investigation-transcriptional-role-runx1-intronic-silencer-crisprcas9

Investigation of the Transcriptional Role of a <em>RUNX1</em> Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...

The Chinese University of Hong Kong

YchF proteins are a group of mysterious but ubiquitous unconventional G-proteins found in all kingdoms of life except Archaea. Their functions have been documented in microorganisms, protozoa and human, but those of plant YchF homologues are largely unknown. Our group has previously shown that OsYchF1 and its interacting protein, OsGAP1, play opposite roles in plant defense responses. OsGAP1 was found to stimulate the GTPase/ATPase activities of OsYchF1 and regulate its subcellular localization. In this report, we demonstrate that both OsYchF1 and OsGAP1 are localized mainly in the cytosol under NaCl treatment. The ectopic expression of OsYchF1 in transgenic Arabidopsis thaliana leads to reduced tolerance towards salinity stress, while the ectopic expression of OsGAP1 has the opposite effect. Similar results were also obtained with the Arabidopsis homologues, AtYchF1 and AtGAP1, by using AtGAP1 overexpressors and underexpressors, as well as an AtYchF1-knockdown mutant. OsYchF1 and OsGAP1 also exhibit highly significant effects on salinity-induced oxidative stress tolerance. The expression of OsYchF1 suppresses the anti-oxidation enzymatic activities and increases lipid peroxidation in transgenic Arabidopsis, and leads to the accumulation of reactive oxygen species (ROS) in tobacco BY-2 cells, while the ectopic expression of OsGAP1 has the opposite effects in these two model systems. 

The unconventional P-loop NTPase OsYchF1 and its regulator OsGAP1 play opposite roles in salinity stress tolerance.

The C2 domain is one of the most diverse phospholipid-binding domains mediating cellular signaling. One group of C2-domain proteins are plant-specific and are characterized by their small sizes and simple structures. We have previously reported that a member of this group, OsGAP1, is able to alleviate salt stress and stimulate defense responses, and bind to both phospholipids and an unconventional G-protein, OsYchF1. Here we solved the crystal structure of OsGAP1 to a resolution of 1.63 Å. Using site-directed mutagenesis, we successfully differentiated between the clusters of surface residues that are required for binding to phospholipids versus OsYchF1, which, in turn, is critical for its role in stimulating defense responses. On the other hand, the ability to alleviate salt stress by OsGAP1 is dependent only on its ability to bind OsYchF1 and is independent of its phospholipid-binding activity. 

Site-directed Mutagenesis Shows the Significance of Interactions with Phospholipids and the G-protein OsYchF1 for the Physiological Functions of the Rice GTPase-activating Protein 1 (OsGAP1).

G proteins are involved in almost all aspects of the cellular regulatory pathways through their ability to bind and hydrolyze GTP. The YchF subfamily, interestingly, possesses the unique ability to bind both ATP and GTP, and is possibly an ancestral form of G proteins based on phylogenetic studies and is present in all kingdoms of life. However, the biological significance of such a relaxed ligand specificity has long eluded researchers. Here, we have elucidated the different conformational changes caused by the binding of a YchF homolog in rice (OsYchF1) to ATP versus GTP by X-ray crystallography. Furthermore, by comparing the 3D relationships of the ligand position and the various amino acid residues at the binding sites in the crystal structures of the apo-bound and ligand-bound versions, a mechanism for the protein's ability to bind both ligands is revealed. Mutation of the noncanonical G4 motif of the OsYchF1 to the canonical sequence for GTP specificity precludes the binding/hydrolysis of ATP and prevents OsYchF1 from functioning as a negative regulator of plant-defense responses, while retaining its ability to bind/hydrolyze GTP and its function as a negative regulator of abiotic stress responses, demonstrating the specific role of ATP-binding/hydrolysis in disease resistance. This discovery will have a significant impact on our understanding of the structure-function relationships of the YchF subfamily of G proteins in all kingdoms of life. 

ATP binding by the P-loop NTPase OsYchF1 (an unconventional G protein) contributes to biotic but not abiotic stress responses.

Platelet concentrates (PC) generally refers to a group of products that are prepared from autologous blood intended to enhance healing activities. PC therapy is now very popular in treating musculoskeletal injuries; however, inconsistent clinical results urge the need to understand the working mechanism of PC. It is generally believed that the platelet-derived bioactive factors are the active constituents, and their bioavailability in the vicinity of the lesion sites determines the treatment efficacies. Therefore, the composition, localisation, and duration of the action of PC would be key determinants. In this review, we discuss how different preparations and delivery methods of PC would affect the treatment outcomes with respect to clinical evidence about PC therapy for osteoarthritis, tendinopathies, rotator cuff tears, anterior cruciate ligament injuries, and bone fractures. This review can be used as a quick guide for the use of PC therapy and provide insights for the further optimisation of the therapy in the near future.

Yuk-Lin Yung

Blood Cancer Cytogenetics and Genomics Laboratory,
Department of Anatomical and Cellular Pathology,
Prince of Wales Hospital,
Blood Cancer Cytogenetics and Genomics Laboratory, Department of Anatomical and Cellular Pathology, Prince of Wales Hospital

The Chinese University of Hong Kong

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells