The Cajal body protein coilin is a regulator of the miR-210 hypoxamiR and influences MIR210HG alternative splicing.

Hypoxia is a severe stressor to cellular homeostasis. At the cellular level, low oxygen triggers the transcription of a variety of genes supporting cell survival and oxygen homeostasis mediated by transcription factors, such as hypoxia-inducible factors (HIFs). Among many determinants dictating cell responses to hypoxia and HIFs are microRNAs (miRNAs). Cajal bodies (CBs), subnuclear structures involved in ribonucleoprotein biogenesis, have been recently proven to contribute to miRNA processing and biogenesis but have not been studied under hypoxia. Here, we show, for the first time, a hypoxia-dependent increase in CB number in WI-38 primary fibroblasts, which normally have very few CBs. Additionally, the CB marker protein coilin is upregulated in hypoxic WI-38 cells. However, the hypoxic coilin upregulation was not seen in transformed cell lines. Furthermore, we found that coilin is needed for the hypoxic induction of a well-known hypoxia-induced miRNA (hypoxamiR), miR-210, as well as for the hypoxia-induced alternative splicing of the miR-210 host gene, MIR210HG. These findings provide a new link in the physiological understanding of coilin, CBs and miRNA dysregulation in hypoxic pathology.

Coilin as a regulator of NF-kB mediated inflammation in preeclampsia.

The nuclear factor-Kappa B (NF-κB) pathway is a crucial mediator of inflammatory signaling. Aberrant activation of NF-κB is associated with several disorders including preeclampsia (PE). Many regulators of the NF-κB pathway have been identified, including microRNAs (miRNAs). Specifically, miR-517-3p targets mRNA encoding TNFAIP3 Interacting Protein 1 (TNIP1), an inhibitor of NF-κB signaling. Activation of NF-κB increases production of the cytokine TNF superfamily member 15 (TNFSF15), leading to the upregulation of anti-angiogenic soluble vascular endothelial growth factor receptor 1 (sFlt-1). We have previously observed that Cajal bodies (CBs), subnuclear domains, are associated with the chromosome 19 miRNA gene cluster (C19MC), which encodes miR-517-3p. We have also found that coilin, the CB marker protein, is a positive regulator of miRNA biogenesis. Here we report that coilin is a regulator of miR-517-3p, sFlt-1, TNIP1, TNFSF15 and NF-κB activation, and this regulation is influenced by hypoxia. We also report that coilin and CBs are induced in the reduced uterine perfusion pressure (RUPP) rat model of PE. Collectively, the data presented here implicate coilin as a novel regulator of NF-κB activation and sFlt-1 upregulation.

Coilin enhances phosphorylation and stability of DGCR8 and promotes miRNA biogenesis.

MicroRNAs (miRNAs) are ∼22 nt small noncoding RNAs that control gene expression at the posttranscriptional level through translational inhibition and destabilization of their target mRNAs. The biogenesis of miRNAs involves a series of processing steps beginning with cropping of the primary miRNA transcript by the Microprocessor complex, which is composed of Drosha and DGCR8. Here we report a novel regulatory interaction between the Microprocessor components and coilin, the Cajal body (CB) marker protein. Coilin knockdown causes alterations in the level of primary and mature miRNAs, let-7a and miR-34a, and their miRNA targets, HMGA2 and Notch1, respectively. We also found that coilin knockdown affects the levels of DGCR8 and Drosha in cells with (HeLa) and without (WI-38) CBs. To further explore the role of coilin in miRNA biogenesis, we conducted a series of coimmunoprecipitation experiments using coilin and DGCR8 constructs, which revealed that coilin and DGCR8 can form a complex. Additionally, our results indicate that phosphorylation of DGCR8, which has been shown to increase protein stability, is impacted by coilin knockdown. Collectively, our results implicate coilin as a member of the regulatory network governing miRNA biogenesis.

Cisplatin and phenanthriplatin modulate long-noncoding RNA expression in A549 and IMR90 cells revealing regulation of microRNAs, Wnt/ß-catenin and TGF-ß signaling.

The monofunctional platinum(II) complex, phenanthriplatin, acts by blocking transcription, but its regulatory effects on long-noncoding RNAs (lncRNAs) have not been elucidated relative to traditional platinum-based chemotherapeutics, e.g., cisplatin. Here, we treated A549 non-small cell lung cancer and IMR90 lung fibroblast cells for 24 h with either cisplatin, phenanthriplatin or a solvent control, and then performed microarray analysis to identify regulated lncRNAs. RNA22 v2 microRNA software was subsequently used to identify microRNAs (miRNAs) that might be suppressed by the most regulated lncRNAs. We found that miR-25-5p, -30a-3p, -138-5p, -149-3p, -185-5p, -378j, -608, -650, -708-5p, -1253, -1254, -4458, and -4516, were predicted to target the cisplatin upregulated lncRNAs, IMMP2L-1, CBR3-1 and ATAD2B-5, and the phenanthriplatin downregulated lncRNAs, AGO2-1, COX7A1-2 and SLC26A3-1. Then, we used qRT-PCR to measure the expression of miR-25-5p, -378j, -4516 (A549) and miR-149-3p, -608, and -4458 (IMR90) to identify distinct signaling effects associated with cisplatin and phenanthriplatin. The signaling pathways associated with these miRNAs suggests that phenanthriplatin may modulate Wnt/ß-catenin and TGF-ß signaling through the MAPK/ERK and PTEN/AKT pathways differently than cisplatin. Further, as some of these miRNAs may be subject to dissimilar lncRNA targeting in A549 and IMR90 cells, the monofunctional complex may not cause toxicity in normal lung compared to cancer cells by acting through distinct lncRNA and miRNA networks.

Molecular determinants that govern scaRNA processing by Drosha/DGCR8.

The Cajal body (CB) is a subnuclear domain that participates in the biogenesis of many different types of ribonucleoproteins (RNPs), including small nuclear RNPs (snRNPs), small Cajal body-specific RNPs (scaRNPs) and telomerase. Most scaRNAs, the RNA component of scaRNPs, accumulate in CBs. However, there are three scaRNAs (scaRNA 2, 9, and 17) that are known to be processed into small, nucleolar-enriched fragments. Evidence suggests that these fragments are packaged into a new class of RNPs, called regulatory RNPs (regRNPs), and may modify small nucleolar RNP (snoRNP) activity, thus playing a role in rRNA modification. However, the mechanism by which these fragments are produced is unknown. Previous work has reported the involvement of Drosha and DGCR8 in the cleavage of primary-scaRNA9. Here, we expand on that knowledge by identifying sequence elements necessary for the efficient production of these RNA fragments and demonstrate that primary scaRNA 2 and 17 are also processed by the Drosha-DGCR8 complex. Collectively, our work establishes new factors in the scaRNP biogenesis pathway and adds to the ever-expanding list of noncanonical functions for the microprocessor complex.