Redox-modulated SNX25 as a novel regulator of GPCR-G protein signaling from endosomes.

GPCR-G protein signaling from endosomes plays a crucial role in various physiological and pathological processes. However, the mechanism by which endosomal G protein signaling is terminated remains largely unknown. In this study, we aimed to investigate the regulatory mechanisms involved in terminating the signaling of Gα subunits from endosomes. Through structural analysis and cell-based assays, we have discovered that SNX25, a protein that targets endosomes via its PXA or PXC domain, interacts with regulator of G protein signaling (RGS) proteins (including RGS2, RGS4, RGS8, and RGS17) in a redox-regulated manner. The interaction between SNX25 and these RGS proteins enhances their GTPase-accelerating activity towards Gαi/q and their ability to bind GDP-bound (inactive form) Gαi/q. As a result, SNX25 recruits these RGS proteins to endosomes, leading to the termination of endosomal Gαi/q signaling. Furthermore, we have found that the SNX25/RGS complex also exerts a negative regulatory effect on Gαi/q signaling from the plasma membrane. This is achieved by recruiting Gαi/q to endosomes and preventing its activation on the plasma membrane. Our findings shed light on the previously unknown role of redox-modulated SNX25 in inhibiting Gαi/q signaling, thereby uncovering a novel mechanism for terminating Gαi/q signaling from endosomes. Importantly, this study expands our understanding of the regulation of GPCR-Gαi/q signaling beyond the plasma membrane.

HNRNPA2B1 as a trigger of RNA switch modulates the miRNA-mediated regulation of CDK6.

The functional inactivation of tumor suppressor microRNA (miRNA) is closely related to the tumorigenesis of cancer. There are instances where the miRNA and the corresponding target both exist in a cell, but the target gene silencing do not occur as expected. Herein, we found that both miR-506 and its target CDK6 are highly co-expressed in lung cancer cells. Sequence analyses suggested that a miR-506 binding site (1648-1654) and a cis-element (1785-1795) for binding by heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1) are evolutionarily conserved and forms a stem structure in the 3' untranslated region (3'UTR) of CDK6. Furthermore, HNRNPA2B1 can bind to the stem structure to denature it and recruit the RNA helicase DExH-box helicase 9 (DHX9) to the 3'UTR, which ultimately facilitates miRNAs-mediated CDK6 silencing. These results indicate that the cis-element of the 3'UTR of CDK6, where HNRNPA2B1 binds, serves as an RNA switch to regulate miRNAs' function in cancer cells.

Capture of the newly transcribed RNA interactome using click chemistry.

Application of synthetic nucleoside analogues to capture newly transcribed RNAs has unveiled key features of RNA metabolism. Whether this approach could be adapted to isolate the RNA-bound proteome (RNA interactome) was, however, unexplored. We have developed a new method (capture of the newly transcribed RNA interactome using click chemistry, or RICK) for the systematic identification of RNA-binding proteins based on the incorporation of 5-ethynyluridine into newly transcribed RNAs followed by UV cross-linking and click chemistry-mediated biotinylation. The RNA-protein adducts are then isolated by affinity capture using streptavidin-coated beads. Through high-throughput RNA sequencing and mass spectrometry, the RNAs and proteins can be elucidated globally. A typical RICK experimental procedure takes only 1 d, excluding the steps of cell preparation, 5-ethynyluridine labeling, validation (silver staining, western blotting, quantitative reverse-transcription PCR (qRT-PCR) or RNA sequencing (RNA-seq)) and proteomics. Major advantages of RICK are the capture of RNA-binding proteins interacting with any type of RNA and, particularly, the ability to discern between newly transcribed and steady-state RNAs through controlled labeling. Thanks to its versatility, RICK will facilitate the characterization of the total and newly transcribed RNA interactome in different cell types and conditions.

Molecular Basis for PI(3,5)P2 Recognition by SNX11, a Protein Involved in Lysosomal Degradation and Endosome Homeostasis Regulation.

Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) is an essential phosphoinositide required for endosome homeostasis and sorting for lysosomal degradation; however, the underlying mechanisms, especially in mammals, remain elusive or unexplored. Here we determined a structure of PI(3,5)P2 bound to Sorting Nexin 11 (SNX11) with an opened PPII-C loop. We also obtained an SNX11 structure with its PPII-C in "closed" form that serves as a potential PI3P-binding model. In addition, our results reveal that SNX11 can interact with the V1D subunit of vacuolar H+-ATPase (V-ATPase), which provides a link between PI(3,5)P2 and human V-ATPase and further evidence for their roles in the endosome homeostasis regulation. Lastly, a new apo-form structure of SNX11, combined with molecular dynamics (MD) studies, indicates that the α5 helix can unfold from the PX domain of SNX11 when targeting the membrane or interacting with its partner. Taken together, these findings identify a novel PI(3,5)P2 effector, which will shed light on the PIs recognizing mechanism and the understanding of the downstream sorting events triggered by different PI binding.

Concurrent binding to DNA and RNA facilitates the pluripotency reprogramming activity of Sox2.

Some transcription factors that specifically bind double-stranded DNA appear to also function as RNA-binding proteins. Here, we demonstrate that the transcription factor Sox2 is able to directly bind RNA in vitro as well as in mouse and human cells. Sox2 targets RNA via a 60-amino-acid RNA binding motif (RBM) positioned C-terminally of the DNA binding high mobility group (HMG) box. Sox2 can associate with RNA and DNA simultaneously to form ternary RNA/Sox2/DNA complexes. Deletion of the RBM does not affect selection of target genes but mitigates binding to pluripotency related transcripts, switches exon usage and impairs the reprogramming of somatic cells to a pluripotent state. Our findings designate Sox2 as a multi-functional factor that associates with RNA whilst binding to cognate DNA sequences, suggesting that it may co-transcriptionally regulate RNA metabolism during somatic cell reprogramming.

Capturing the interactome of newly transcribed RNA.

We combine the labeling of newly transcribed RNAs with 5-ethynyluridine with the characterization of bound proteins. This approach, named capture of the newly transcribed RNA interactome using click chemistry (RICK), systematically captures proteins bound to a wide range of RNAs, including nascent RNAs and traditionally neglected nonpolyadenylated RNAs. RICK has identified mitotic regulators amongst other novel RNA-binding proteins with preferential affinity for nonpolyadenylated RNAs, revealed a link between metabolic enzymes/factors and nascent RNAs, and expanded the known RNA-bound proteome of mouse embryonic stem cells. RICK will facilitate an in-depth interrogation of the total RNA-bound proteome in different cells and systems.

The p53-induced lincRNA-p21 derails somatic cell reprogramming by sustaining H3K9me3 and CpG methylation at pluripotency gene promoters.

Recent studies have boosted our understanding of long noncoding RNAs (lncRNAs) in numerous biological processes, but few have examined their roles in somatic cell reprogramming. Through expression profiling and functional screening, we have identified that the large intergenic noncoding RNA p21 (lincRNA-p21) impairs reprogramming. Notably, lincRNA-p21 is induced by p53 but does not promote apoptosis or cell senescence in reprogramming. Instead, lincRNA-p21 associates with the H3K9 methyltransferase SETDB1 and the maintenance DNA methyltransferase DNMT1, which is facilitated by the RNA-binding protein HNRNPK. Consequently, lincRNA-p21 prevents reprogramming by sustaining H3K9me3 and/or CpG methylation at pluripotency gene promoters. Our results provide insight into the role of lncRNAs in reprogramming and establish a novel link between p53 and heterochromatin regulation.

Tumor-suppressive microRNA-449a induces growth arrest and senescence by targeting E2F3 in human lung cancer cells.

MicroRNA-449a (miR-449a) was significantly downregulated in 156 lung cancer tissues (p<0.001). We found that the low expression of miR-449a was highly correlated with cancer recurrence and survival of lung cancer patients. The transient introduction of miR-449a caused cell cycle arrest and cell senescence in A549 and 95D cells. Further studies revealed that E2F3 was a direct target of miR-449a in lung cancer cells. miR-449a also suppressed tumor formation in vivo in nude mice. These results suggest that miR-449a plays an important role in lung cancer tumorigenesis and that miR-449a might predict cancer recurrence and survival of lung cancer patients.

MicroRNA-192 targeting retinoblastoma 1 inhibits cell proliferation and induces cell apoptosis in lung cancer cells.

microRNAs play an important roles in cell growth, differentiation, proliferation and apoptosis. They can function either as tumor suppressors or oncogenes. We found that the overexpression of miR-192 inhibited cell proliferation in A549, H460 and 95D cells, and inhibited tumorigenesis in a nude mouse model. Both caspase-7 and the PARP protein were activated by the overexpression of miR-192, thus suggesting that miR-192 induces cell apoptosis through the caspase pathway. Further studies showed that retinoblastoma 1 (RB1) is a direct target of miR-192. Over-expression of miR-192 decreased RB1 mRNA and protein levels and repressed RB1-3'-UTR reporter activity. Knockdown of RB1 using siRNA resulted in a similar cell morphology as that observed for overexpression of miR-192. Additionally, RB1-siRNA treatment inhibited cell proliferation and induced cell apoptosis in lung cancer cells. Analysis of miRNA expression in clinical samples showed that miR-192 is significantly downregulated in lung cancer tissues compared to adjacent non-cancerous lung tissues. In conclusion, our results demonstrate that miR-192 is a tumor suppressor that can target the RB1 gene to inhibit cell proliferation and induce cell apoptosis in lung cancer cells. Furthermore, miR-192 was expressed at low levels in lung cancer samples, indicating that it might be a promising therapeutic target for lung cancer treatment.

Gibberellin 20-oxidase promotes initiation and elongation of cotton fibers by regulating gibberellin synthesis.

Cotton is the leading natural fiber, and gibberellin (GA) is a phytohormone involved in the development of cotton fibers. However, it is largely unknown how the GA content in ovules and fibers is regulated and how the endogenous GA concentration affects fiber development. To address these questions, three GA 20-oxidase homologous genes (GhGA20ox1-3) were cloned and the endogenous bioactive GA content in developing ovules and fibers determined by liquid chromatography-electrospray ionization-mass spectrometry. Real-time reverse transcription PCR (RT-PCR) revealed that GhGA20ox1 expressed preferentially in elongating fibers and that the expression level varied with the endogenous GA content consistently, while GhGA20ox2 and GhGA20ox3 transcripts accumulated mainly in ovules. The GA accumulation kinetics as well as the GhGA20ox expression differed in ovules and the attached fibers, suggesting relatively independent GA regulation system in these two sites. Transgenic cotton, over-expressing GhGA20ox1, showed GA over-production phenotypes with increased endogenous GA levels (especially GA(4)) in fibers and ovules. It also produced significantly more fiber initials per ovule, and fiber lengths was increased compared with the control, which demonstrates that up-regulation of the GhGA20ox1 gene promoted fiber initiation and elongation. Our results suggest that GA 20-oxidase is involved in fiber development by regulating GA levels, and corresponding genes might be employed as target genes for the manipulation of fiber initiation and elongation in cotton.

Cotton flavonoid structural genes related to the pigmentation in brown fibers.

Five flavonoid structural genes, encoding chalcone isomerase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, anthocyanidin synthase, and anthocyanidin reductase, were cloned from a brown-fiber cotton line (T586). The predicted proteins of these genes exhibit high sequence similarity with corresponding enzymes from various plants. RT-PCR analysis showed these genes are developmentally co-regulated and preferentially expressed in developing fibers of T586. Expression analyses and dimethylaminocinnaldehyde staining demonstrated that high transcript levels of these genes in developing fibers and presence of proanthocyanidins in mature fibers co-segregated with brown fiber in a recombination inbred line population. Our results indicated that the cloned flavonoid structural genes and proanthocyanidins were involved in the pigmentation in brown cotton fibers.

Asymmetric overlap extension PCR method bypassing intermediate purification and the amplification of wild-type template in site-directed mutagenesis.

By combining asymmetric PCR and overlap extension, we developed a novel asymmetric overlap extension PCR (AOE-PCR) method for site-directed mutagenesis which bypassed the need for intermediate purification and excluded the amplification of a wild-type template. This method was used to introduce single base mutations into a small GTPase gene from cotton and to simultaneously introduce two mutations just by repeating this method using the first round AOE-PCR products as template. Our results suggested that the AOE-PCR method represents a valuable improvement of the original overlap extension PCR for site-directed mutagenesis.