Toehold mediated strand displacement to measure released product from self-cleaving ribozymes

This paper presents a probe comprising a fluorophore and a quencher, enabling measurement of released product from self-cleaving hammerhead ribozyme, without labeled RNA molecules, regular sampling or use of polyacrylamide gels. The probe is made of two DNA strands; one strand is labelled with a fluorophore at its 5′-end, while the other strand is labelled with a quencher at its 3′-end. These two DNA strands are perfectly complementary, but with a 3′-overhang of the fluorophore strand. These unpaired nucleotides act as a toehold, which is utilized by a detached cleaved fragment (coming from a self-cleaving hammerhead ribozyme) as the starting point for a strand displacement reaction. This reaction causes the separation of the fluorophore strand from the quencher strand, culminating in fluorescence, detectable in a plate reader. Notably, the emitted fluorescence is proportional to the amount of detached cleaved-off RNAs, displacing the DNA quencher strand. This method can replace or complement radio-hazardous unstable 32P as a method of measurement of the product release from ribozyme cleavage reactions; it also eliminates the need for polyacrylamide gels, for the same purpose. Critically, this method allows to distinguish between the total amount of cleaved ribozymes and the amount of detached fragments, resulting from that cleavage reaction.

Isotope-Labeled RNA Building Blocks for NMR Structure and Dynamics Studies

RNA structural research lags behind that of proteins, preventing a robust understanding of RNA functions. NMR spectroscopy is an apt technique for probing the structures and dynamics of RNA molecules in solution at atomic resolution. Still, RNA analysis by NMR suffers from spectral overlap and line broadening, both of which worsen for larger RNAs. Incorporation of stable isotope labels into RNA has provided several solutions to these challenges. In this review, we summarize the benefits and limitations of various methods used to obtain isotope-labeled RNA building blocks and how they are used to prepare isotope-labeled RNA for NMR structure and dynamics studies.

Specific Recognition of a Stem-Loop RNA Structure by the Alphavirus Capsid Protein

Alphaviruses are small enveloped viruses with positive-sense RNA genomes. During infection, the alphavirus capsid protein (Cp) selectively packages and assembles with the viral genomic RNA to form the nucleocapsid core, a process critical to the production of infectious virus. Prior studies of the alphavirus Semliki Forest virus (SFV) showed that packaging and assembly are promoted by Cp binding to multiple high affinity sites on the genomic RNA. Here, we developed an in vitro Cp binding assay based on fluorescently labeled RNA oligos. We used this assay to explore the RNA sequence and structure requirements for Cp binding to site #1, the top binding site identified on the genomic RNA during all stages of virus assembly. Our results identify a stem-loop structure that promotes specific binding of the SFV Cp to site #1 RNA. This structure is also recognized by the Cps of the related alphaviruses chikungunya virus and Ross River virus.

VEGF-mediated angiogenesis in retinopathy of prematurity is co-regulated by miR-17-5p and miR-20a-5p

The microRNAs miR-17-5p and miR-20a-5p play important roles on angiogenesis; however, it is arguable whether they regulate the formation of retinal blood vessels in retinopathy of prematurity (ROP). We used a mouse model of oxygen-induced retinopathy (OIR) to simulate the development of retinas in mice suffering from ROP, and the expression levels of miR-20a-5p, miR-17-5p, hypoxia-inducible factor 1-alpha (HIF-1α), and vascular endothelial growth factor (VEGF) were measured by RT-qPCR and Western blotting. Cell proliferation, apoptosis, and angiogenesis in the OIR model mice were measured using MTT assays, flow cytometry, and Matrigel assays, respectively. The interaction between HIF-1α/VEGF and miR-20a-5p/miR-17-5p were further validated using dual-luciferase reporter assays, biotin-labeled RNA-pulldown, and RNA immunoprecipitation (RIP) assays. In our OIR model, retinal angiogenesis in the mice was associated with down-regulation of miR-20a-5p and miR-17-5p, as well as up-regulation of HIF-1α and VEGF. In addition, the miR-20a-5p and miR-17-5p inhibited cell proliferation and angiogenesis through regulating HIF-1α and VEGF in the retinal cells of the OIR model mice. Moreover, it was found that miR-20a-5p and miR-17-5p bind to HIF-1α and VEGF at the 3′UTR, and there was a combined effect between miR-20a-5p and miR-17-5p on the regulation of HIF-1α and VEGF. It is worth noting that miR-17-5p and miR-20a-5p can preferentially regulate HIF-1α, then act on VEGF, thereby affecting the angiogenesis associated with ROP.

Circ_0116061 regulated the proliferation, apoptosis, and inflammation of osteoarthritis chondrocytes through regulating the miR-200b-3p/SMURF2 axis

Background Circular RNA (circRNA) has been shown to be associated with osteoarthritis (OA) progression. Circ_0116061 has been found to be highly expressed in OA cartilage tissues, but its role and mechanism in OA progression remain unclear. Methods Expression levels of circ_0116061, microRNA (miR)-200b-5p, and Smad ubiquitin regulatory factor 2 (SMURF2) were detected using quantitative real-time PCR. The proliferation and apoptosis of cells were measured using cell counting kit 8 (CCK8) assay, colony formation assay, and flow cytometry. Furthermore, the protein levels of proliferation-related marker, apoptosis-related markers, inflammatory factors, and SMURF2 were tested using western blot (WB) analysis. In addition, the interaction between miR-200b-3p and circ_0116061 or SMURF2 was examined using dual-luciferase reporter assay and biotin-labeled RNA pull-down assay. Results Circ_0116061 and SMURF2 were highly expressed, and miR-200b-3p was lowly expressed in OA cartilage tissues. Knockdown of circ_0116061 could promote the proliferation and inhibit the apoptosis and inflammation of OA chondrocytes. MiR-200b-3p could be sponged by circ_0116061, and its inhibitor could reverse the regulation of circ_0116061 silencing on the biological functions of OA chondrocytes. SMURF2 was a target of miR-200b-3p, and its expression was positively regulated by circ_0116061. Silencing of SMURF2 also could enhance the proliferation and suppress the apoptosis and inflammation of OA chondrocytes. Furthermore, the regulation of circ_0116061 silencing on the biological functions of OA chondrocytes also could be reversed by SMURF2 overexpression. Conclusion Our data showed that circ_0116061 might regulate the miR-200b-3p/SMURF2 axis to promote the progression of OA.

Measurement of Kinetics of Hammerhead Ribozyme Cleavage Reactions using Toehold Mediated Strand Displacement

ABSTRACTThis paper presents a probe comprising a fluorophore and a quencher, enabling measurement of hammerhead ribozyme cleavage reactions, without labeled RNA molecules, regular sampling or use of polyacrylamide gels. The probe is made of two DNA strands; one strand is labelled with a fluorophore at its 5’-end, while the other strand is labelled with a quencher at its 3’-end. These two DNA strands are perfectly complementary, but with a 3’-overhang of the fluorophore strand. These unpaired nucleotides act as a toehold, which is utilized by a detached cleaved fragment (coming from a self-cleaving hammerhead ribozyme) as the starting point for a strand displacement reaction. This reaction causes the separation of the fluorophore strand from the quencher strand, culminating in fluorescence, detectable in a plate reader. Notably, the emitted fluorescence is proportional to the amount of detached cleaved-off RNAs, displacing the DNA quencher strand. This method can replace or complement radio-hazardous unstable 32P as a method of measurement of the kinetics of ribozyme cleavage reactions; it also eliminates the need for polyacrylamide gels, for the same purpose. Critically, this method allows to distinguish between the total amount of cleaved ribozymes and the amount of detached fragments, resulting from that cleavage reaction.

Towards safer stable isotope probing — effect of formamide on the separation of isotope-labeled and unlabeled Escherichia coli RNA by isopycnic density ultracentrifugation

RNA-based stable isotope probing (RNA-SIP) is used in molecular microbial ecology to link the identity of microorganisms in a complex community with the assimilation of a distinct substrate. The technique is highly dependent on a reliable separation of isotopic-labeled RNA from unlabeled RNA by isopycnic density gradient ultracentrifugation. Here we show that 13C-labeled and unlabeled Escherichia coli RNA can be sufficiently separated by isopycnic ultracentrifugation even in the absence of formamide. However, a slightly lower starting density is needed to obtain a distribution pattern similar to that obtained when formamide was used. Hence, the commonly used addition of formamide to the centrifugation solution might not be needed to separate 13C-labeled RNA from unlabeled RNA, but this must be verified for more complex environmental mixtures of RNA. Clearly, an omission of formamide would increase the safety of RNA-SIP analyses.

Long-TUC-seq is a robust method for quantification of metabolically labeled full-length isoforms

ABSTRACTThe steady state expression of each gene is the result of a dynamic transcription and degradation of that gene. While regular RNA-seq methods only measure steady state expression levels, RNA-seq of metabolically labeled RNA identifies transcripts that were transcribed during the window of metabolic labeling. Whereas short-read RNA sequencing can identify metabolically labeled RNA at the gene level, long-read sequencing provides much better resolution of isoform-level transcription. Here we combine thiouridine-to-cytosine conversion (TUC) with PacBio long-read sequencing to study the dynamics of mRNA transcription in the GM12878 cell line. We show that using long-TUC-seq, we can detect metabolically labeled mRNA of distinct isoforms more reliably than using short reads. Long-TUC-seq holds the promise of capturing isoform dynamics robustly and without the need for enrichment.