A nuclear function for an oncogenic microRNA as a modulator of snRNA and splicing

Background miRNAs are regulatory transcripts established as repressors of mRNA stability and translation that have been functionally implicated in carcinogenesis. miR-10b is one of the key onco-miRs associated with multiple forms of cancer. Malignant gliomas exhibit particularly striking dependence on miR-10b. However, despite the therapeutic potential of miR-10b targeting, this miRNA’s poorly investigated and largely unconventional properties hamper the clinical translation. Methods We utilized Covalent Ligation of Endogenous Argonaute-bound RNAs and their high-throughput RNA sequencing to identify miR-10b interactome and a combination of biochemical and imaging approaches for target validation. They included Crosslinking and RNA immunoprecipitation with spliceosomal proteins, a combination of miRNA FISH with protein immunofluorescence in glioma cells and patient-derived tumors, native Northern blotting, and the transcriptome-wide analysis of alternative splicing. Results We demonstrate that miR-10b binds to U6 snRNA, a core component of the spliceosomal machinery. We provide evidence of the direct binding between miR-10b and U6, in situ imaging of miR-10b and U6 co-localization in glioma cells and tumors, and biochemical co-isolation of miR-10b with the components of the spliceosome. We further demonstrate that miR-10b modulates U6 N-6-adenosine methylation and pseudouridylation, U6 binding to splicing factors SART3 and PRPF8, and regulates U6 stability, conformation, and levels. These effects on U6 result in global splicing alterations, exemplified by the altered ratio of the isoforms of a small GTPase CDC42, reduced overall CDC42 levels, and downstream CDC42 -mediated effects on cell viability. Conclusions We identified U6 snRNA, the key RNA component of the spliceosome, as the top miR-10b target in glioblastoma. We, therefore, present an unexpected intersection of the miRNA and splicing machineries and a new nuclear function for a major cancer-associated miRNA.

A nuclear function for an oncogenic microRNA as a modulator of snRNA and splicing

miRNAs are regulatory transcripts established as repressors of mRNA stability and translation. Here we demonstrate that a key onco-miRNA, miR-10b, binds to U6 snRNA, a core component of the spliceosomal machinery. We provide evidence of direct binding between miR-10b and U6, in situ visualizations of miR-10b and U6 co-localization in glioma cells and patient-derived tumor tissues, and biochemical co-isolation of miR-10b with the components of the spliceosome. We further demonstrate that miR-10b modulates U6 N-6-adenosine methylation and pseudouridylation, U6 binding to splicing factors SART3 and PRPF8, and regulates U6 stability, conformation, and levels. These effects on U6 result in splicing alterations, illustrated by the altered ratio of the isoforms of a small GTPase CDC42, reduced overall CDC42 levels, and downstream CDC42 -mediated effects on cell viability. We, therefore, present an unexpected intersection of the miRNA and splicing machineries and a new nuclear function for a major cancer-associated miRNA.

Nuclear cGAS: sequestration and beyond

The cyclic GMP-AMP (cGAMP) synthase (cGAS) has been identified as a cytosolic double stranded DNA sensor that plays a pivotal role in the type I interferon and inflammation responses via the STING-dependent signaling pathway. In the past several years, a growing body of evidence has revealed that cGAS is also localized in the nucleus where it is associated with distinct nuclear substructures such as nucleosomes, DNA replication forks, the double-stranded breaks, and centromeres, suggesting that cGAS may have other functions in addition to its role in DNA sensing. However, while the innate immune function of cGAS is well established, the non-canonical nuclear function of cGAS remains poorly understood. Here, we review our current understanding of the complex nature of nuclear cGAS and point to open questions on the novel roles and the mechanisms of action of this protein as a key regulator of cell nuclear function, beyond its well-established role in dsDNA sensing and innate immune response.

The mechanistic role of alpha-synuclein in the nucleus: impaired nuclear function caused by familial Parkinson’s disease SNCA mutations

Alpha-synuclein SNCA has been implicated in the etiology of Parkinson’s disease (PD); however, the normal function of alpha-synuclein protein and the pathway that mediates its pathogenic effect is yet to be discovered. We investigated the mechanistic role of SNCA in the nucleus utilizing isogenic human-induced pluripotent stem cells-derived neurons from PD patients with autosomal dominant mutations, A53T and SNCA-triplication, and their corresponding corrected lines by genome- and epigenome-editing. Comparisons of shape and integrity of the nuclear envelope and its resistance to stresses found that both mutations result in similar nuclear envelope perturbations that were reversed in the isogenic mutation-corrected cells. Further mechanistic studies showed that SNCA mutation has adverse effects on the nucleus by trapping Ras-related nuclear protein (RAN) and preventing it from transporting key nuclear proteins such as, DNMT3A, for maintaining normal nuclear function. For the first time, we proposed that α-syn interacts with RAN and normally functions in the nucleocytoplasmic transport while exerts its pathogenic effect by sequestering RAN. We suggest that defects in the nucleocytoplasmic transport components may be a general pathomechanistic driver of neurodegenerative diseases.

A single-cell method to map higher-order 3D genome organization in thousands of individual cells reveals structural heterogeneity in mouse ES cells

ABSTRACTIn eukaryotes, the nucleus is organized into a three dimensional structure consisting of both local interactions such as those between enhancers and promoters, and long-range higher-order structures such as nuclear bodies. This organization is central to many aspects of nuclear function, including DNA replication, transcription, and cell cycle progression. Nuclear structure intrinsically occurs within single cells; however, measuring such a broad spectrum of 3D DNA interactions on a genome-wide scale and at the single cell level has been a great challenge. To address this, we developed single-cell split-pool recognition of interactions by tag extension (scSPRITE), a new method that enables measurements of genome-wide maps of 3D DNA structure in thousands of individual nuclei. scSPRITE maximizes the number of DNA contacts detected per cell enabling high-resolution genome structure maps within each cells and is easy-to-use and cost-effective. scSPRITE accurately detects chromosome territories, active and inactive compartments, topologically associating domains (TADs), and higher-order structures within single cells. In addition, scSPRITE measures cell-to-cell heterogeneity in genome structure at different levels of resolution and shows that TADs are dynamic units of genome organization that can vary between different cells within a population. scSPRITE will improve our understanding of nuclear architecture and its relationship to nuclear function within an individual nucleus from complex cell types and tissues containing a diverse population of cells.

Specific Roles of HSP27 S15 Phosphorylation Augmenting the Nuclear Function of HER2 to Promote Trastuzumab Resistance

Trastuzumab (TZMB) is widely used as first line therapy for breast cancer (BC) patients overexpressing human epidermal growth factor receptor 2 (HER2). Despite its clinical benefits, many patients suffer from primary or secondary resistance to this drug within one year. As diverse molecular mechanisms occur contemporaneously during the resistance development, we focused on elucidating the role of heat shock protein 27 (HSP27) in TZMB-resistance, as this protein simultaneously regulates the function of diverse client molecules that are involved in the resistance mechanism. By extensively utilizing TZMB-refractory breast cancer cell lines transduced with diverse phosphovariants of HSP27, our study newly revealed that specific phosphorylation of HSP27 at S15 promoted its S78 phosphorylation and served as key mediator to promote direct interactions that increase the stability of HER2 and protein kinase B (AKT). This phosphorylation promoted nuclear translocation of HER2, enhancing the distinct nuclear function of HER2 that promoted AKT activation and cyclin D1 expression. Co-administration of TZMB and a functional inhibitor of HSP27, J2, significantly reduced the S15/78 phosphorylation of HSP27, which downregulated HER2 and its downstream signals, sensitizing TZMB-refractory cell, and JIMT1-xenograft mouse models to TZMB. Collectively, p-HSP27S15 could serve as a valuable predictive marker and also a therapeutic target for TZMB-resistance.