RNA G-quadruplexes (rG4s) have functional roles in many cellular processes in diverse organisms. ... more 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 (ncR-NAs), 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.
RNA G-quadruplexes (rG4s) have functional roles in many cellular processes in diverse organisms. ... more 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 in vivo data to support that NONO protein recognizes MALAT1 lncRNA via rG4 motifs. Notably, we demonstrate that rG4s in MALAT1 lncRNA can be targeted by 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, reveal...
Skeletal muscle has a remarkable ability to regenerate owing to its resident stem cells (also cal... more 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 regulat...
G-quadruplexes (G4s) are non-classical DNA or RNA secondary structures that have been first obser... more 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.
Skeletal muscle has a remarkable ability to regenerate owing to its resident stem cells, also cal... more Skeletal muscle has a remarkable ability to regenerate owing to its resident stem cells, also called satellite cells (SCs), that are normally quiescent. When stimulated by damage, SCs 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 quadruplex (rG4) structures, is essential for muscle regeneration by regulating SC expansion. We find that Dhx36 (initially named RHAU) is barely expressed at quiescence and 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 revealed Dhx36 binding predominantly to rG4 structures at various regions of mRNAs, while integrated polysome profiling showed that Dhx36 promotes mRNA translation via 5’UTR rG4 binding. Furthermore, we demonstrate that Dhx36 specifically regulates the tra...
Aim: To identify and characterize new mycobacterium pyrazinamide (PZA) resistance genes in additi... more 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.
An amendment to this paper has been published and can be accessed via a link at the top of the pa... more An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Based on target-induced catalytic hairpin assembly and two fluorescent dyes, we have developed a ... more Based on target-induced catalytic hairpin assembly and two fluorescent dyes, we have developed a new label-free and ratiometric detection method for microRNAs.
cDNA library preparation is important for many high-throughput sequencing applications, such as R... more 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. Multitudes of cDNA library preparation methods have been developed as a result of the recent exploration and investigation of the novel features of RNA, such as RNA structures 1-4. However, most of these methods require high RNA input (microgram of RNA), numerous processing and purification steps, and thus long cDNA library preparation time (few days) 5-9. As such, a simple and efficient cDNA library preparation is critical to the further improvement of existing protocols, development of new transcriptome-wide methods and many other biochemical assays. Recently, we have developed RNA G-quadruplex structure sequencing (rG4-seq), and reported the prevalent in vitro formation of RNA G-quadruplex structures in the human transcriptome 9 , providing a useful resource for further in vivo rG4 structural and functional characterization. The rG4-seq protocol requires high input of RNA (~500 ng RNA), and lengthy library preparation (~1.5 days) 9,10. In addition, an extensive assessment on the procedures of the experimental pipeline of rG4-seq is currently lacking. In this study, we have systematically evaluated and optimized the 5 key experimental steps of the experimental pipeline in rG4-seq, namely 3′ dephosphorylation, 3′-adapter ligation, excess 3′-adapter digestion and removal, reverse transcription, and 5′-adapter ligation (Fig. 1). We applied the improved methods to fragmented cellular RNA and generated two new cDNA libraries using reduced RNA inputs (~250 ng and ~50 ng RNA). The new libraries were found to have lower transcript abundance variations and lower 5′ and 3′ transcript coverage bias when compared to prior arts 9. Moreover, the total time of the library preparation has been greatly reduced to several hours.
RNA G-quadruplexes (rG4s) have functional roles in many cellular processes in diverse organisms. ... more 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 (ncR-NAs), 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.
RNA G-quadruplexes (rG4s) have functional roles in many cellular processes in diverse organisms. ... more 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 in vivo data to support that NONO protein recognizes MALAT1 lncRNA via rG4 motifs. Notably, we demonstrate that rG4s in MALAT1 lncRNA can be targeted by 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, reveal...
Skeletal muscle has a remarkable ability to regenerate owing to its resident stem cells (also cal... more 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 regulat...
G-quadruplexes (G4s) are non-classical DNA or RNA secondary structures that have been first obser... more 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.
Skeletal muscle has a remarkable ability to regenerate owing to its resident stem cells, also cal... more Skeletal muscle has a remarkable ability to regenerate owing to its resident stem cells, also called satellite cells (SCs), that are normally quiescent. When stimulated by damage, SCs 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 quadruplex (rG4) structures, is essential for muscle regeneration by regulating SC expansion. We find that Dhx36 (initially named RHAU) is barely expressed at quiescence and 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 revealed Dhx36 binding predominantly to rG4 structures at various regions of mRNAs, while integrated polysome profiling showed that Dhx36 promotes mRNA translation via 5’UTR rG4 binding. Furthermore, we demonstrate that Dhx36 specifically regulates the tra...
Aim: To identify and characterize new mycobacterium pyrazinamide (PZA) resistance genes in additi... more 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.
An amendment to this paper has been published and can be accessed via a link at the top of the pa... more An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Based on target-induced catalytic hairpin assembly and two fluorescent dyes, we have developed a ... more Based on target-induced catalytic hairpin assembly and two fluorescent dyes, we have developed a new label-free and ratiometric detection method for microRNAs.
cDNA library preparation is important for many high-throughput sequencing applications, such as R... more 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. Multitudes of cDNA library preparation methods have been developed as a result of the recent exploration and investigation of the novel features of RNA, such as RNA structures 1-4. However, most of these methods require high RNA input (microgram of RNA), numerous processing and purification steps, and thus long cDNA library preparation time (few days) 5-9. As such, a simple and efficient cDNA library preparation is critical to the further improvement of existing protocols, development of new transcriptome-wide methods and many other biochemical assays. Recently, we have developed RNA G-quadruplex structure sequencing (rG4-seq), and reported the prevalent in vitro formation of RNA G-quadruplex structures in the human transcriptome 9 , providing a useful resource for further in vivo rG4 structural and functional characterization. The rG4-seq protocol requires high input of RNA (~500 ng RNA), and lengthy library preparation (~1.5 days) 9,10. In addition, an extensive assessment on the procedures of the experimental pipeline of rG4-seq is currently lacking. In this study, we have systematically evaluated and optimized the 5 key experimental steps of the experimental pipeline in rG4-seq, namely 3′ dephosphorylation, 3′-adapter ligation, excess 3′-adapter digestion and removal, reverse transcription, and 5′-adapter ligation (Fig. 1). We applied the improved methods to fragmented cellular RNA and generated two new cDNA libraries using reduced RNA inputs (~250 ng and ~50 ng RNA). The new libraries were found to have lower transcript abundance variations and lower 5′ and 3′ transcript coverage bias when compared to prior arts 9. Moreover, the total time of the library preparation has been greatly reduced to several hours.
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