not efficiently eliminate all linear RNAs. Here, we describe a novel method for the isolation of highly pure circRNA populations involving RNase R treatment followed by Polyadenylation and poly(A)+ RNA Depletion (RPAD), which removes linear RNA to near completion. High-throughput sequencing of RNA prepared using RPAD from human cervical carcinoma HeLa cells and mouse C2C12 myoblasts led to two surprising discoveries: (i) many exonic circRNA (EcircRNA) isoforms share an identical backsplice sequence but have different body sizes and sequences, and (ii) thousands of novel intronic circular RNAs (IcircRNAs) are expressed in cells. In sum, isolating high-purity circRNAs using the RPAD method can enable quantitative and qualitative analyses of circRNA types and sequence composition, paving the way for the elucidation of circRNA functions.