circRNA N6-methyladenosine methylation in preeclampsia and the potential role of N6-methyladenosine-modified circPAPPA2 in trophoblast invasion

Here, we performed N6-methyladenosine (m6A) RNA sequencing to determine the circRNA m6A methylation changes in the placentas during the pathogenesis of preeclampsia (PE). We verified the expression of the circRNA circPAPPA2 using quantitative reverse transcription-PCR. An invasion assay was carried out to identify the role of circPAPPA2 in the development of PE. Mechanistically, we investigated the cause of the altered m6A modification of circPAPPA2 through overexpression and knockdown cell experiments, RNA immunoprecipitation, fluorescence in situ hybridization and RNA stability experiments. We found that increases in m6A-modified circRNAs are prevalent in PE placentas and that the main changes in methylation occur in the 3’UTR and near the start codon, implicating the involvement of these changes in PE development. We also found that the levels of circPAPPA2 are decreased but that m6A modification is augmented. Furthermore, we discovered that methyltransferase‑like 14 (METTL14) increases the level of circPAPPA2 m6A methylation and that insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) maintains circPAPPA2 stability. Decreases in IGF2BP3 levels lead to declines in circPAPPA2 levels. In summary, we provide a new vision and strategy for the study of PE pathology and report that placental circRNA m6A modification appears to be an important regulatory mechanism.

A genetically-encoded crosslinker screen identifies SERBP1 as a PKCε substrate influencing translation and cell division

The PKCε-regulated genome protective pathway provides transformed cells a failsafe to successfully complete mitosis. Despite the necessary role for Aurora B in this programme, it is unclear whether its requirement is sufficient or if other PKCε cell cycle targets are involved. To address this, we developed a trapping strategy using UV-photocrosslinkable amino acids encoded in the PKCε kinase domain. The validation of the mRNA binding protein SERBP1 as a PKCε substrate revealed a series of mitotic events controlled by the catalytic form of PKCε. PKCε represses protein translation, altering SERBP1 binding to the 40 S ribosomal subunit and promoting the assembly of ribonucleoprotein granules containing SERBP1, termed M-bodies. Independent of Aurora B, SERBP1 is shown to be necessary for chromosome segregation and successful cell division, correlating with M-body formation. This requirement for SERBP1 demonstrates that Aurora B acts in concert with translational regulation in the PKCε-controlled pathway exerting genome protection.

Regulation of Neuroendocrine Plasticity by the RNA-Binding Protein ZFP36L1

Some small cell lung cancers (SCLCs) are highly sensitive to inhibitors of the histone demethylase LSD1. LSD1 inhibitors are thought to induce their anti-proliferative effects by blocking neuroendocrine differentiation, but the mechanisms by which LSD1 controls the SCLC neuroendocrine phenotype are not well understood. To identify genes required for LSD1 inhibitor sensitivity in SCLC, we performed a positive selection genome-wide CRISPR/Cas9 loss of function screen and found that ZFP36L1, an mRNA-binding protein that destabilizes mRNAs, is required for LSD1 inhibitor sensitivity. LSD1 binds and represses ZFP36L1 and upon LSD1 inhibition, ZFP36L1 expression is restored, which is sufficient to block the SCLC neuroendocrine differentiation phenotype and induce a non-neuroendocrine inflammatory phenotype. Mechanistically, ZFP36L1 binds and destabilizes SOX2 and INSM1 mRNAs, two transcription factors that are required for SCLC neuroendocrine differentiation. This work identifies ZFP36L1 as an LSD1 target gene that controls the SCLC neuroendocrine phenotype and demonstrates that modulating mRNA stability of lineage transcription factors controls neuroendocrine to non-neuroendocrine plasticity.

m6A reader IGF2BP2-stabilized CASC9 accelerates glioblastoma aerobic glycolysis by enhancing HK2 mRNA stability

N6-methyladenosine (m6A) has been identified to exert critical roles in human cancer; however, the regulation of m6A modification on glioblastoma multiforme (GBM) and long non-coding RNA (lncRNA) CASC9 (cancer susceptibility 9) is still unclear. Firstly, MeRIP-Seq revealed the m6A profile in the GBM. Moreover, the m6A-related lncRNA CASC9 expression was significantly elevated in the GBM tissue and its ectopic high expression was associated with poor survival, acting as an independent prognostic factor for GBM patients. Functionally, the aerobic glycolysis was promoted in the CASC9 overexpression transfection, which was inhibited in CASC9 knockdown in GBM cells. Mechanistically, m6A reader IGF2BP2 (insulin-like growth factor 2 mRNA binding protein 2) could recognize the m6A site of CASC9 and enhance its stability, then CASC9 cooperated with IGF2BP2, forming an IGF2BP2/CASC9 complex, to increase the HK2 (Hexokinase 2) mRNA stability. Our findings reveal that CASC9/IGF2BP2/HK2 axis promotes the aerobic glycolysis of GBM.

IGF2BP3 expression associates with survival in triple negative breast cancer.

We mined published microarray data (1) to understand the most significant gene expression differences in the tumors of triple negative breast cancer patients based on survival following treatment: dead or alive. We observed significant transcriptome-wide differential expression of insulin-like growth factor 2 mRNA binding protein 3, encoded by IGF2BP3 when comparing the primary tumors of triple negative breast cancer patients dead or alive. Importantly, IGF2BP3 expression was correlated with post-progression survival in basal subtype breast cancer, a molecular subtype sharing significant overlap with triple negative breast cancer. IGF2BP3 may be of relevance as a biomarker or as a molecule of interest in understanding the etiology or progression of triple negative breast cancer.