Identification and targeting of G-quadruplex structures in MALAT1 long non-coding RNA.

RNA G-quadruplexes (rG4s) have functional roles in many cellular processes in diverse organisms. While a number of rG4 examples have been reported in coding messenger RNAs (mRNA), so far only limited works have studied rG4s in non-coding RNAs (ncRNAs), especially in long non-coding RNAs (lncRNAs) that are of emerging interest and significance in biology. Herein, we report that MALAT1 lncRNA contains conserved rG4 motifs, forming thermostable rG4 structures with parallel topology. We also show that rG4s in MALAT1 lncRNA can interact with NONO protein with high specificity and affinity in vitro and in nuclear cell lysate, and we provide cellular data to support that NONO protein recognizes MALAT1 lncRNA via rG4 motifs. Notably, we demonstrate that rG4s in MALAT1 lncRNA can be targeted by the rG4-specific small molecule, peptide, and L-aptamer, leading to the dissociation of MALAT1 rG4-NONO protein interaction. Altogether, this study uncovers new and important rG4s in MALAT1 lncRNAs, reveals their specific interactions with NONO protein, offers multiple strategies for targeting MALAT1 and its RNA-protein complex via its rG4 structure and illustrates the prevalence and significance of rG4s in ncRNAs.

Peptides Selected by G4-mRNA Display-Seq Enable RNA G-Quadruplex Recognition and Gene Regulation.

G-quadruplexes (G4s) are noncanonical secondary structures that play critical roles in both chemistry and biology. Although several approaches have been developed for G4 targeting, such as chemicals and antibodies, there is currently no general and efficient platform for G4-specific peptides. In this study, we developed a new platform, G4-mRNA display-Seq, for selecting peptides that specifically recognize the G4 target of interest. By using an RNA G4 (rG4) found in human telomerase RNA (hTERC) as the target, we have identified a novel short peptide, namely, peptide 11 (pep11), which displays high affinity and selectivity to hTERC rG4. Furthermore, we designed tandem and cyclic versions of pep11 and found that both modified versions exhibit stronger binding affinity with preferential rG4 selectivity. Notably, we have demonstrated that these peptides can negatively regulate gene expression by targeting rG4. Our results provide a universal platform for the discovery of G4-targeting peptides and demonstrate the ability of these peptides to regulate G4-mediated gene functions.

RNA G-quadruplexes (rG4s): genomics and biological functions.

G-quadruplexes (G4s) are non-classical DNA or RNA secondary structures that have been first observed decades ago. Over the years, these four-stranded structural motifs have been demonstrated to have significant regulatory roles in diverse biological processes, but challenges remain in detecting them globally and reliably. Compared to DNA G4s (dG4s), the study of RNA G4s (rG4s) has received less attention until recently. In this review, we will summarize the innovative high-throughput methods recently developed to detect rG4s on a transcriptome-wide scale, highlight the many novel and important functions of rG4 being discovered in vivo across the tree of life, and discuss the key biological questions to be addressed in the near future.

Effect of RNA sequence context and stereochemistry on G-quadruplex-RHAU53 interaction.

RNA G-quadruplex (rG4) structure and its association with rG4-binding proteins/peptides are important for its function. However, there is very limited study that investigates what factors are involved in rG4 that drive the rG4-protein/peptide interaction. Here we study and uncover the effect of RNA sequence context and stereochemistry on G-quadruplex-peptide interaction. Using rG4-binding RHAU53 peptide as an example, we report that the number of G-quartet, thermostability, overhanging nucleotides, and RNA base chirality have an impact on rG4-RHAU53 binding. Notably, our data also demonstrate that RHAU53 preferentially binds to 5' G-quartet over 3' G-quartet, and showcase that RHAU53 interacts with unnatural L-rG4 for the first time. Our findings reported here offer unique insights to the potential development of targeting tools that recognize rG4 structure and rG4-binding peptide/protein.

Translational control by DHX36 binding to 5'UTR G-quadruplex is essential for muscle stem-cell regenerative functions.

Skeletal muscle has a remarkable ability to regenerate owing to its resident stem cells (also called satellite cells, SCs). SCs are normally quiescent; when stimulated by damage, they activate and expand to form new fibers. The mechanisms underlying SC proliferative progression remain poorly understood. Here we show that DHX36, a helicase that unwinds RNA G-quadruplex (rG4) structures, is essential for muscle regeneration by regulating SC expansion. DHX36 (initially named RHAU) is barely expressed at quiescence but is highly induced during SC activation and proliferation. Inducible deletion of Dhx36 in adult SCs causes defective proliferation and muscle regeneration after damage. System-wide mapping in proliferating SCs reveals DHX36 binding predominantly to rG4 structures at various regions of mRNAs, while integrated polysome profiling shows that DHX36 promotes mRNA translation via 5'-untranslated region (UTR) rG4 binding. Furthermore, we demonstrate that DHX36 specifically regulates the translation of Gnai2 mRNA by unwinding its 5' UTR rG4 structures and identify GNAI2 as a downstream effector of DHX36 for SC expansion. Altogether, our findings uncover DHX36 as an indispensable post-transcriptional regulator of SC function and muscle regeneration acting through binding and unwinding rG4 structures at 5' UTR of target mRNAs.

Mycobacterium smegmatis msmeg_3314 is involved in pyrazinamide and fluoroquinolones susceptibility via NAD+/NADH dysregulation.

Aim: To identify and characterize new mycobacterium pyrazinamide (PZA) resistance genes in addition to pncA, rpsA and panD. Materials & methods: To screen a Tn7 M. smegmatis mc2155 transposon library using 50 µM PZA and a PZA hypersensitive mutant (M492) was obtained. MIC was further used to confirm the hypersensitivity of M492 mutant by culturing the mutant in Middlebrook 7H9 liquid medium at 37°C. Results:msmeg_3314 is the gene underlying the hypersensitive phenotype of mutant M492. The observed resistance to PZA and fluoroquinolones involved the alteration of Mycobacterium cell wall permeability and the dissipation of the proton motive force. NAD+/NADH dysregulation and attenuated glyoxylate shunt might underlie the declined scavenging capacity of reactive oxygen species in the msmeg_3314-deficient mutants. Conclusion:msmeg_ 3314 is a novel gene involved in pyrazinamide resistance and might be a new candidate for drugs target.

Author Correction: Systematic evaluation and optimization of the experimental steps in RNA G-quadruplex structure sequencing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Systematic evaluation and optimization of the experimental steps in RNA G-quadruplex structure sequencing.

cDNA library preparation is important for many high-throughput sequencing applications, such as RNA G-quadruplex structure sequencing (rG4-seq). A systematic evaluation of the procedures of the experimental pipeline, however, is lacking. Herein, we perform a comprehensive assessment of the 5 key experimental steps involved in the cDNA library preparation of rG4-seq, and identify better reaction conditions and/or enzymes to carry out each of these key steps. Notably, we apply the improved methods to fragmented cellular RNA, and show reduced RNA input requirement, lower transcript abundance variations between biological replicates, as well as lower transcript coverage bias when compared to prior arts. In addition, the time to perform these steps is substantially reduced to hours. Our method and results can be directly applied in protocols that require cDNA library preparation, and provide insights to the further development of simple and efficient cDNA library preparation for different biological applications.

Preliminary evaluation of discomfort glare from organic light-emitting diode and edge-lit light-emitting diode lighting panels.

The organic light-emitting diode (OLED) is an area light source, and its primary competing technology is the edge-lit light-emitting diode (LED) panel. Both technologies are similar in shape and appearance, but there is little understanding of how people perceive discomfort glare (DG) from area sources. The objective of this study was to evaluate the DG of these two technologies under similar operating conditions. Additionally, two existing DG models were compared to evaluate the correlation between predicted values and observed values. In an earlier study, we found no statistically significant difference in human response in terms of DG between OLED and edge-lit LED panels when the two sources produced the same luminous stimulus. The range of testing stimulus was expanded to test different panel luminances at three background illuminations. The results showed no difference in perceived glare between the panels, and, as the background illumination increased, the perceived glare decreased. In other words, both appeared equally glary beyond a certain luminance and background illumination. We then compared two existing glare models with the observed values and found that one model showed a good estimation of how humans perceive DG. That model was further modified to increase its power.

[Hemodynamic effects of aminophylline and nifedipine in patients with high altitude pulmonary edema].

AIM: To evaluate the hemodynamic effects of aminophylline and nifedipine in patients with HAPE. METHODS: 10 patients with HAPE undergone Swan-Ganz catheter. The parameters of hemodynamics and arterial blood gases in HAPE were measured before and after administration of nifedipine 20 mg sublingually and aminophylline 0.25 g intravenously respectively. RESULTS: After administering 0.25 g aminophylline the mPAP and PVR significantly decreased, the cardiac output and the level of PaO2, SaO2 increased obviously, the mSAP, HR did not change so much. After using 20 mg nifedipine, the mPAP, PVR and mSAP also decreased, while the cardiac output, HR and the level of PaO2, SaO2 did not show any changes. CONCLUSION: Both of aminophylline and nifedipine can attenuate pulmonary hypertension in patients with HAPE, but the effect of aminophylline was better than the effect of nifedipine.