Deciphering the Functional Long Non-Coding RNAs Derived from MicroRNA Loci.

Albeit the majority of eukaryotic genomes can be pervasively transcribed to a diverse population of lncRNAs and various subtypes of lncRNA are discovered. However, the genome-wide study of miRNA-derived lncRNAs is still lacking. Here, it is reported that over 800 miRNA gene-originated lncRNAs (molncRNAs) are generated from miRNA loci. One of them, molnc-301b from miR-301b and miR-130b, functions as an "RNA decoy" to facilitate dissociation of the chromatin remodeling protein SMARCA5 from chromatin and thereby sequester transcription and mRNA translation. Specifically, molnc-301b attenuates erythropoiesis by mitigating the transcription of erythropoietic and translation-associated genes, such as GATA1 and FOS. In addition, a useful and powerful CRISPR screen platform to characterize the biological functions of molncRNAs at large-scale and single-cell levels is established and 29 functional molncRNAs in hematopoietic cells are identified. Collectively, the focus is on miRNA-derived lncRNAs, deciphering their landscape during normal hematopoiesis, and comprehensively evaluating their potential roles.

LncReader: identification of dual functional long noncoding RNAs using a multi-head self-attention mechanism.

Long noncoding ribonucleic acids (RNAs; LncRNAs) endowed with both protein-coding and noncoding functions are referred to as 'dual functional lncRNAs'. Recently, dual functional lncRNAs have been intensively studied and identified as involved in various fundamental cellular processes. However, apart from time-consuming and cell-type-specific experiments, there is virtually no in silico method for predicting the identity of dual functional lncRNAs. Here, we developed a deep-learning model with a multi-head self-attention mechanism, LncReader, to identify dual functional lncRNAs. Our data demonstrated that LncReader showed multiple advantages compared to various classical machine learning methods using benchmark datasets from our previously reported cncRNAdb project. Moreover, to obtain independent in-house datasets for robust testing, mass spectrometry proteomics combined with RNA-seq and Ribo-seq were applied in four leukaemia cell lines, which further confirmed that LncReader achieved the best performance compared to other tools. Therefore, LncReader provides an accurate and practical tool that enables fast dual functional lncRNA identification.

METTL3 preferentially enhances non-m6A translation of epigenetic factors and promotes tumourigenesis.

METTL3 encodes the predominant catalytic enzyme to promote m6A methylation in nucleus. Recently, accumulating evidence has shown the expression of METTL3 in cytoplasm, but its function is not fully understood. Here we demonstrated an m6A-independent mechanism for METTL3 to promote tumour progression. In gastric cancer, METTL3 could not only facilitate cancer progression via m6A modification, but also bind to numerous non-m6A-modified mRNAs, suggesting an unexpected role of METTL3. Mechanistically, cytoplasm-anchored METTL3 interacted with PABPC1 to stabilize its association with cap-binding complex eIF4F, which preferentially promoted the translation of epigenetic factors without m6A modification. Clinical investigation showed that cytoplasmic distributed METTL3 was highly correlated with gastric cancer progression, and this finding could be expanded to prostate cancer. Therefore, the cytoplasmic METTL3 enhances the translation of epigenetic mRNAs, thus serving as an oncogenic driver in cancer progression, and METTL3 subcellular distribution can assist diagnosis and predict prognosis for patients with cancer.

A global screening identifies chromatin-enriched RNA-binding proteins and the transcriptional regulatory activity of QKI5 during monocytic differentiation.

BACKGROUND: Cellular RNA-binding proteins (RBPs) have multiple roles in post-transcriptional control, and some are shown to bind DNA. However, the global localization and the general chromatin-binding ability of RBPs are not well-characterized and remain undefined in hematopoietic cells. RESULTS: We first provide a full view of RBPs' distribution pattern in the nucleus and screen for chromatin-enriched RBPs (Che-RBPs) in different human cells. Subsequently, by generating ChIP-seq, CLIP-seq, and RNA-seq datasets and conducting combined analysis, the transcriptional regulatory potentials of certain hematopoietic Che-RBPs are predicted. From this analysis, quaking (QKI5) emerges as a potential transcriptional activator during monocytic differentiation. QKI5 is over-represented in gene promoter regions, independent of RNA or transcription factors. Furthermore, DNA-bound QKI5 activates the transcription of several critical monocytic differentiation-associated genes, including CXCL2, IL16, and PTPN6. Finally, we show that the differentiation-promoting activity of QKI5 is largely dependent on CXCL2, irrespective of its RNA-binding capacity. CONCLUSIONS: Our study indicates that Che-RBPs are versatile factors that orchestrate gene expression in different cellular contexts, and identifies QKI5, a classic RBP regulating RNA processing, as a novel transcriptional activator during monocytic differentiation.

A comprehensive RNA editome reveals that edited Azin1 partners with DDX1 to enable hematopoietic stem cell differentiation.

Adenosine-to-inosine RNA editing and the catalyzing enzyme adenosine deaminase are both essential for hematopoietic development and differentiation. However, the RNA editome during hematopoiesis and the underlying mechanisms are poorly defined. Here, we sorted 12 murine adult hematopoietic cell populations at different stages and identified 30 796 editing sites through RNA sequencing. The dynamic landscape of the RNA editome comprises stage- and group-specific and stable editing patterns, but undergoes significant changes during lineage commitment. Notably, we found that antizyme inhibitor 1 (Azin1) was highly edited in hematopoietic stem and progenitor cells (HSPCs). Azin1 editing results in an amino acid change to induce Azin1 protein (AZI) translocation to the nucleus, enhanced AZI binding affinity for DEAD box polypeptide 1 to alter the chromatin distribution of the latter, and altered expression of multiple hematopoietic regulators that ultimately promote HSPC differentiation. Our findings have delineated an essential role for Azin1 RNA editing in hematopoietic cells, and our data set is a valuable resource for studying RNA editing on a more general basis.

mRNA-Activated Multifunctional DNAzyme Nanotweezer for Intracellular mRNA Sensing and Gene Therapy.

Deoxyribozyme (DNAzyme) is regarded as a promising gene therapy drug. However, poor cellular uptake efficacy and low biological stability limit the utilization of DNAzyme in gene therapy. Here, we report a well-known programmable DNAzyme-based nanotweezer (DZNT) that provides a new strategy for the detection of TK1 mRNA and survivin mRNA-targeted gene silencing therapy. At the end of the DZNT arm, there are two functionalized single-stranded DNA and each consists of two parts: the segment complementary to TK1 mRNA and the split-DNAzyme segment. The hybridization with intracellular TK1 mRNA enables the imaging of TK1 mRNA. Meanwhile, the hybridization draws the split-DNAzyme close to each other and activates DNAzyme to cleave the survivin mRNA to realize gene silencing therapy. The results demonstrate that the DZNT nanocarrier has excellent cell penetration, good biocompatibility, and noncytotoxicity. DZNT can image intracellular biomolecule TK1 mRNA with a high contrast. Furthermore, the split-DNAzyme can efficiently cleave the survivin mRNA with the aid of TK1 mRNA commonly present in cancer cells, accordingly can selectively kill cancer cells, and has no harm to normal cells. Taken together, the multifunctional programmable DZNT provides a promising platform for the early diagnosis of tumors and gene therapy.

Biomineralized metal-organic framework nanoparticles enable a primer exchange reaction-based DNA machine to work in living cells for imaging and gene therapy.

Sensitive tumor imaging and precise tumor therapy play critical roles in the cancer combat. Herein, we build a DNA machine based on a primer exchange reaction (PER) for mRNA imaging and gene therapy. By using zeolitic imidazolate framework-8 nanoparticles (ZIF-8 NPs) to co-deliver the components including a primer, hairpin and strand displacing polymerase to the living cells, the PER-based DNA machine can be initiated by intracellular survivin mRNA and continuously produce Bcl-2 antisense DNA (ASD), which enables the DNA machine not only to image survivin mRNA but also to implement gene therapy. The results demonstrate that ZIF-8 NPs can protect the polymerases and nucleic acid probes from protease attack and nuclease degradation. After internalization, pH-responsive ZIF-8 NPs can efficiently release cargos from endo-lysosomes due to the protonation effect. The intracellular PER-based DNA machine has been demonstrated to be able to sensitively image survivin mRNA expression levels and selectively kill the cancer cells and has no effect on the normal cells. The PER-based DNA machine may provide a promising platform for early stage tumor diagnosis and more precise tumor therapy.

Engineering a Biodegradable Nanocarrier for Enhancing the Response of T98G Cells to Temozolomide.

Temozolomide (TMZ), the most common DNA alkylating agent, is predominantly mediated by O6-methylguanine DNA lesions for the treatment of glioblastoma (GBM). When O6-methylguanine-DNA methyltransferase (MGMT) is present, TMZ-induced O6-methylguanine lesions are repaired, resulting in the emergence of resistance to chemotherapy. Herein, we attempted to enhance the response of T98G cells to TMZ by gene silencing of MGMT. In this work, we developed transition metal manganese (Mn)-doped mesoporous silica nanoparticles (MSNs) as a carrier system for the co-delivery of TMZ and 10-23 DNAzyme, and realized gene silencing to enhance the TMZ sensitivity in T98G cells. The intelligent theranostic platform based on manganese-doped mesoporous silica nanoparticles (Mn-MSNs) can be decomposed and release chemotherapy drugs under acidic pH and reducing conditions. Meanwhile, the produced Mn2+ could act as a cofactor of 10-23 DNAzyme to effectively cleave MGMT mRNA, knock down MGMT protein, and sensitize T98G cells to TMZ-induced apoptosis. By co-delivering TMZ and 10-23 DNAzyme employing Mn-MSNs, the concentrations of TMZ that needed to inhibit cell growth by 50% (IC50 values) decreased (by more than 3.8-fold) compared with free TMZ. This work shows that the designed platform holds great promise for advancing the treatment of drug-resistant cancer.

A spatial-confinement hairpin cascade reaction-based DNA tetrahedral amplifier for mRNA imaging in live cells.

The three-dimensional (3D) DNA nanostructure has been got much attention due to its excellent biocompatibility, enhanced structural stability, highly programmable and perfect cell-delivery performance. Here, a novel 3D DNA tetrahedron amplifier (DTA) has been developed for rapid and efficient mRNA imaging in living cells using target catalyzing spatial-confinement hairpin DNA assembly cascade reaction inside the DNA nanostructure. The DTA was constructed by assembling a DNA tetrahedron with four DNA strands at first, and then by assembling two metastable DNA hairpins H1 (Cy5) and H2 (Cy3) at specific locations of the DNA tetrahedron. In the presence of target mRNA, the catalyzed hairpin assembly (CHA) reaction on the DTA could be triggered and a H1-H2 duplexes nanostructure could be formed, which would obtain a significant fluorescence resonance energy transfer (FRET) signal, and release the target mRNA could trigger next H1-H2 duplexes formation. Due to the 3D DNA tetrahedral spatial-confinement effect, the circular reaction of DTA could achieve rapid and efficient amplification detection of target mRNA in living cells. Moreover, the DTA show excellent structural stability and non-cytotoxicity. This strategy presents a versatile method for the ultrasensitive detection of biomarkers in living system and gains a deeper development of the DNA nanostructures in biomedical functions.

An enzyme-initiated DNAzyme motor for RNase H activity imaging in living cell.

In this work, we have developed a novel DNAzyme motor initiated by endogenous enzyme for sensitive imaging of intracellular RNase H activity.

Aptamer-Functionalized DNA Origami for Targeted Codelivery of Antisense Oligonucleotides and Doxorubicin to Enhance Therapy in Drug-Resistant Cancer Cells.

Drug resistance is a major obstacle to the efficient therapy of drug-resistant cancer. To overcome this problem, we constructed a multifunctional DNA origami-based nanocarrier for codelivery of a chemotherapeutic drug (doxorubicin, Dox) and two different antisense oligonucleotides (ASOs; B-cell lymphoma 2 (Bcl2) and P-glycoprotein (P-gp)) into drug-resistant cancer cells for enhanced therapy. To increase the targeting ability of origami, staple strands with 5'-end extended MUC1 sequences were used in the preparation of aptamer-functionalized origami carrying ASOs (Apt-origami-ASO). Dox-loaded Apt-origami-ASO (Apt-Dox-origami-ASO) was prepared by electrostatic adsorption of Dox in origami. Atomic force microscopy (AFM) images demonstrated the successful preparation of Apt-origami-ASO. In vitro studies showed that the Apt-Dox-origami-ASO (Apt-DOA) could controllably release Dox in pH 5.0 phosphate-buffered saline (PBS) buffer and release ASOs in response to glutathione. Further experiments revealed that the origami could protect ASOs against nuclease degradation in 10% FBS. Confocal imaging showed that the Apt-DOA nanocarrier could efficiently enter the Hela/adriamycin (ADR) cells and escape from lysosomes for codelivery of Dox and ASOs into the cytoplasm. The quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot assays testified the efficient silencing of Bcl2 and P-gp mRNA and downregulation of the corresponding protein expressions by Apt-DOA in Hela/ADR cells. Moreover, with the synergetic effect by codelivery of multi-ASOs and Dox, the anticancer assay showed that Apt-DOA could circumvent multidrug resistance and significantly enhance cancer therapy in Hela/ADR and MCF-7/ADR cells. Hence, this multifunctional origami-based codelivery nanocarrier presents a new strategy for efficient therapy of drug-resistant cancer.

Biomineralized Metal-Organic Framework Nanoparticles Enable Enzymatic Rolling Circle Amplification in Living Cells for Ultrasensitive MicroRNA Imaging.

The enzymatic amplification strategy in living cells faces challenges of highly efficient intracellular codelivery of amplification reagents including DNA polymerase. In this work, we develop biomineralized metal-organic framework nanoparticles (MOF NPs) as a carrier system for intracellular codelivery of ϕ29 DNA polymerase (ϕ29DP) and nucleic acid probes and realize a polymerization amplification reaction in living cells. A pH-sensitive biodegradable MOF NP of zeolitic imidazolate framework-8 (ZIF-8) is utilized to encapsulate ϕ29DP and adsorb nucleic acid probes. After uptake into cells, the encapsulated ϕ29DP and surface-adsorbed DNA probes are released and escaped from endolysosomes. In the presence of ϕ29DP and deoxyribonucleotide triphosphates (dNTPs), the intracellular miRNA-21 triggers a rolling circle amplification (RCA) reaction and the autonomous synthesized Mg2+-dependent DNAzyme cleaves the fluorogenic substrate, providing a readout fluorescence signal for the monitoring of miRNA-21. This is the first example of the intracellular RCA reaction in living cells. Therefore, the proposed method provides new opportunities for achieving enzymatic amplification reaction in living cells.

A microRNA-triggered self-powered DNAzyme walker operating in living cells.

DNA-based nanomachines have received increasing attention due to their great potential to mimic natural biological motors and create novel modes of motion. Here, we report a DNAzyme-based walking machine, which can operate in living cells after triggered by intracellular miRNA-21. The walking machine is constructed by assembling DNAzyme walking strands and FAM-labeled substrate strands on a single gold nanoparticle (AuNP). The DNAzyme walking strand is first silenced by a blocker strand. After cellular uptake, DNAzyme-based walker can be triggered by intracellular miRNA-21 and autonomously walk along the AuNP-based 3D track fueled by DNAzyme-catalyzed substrate cleavage. Each walking step results in the cleavage of a substrate strand and the release of a FAM-labeled DNA strand, allowing real-time monitoring of the operation of the machine. The DNAzyme-based walking machine has been successfully applied to image and monitor miRNA-21 expression levels in living cells with excellent specificity and reliability. This walking machine would hold great potential in the miRNA associated biological research and disease diagnostics.