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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.

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.

A natural hydrogen donor, i.e., a subfraction of the coker gas oil, was used in thermal upgrading of the Venezuela extra-heavy oil. The role of hydrogen donor was quantitatively studied by a combin...

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.

Summary 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., Graphical abstract, Highlights • Both miR-506 and its target CDK6 are highly co-expressed in lung cancer • HNRNPA2B1 facilitates miR-506-mediated CDK6 silence by switching structure in 3′UTR • HNRNPA2B1 also recruits the DHX9 to the 3′UTR of its targets • HNRNPA2B1 extensively regulates miRNAs-mediated gene silencing through binding to 3′UTR, Biological sciences; Biochemistry; Molecular biology; Cell biology

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.

Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P

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.

A new 2′-OH protecting group, {[2,2-dimethyl-2-(2-nitrophenyl)acetyl]oxy} methyl (DAM), has been successfully applied to RNA oligonucleotides synthesis with high coupling efficiency. The 2′-O-DAM method is fully compatible with reagents and conditions employed in automated solid phase synthesis of RNA. Moreover, it can be easily removed via reduction in near-neutral solution.

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.

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.

MicroRNA-449a (miR-449a) was significantly downregulated in 156 lung cancer tissues (p