Alsudani, H. | Search bioRxiv

bioRxiv2020

The human Origin Recognition Complex is essential for pre-RC assembly, mitosis and maintenance of nuclear structure.

The Origin Recognition Complex (ORC) cooperates with CDC6, MCM2-7, and CDT1 to form pre- RC complexes at origins of DNA replication. Here we report tiling-sgRNA CRISPR screens that show that each subunit of ORC and CDC6 are essential in human cells. Using an auxin-inducible degradation system, stable cell lines were created that ablate ORC2 rapidly, revealing multiple cell division cycle phenotypes. The primary defect in the absence of ORC2 was cells encountering difficulty in initiating DNA replication or progressing through the cell division cycle due to reduced MCM2-7 loading onto chromatin in G1 phase. The nuclei of ORC2 deficient cells were also large, with decompacted heterochromatin. Some ORC2 deficient cells that completed DNA replication entered into, but never exited mitosis. ORC1 knockout cells also demonstrated extremely slow cell proliferation and abnormal cell and nuclear morphology. Thus, ORC proteins and CDC6 are indispensable for normal cellular proliferation and contribute to nuclear organization.

molecular biology↗

bioRxiv2020

MaTAR25 LncRNA Regulates the Tensin1 Gene to Impact Breast Cancer Progression

Misregulation of long non-coding RNA genes has been linked to a wide variety of cancer types. Here we report on Mammary Tumor Associated RNA 25 (MaTAR25), a nuclear enriched and chromatin associated lncRNA that plays a role in mammary tumor cell proliferation, migration, and invasion, both in vitro and in vivo. MaTAR25 functions by interacting with purine rich element binding protein B (PURB), and associating with a major downstream target gene Tensin 1 (Tns1) to regulate its expression in trans. Knockout of MaTAR25 results in down-regulation of Tns1 leading to a reorganization of the actin cytoskeleton, and a reduction of focal adhesions and microvilli. The human ortholog of MaTAR25, LINC01271, is upregulated with human breast cancer stage and metastasis. SIGNIFICANCELncRNAs have great potential to reveal new regulatory mechanisms of function as well as having exciting therapeutic capacity given their ease of being targeted by nucleic acid drugs. Our study of MaTAR25, and its human ortholog LINC01271, reveal an unexpected function of this lncRNA in breast cancer progression by regulating Tns1 gene expression, whose protein product is a critical component of focal adhesions linking signaling between the extracellular matrix and the actin cytoskeleton. We identified LINC01271 as the human ortholog of MaTAR25, and importantly, increased expression of LINC01271 is associated with poor patient prognosis and cancer metastasis. Our findings demonstrate that LINC01271 represents an exciting therapeutic target to alter breast cancer progression.

cancer biology↗