P68 RNA helicase was identified as being required for unwinding the human let-7 microRNA precursor duplex. Recombinant P68 RNA helicase was shown to unwind the let-7 duplex in vitro. Knockdown of P68 inhibited let-7 microRNA function, indicating P68 is essential for loading let-7 into the silencing complex. This study identifies P68 RNA helicase as playing a key role in the human let-7 microRNA pathway.
The document discusses various genome editing techniques including meganucleases, zinc fingers, TALENs, and CRISPR-Cas9. It provides details on the mechanism of action, design, advantages, and limitations of each technique. Meganucleases were among the earliest tools but were difficult to engineer for new target sites. Zinc fingers and TALENs improved targeting ability but were still complex to design. CRISPR-Cas9 is now the most widely used system due to its simple and affordable design, high efficiency, and ability to minimize off-target effects.
The document discusses various gene editing technologies. It begins by introducing genome/gene editing as a type of genetic engineering that uses engineered nucleases to precisely modify genomes by creating DNA insertions, deletions, or replacements at specific DNA sequences. It then describes three main gene editing systems - zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 system. For each system, it provides details on the nuclease domains, methods for engineering DNA binding specificity, and mechanisms for creating DNA double strand breaks to facilitate gene modifications.
Genome editing tools form the basis for personalized medicine, especially for therapies requiring change in genome. Currently there are four contenders to this – Meganucleases, ZNF Nucleases, TALENs and CRISPRs. Although, the technologies are many, there are very few commercial providers of this technology. This is attributed to the fact that select few possess the intellectual property rights of turning these technologies to valid form of therapy; for example, ZFN patent with Sangamo BioSciences and TALENs with Cellectis, Transposagen and Life Technologies.
Genome editing with engineered nucleasesKrishan Kumar
Genome editing uses engineered nucleases to insert, replace or remove DNA from the genome. These nucleases create targeted double-strand breaks which are repaired through natural DNA repair processes, allowing for changes to the genome sequence. Three main engineered nuclease systems for genome editing are ZFNs, TALENs, and CRISPR-Cas9. CRISPR uses a guide RNA and Cas9 nuclease to make precise cuts at targeted DNA sequences for editing. It has advantages over ZFNs and TALENs in being cheaper, easier to design, and more efficient. Genome editing holds promise for applications in crops, medicine, and research.
Zinc finger nucleases (ZFNs) and transcription activator-like effectors (TALEs) are genome editing tools that use engineered DNA-binding domains to target specific locations in the genome. ZFNs use zinc finger proteins fused to FokI endonuclease domains, while TALEs use transcription activator proteins from bacteria with engineered repeat domains to target DNA. Both can be used to create double-strand breaks and induce genome editing through non-homologous end joining or homology-directed repair. ZFNs and TALEs have applications including nucleases, recombinases, transposases, and artificial transcription factors for genome editing, gene regulation, and protein delivery.
Gene editing application for cancer therapeuticsNur Farrah Dini
The application of TALENs as one of the gene editing tools in order to modify a specific targeted sites on a genome. This method shows a tremendous benefits especially in cancer research.
The document discusses various genome editing techniques including meganucleases, zinc fingers, TALENs, and CRISPR-Cas9. It provides details on the mechanism of action, design, advantages, and limitations of each technique. Meganucleases were among the earliest tools but were difficult to engineer for new target sites. Zinc fingers and TALENs improved targeting ability but were still complex to design. CRISPR-Cas9 is now the most widely used system due to its simple and affordable design, high efficiency, and ability to minimize off-target effects.
The document discusses various gene editing technologies. It begins by introducing genome/gene editing as a type of genetic engineering that uses engineered nucleases to precisely modify genomes by creating DNA insertions, deletions, or replacements at specific DNA sequences. It then describes three main gene editing systems - zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 system. For each system, it provides details on the nuclease domains, methods for engineering DNA binding specificity, and mechanisms for creating DNA double strand breaks to facilitate gene modifications.
Genome editing tools form the basis for personalized medicine, especially for therapies requiring change in genome. Currently there are four contenders to this – Meganucleases, ZNF Nucleases, TALENs and CRISPRs. Although, the technologies are many, there are very few commercial providers of this technology. This is attributed to the fact that select few possess the intellectual property rights of turning these technologies to valid form of therapy; for example, ZFN patent with Sangamo BioSciences and TALENs with Cellectis, Transposagen and Life Technologies.
Genome editing with engineered nucleasesKrishan Kumar
Genome editing uses engineered nucleases to insert, replace or remove DNA from the genome. These nucleases create targeted double-strand breaks which are repaired through natural DNA repair processes, allowing for changes to the genome sequence. Three main engineered nuclease systems for genome editing are ZFNs, TALENs, and CRISPR-Cas9. CRISPR uses a guide RNA and Cas9 nuclease to make precise cuts at targeted DNA sequences for editing. It has advantages over ZFNs and TALENs in being cheaper, easier to design, and more efficient. Genome editing holds promise for applications in crops, medicine, and research.
Zinc finger nucleases (ZFNs) and transcription activator-like effectors (TALEs) are genome editing tools that use engineered DNA-binding domains to target specific locations in the genome. ZFNs use zinc finger proteins fused to FokI endonuclease domains, while TALEs use transcription activator proteins from bacteria with engineered repeat domains to target DNA. Both can be used to create double-strand breaks and induce genome editing through non-homologous end joining or homology-directed repair. ZFNs and TALEs have applications including nucleases, recombinases, transposases, and artificial transcription factors for genome editing, gene regulation, and protein delivery.
Gene editing application for cancer therapeuticsNur Farrah Dini
The application of TALENs as one of the gene editing tools in order to modify a specific targeted sites on a genome. This method shows a tremendous benefits especially in cancer research.
Genome editing technologies allow genetic material to be added, removed or altered at specific locations in an organism's genome. Several approaches exist, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR/Cas9, and base editors. These tools create precise breaks in DNA that can be repaired through non-homologous end joining or homology-directed repair. They enable trait discovery and crop improvement by generating plants with high yield, stress resistance, or other desired properties. While powerful, challenges remain in fully editing complex genomes and reducing off-target mutations.
This document discusses genome editing using the CRISPR-Cas9 system. It begins by introducing three main genome editing technologies - zinc-finger nucleases, TALENs, and the CRISPR-Cas9 system. It then describes the key events in the discovery of CRISPR-Cas9, including its origins as a bacterial defense system. The document outlines the main components of the CRISPR-Cas9 system, including crRNA, tracrRNA, sgRNA, and Cas9. It also summarizes the two main steps in genome editing using CRISPR-Cas9 - knocking out genes and DNA repair. The document concludes by discussing opportunities for applying CRISPR-Cas9 technology across various
Genome editing is one of the most important tools which supports genetic engineering. It is based on the naturally occurring mechanism of DNA recombination which involves the initiation of breaks with the double stranded DNA followed by repair by the endogenous DNA polymerases.
Conventional techniques such as gene knockouts using P-elements and transposable genetic elements have been superseded by more accurate genome editing methods such as TALENs and CRISPR/Cas.
Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cut specific DNA sequences. TALENs are made by fusing a DNA-binding domain from TALE (transcription activator-like effector) proteins with a DNA cleavage domain from the FokI nuclease. This fusion allows TALENs to be targeted to specific DNA sequences and induce double-stranded breaks, making them useful for genome editing applications.
This document describes a study that used transposon mutagenesis and targeted gene deletions to identify genes involved in perchlorate reduction in the bacterium Azospira suillum PS. Transposon mutagenesis identified 18 mutants that were completely unable to grow using perchlorate as an electron acceptor (perchlorate null mutants). Most of these mutants had transposon insertions in genes located within the previously identified perchlorate reduction genomic island. Targeted deletions of each gene within this island identified 8 genes that were essential for perchlorate reduction, including those encoding the key enzymes chlorite dismutase and perchlorate reductase as well as genes for a putative regulatory system. This
This presentation provides an overview of genome editing. It defines genome editing as a technique used to modify DNA within a cell precisely and efficiently using engineered nucleases. The presentation discusses different tools for genome editing including meganucleases, zinc finger nucleases, TALENs, and CRISPR. It explains how these tools work by creating cuts in DNA at specific sequences, and how cells naturally repair this using homologous directed repair or non-homologous end joining. Applications of genome editing discussed include gene therapy, modifying crops and animals, disease treatment, and ecological control.
CRISPR is a novel genome editing tool using Cas9 nuclease guided by CRISPR RNA. The document discusses CRISPR's mechanism and applications in editing plant fungal pathogens. It provides examples where CRISPR was used to modify genes conferring disease resistance in rice, citrus, and viruses. The advantages of CRISPR include specificity, minimizing off-target effects, and applicability across species. Challenges include off-target effects and mosaicism. Highlights of studies editing fungi include creating mutants in Alternaria alternata and Ganoderma lucidum. CRISPR also suppressed pathogenicity in Sclerotinia sclerotiorum by editing oxalate genes.
Genome Editing Techniques by Kainat RamzanKainatRamzan3
Genome technology has revolutionized biological science through techniques of Gene Editing in order to edit any organism's genome.MegNs and zinc-finger nucleases are commonly understood to be used, as is the effector's transcriptional activator-like nucleases. In CRISPR/Cas9, genetic alterations, and gene functionality have become a well-known tool for understanding gene targeting.
This document discusses gene editing applications using CRISPR-Cas9, including in gametes and embryos. It provides background on the development of CRISPR-Cas9 as a gene editing tool. Genome editing has been applied to male and female germ cells in animal models and research embryos to correct genetic mutations. However, human embryo genome editing faces limitations such as mosaicism and off-target effects. While genome editing holds promise for treating genetic diseases, more research is needed to improve specificity and fidelity before clinical applications.
Have you considered that protein over-expression or inefficient mRNA knockdown may be masking physiological effects in your assays? Increasingly scientists are moving to endogenous gene-editing to characterise the function of their genes of interest.
Dr Chris Thorne from Cambridge Biotech Horizon Discovery discusses the ground breaking gene-editing technology CRISPR. The simplicity of experimental design has led to rapid adoption of the technology across the scientific community. However, challenges remain.
This Slidedeck focuses specifically on implementing CRISPR experiments, and explore a number of key considerations crucial to maximising chances of targeting success, whether your goal is to generate a knock-out or a knock-in. Chris also takes a look at some of the alternative uses of CRISPR, including sgRNA genome wide synthetic lethality screens.
The slides aim to support those researchers either planning to or already using CRISPR gene-editing in their lab. Horizon Discovery have also recently launched a program aimed specifically at academic cell biologists to promote the adoption of CRISPR by offering FREE CRISPR Reagents for knock-out cell line generation - more information available here. http://www.horizondiscovery.com/what-we-do/discovery-toolbox/genassist-crispr--raav-genome-editing-tools
TAPPING THE RNA WORLD FOR THERAPEUTICSHasnat Tariq
ASO drugs, Si-RNA, Delivery of si-RNA based drugs, CRISPR-Cas gene editing, mRNA based drugs, Aptamer based therapeutics, RNAI PATHWAY, Aptamer based delivery.
Genome editing is a technique used to precisely modify DNA within a cell. It involves using artificially engineered nucleases called "molecular scissors" to cut DNA at specific locations. This creates breaks that can then be repaired through natural cellular processes, allowing the genome to be altered. Early methods like homologous recombination were inefficient. New tools like zinc finger nucleases, TALENs, and the CRISPR/Cas9 system allow genome editing to be targeted to specific DNA sequences with greater accuracy and efficiency. These programmable nucleases make targeted cuts in the genome that can then be repaired through mechanisms like non-homologous end joining or homology-directed repair. CRISPR/Cas9 has become particularly
The document describes an experiment using CRISPR/Cas9 to introduce mutations into the PNPLA3 gene in HepG2 cells. Sequencing of genomic DNA and mRNA from the cells showed the presence of SNP1, which changes an amino acid, but not SNP2. The researcher will use this sequence as a template to generate four combinations of the SNPs in order to study their effects on fat accumulation when transfected into HepG2 cells. This could provide insight into how genetic variants influence susceptibility to NAFLD in different ethnic groups.
The next generation of crispr–cas technologies and Applicationsiqraakbar8
The document discusses recent advances in CRISPR-Cas gene editing tools, including Cas9, Cas12a, and Cas13a. It describes how these tools work, how they can be used to make various genomic alterations through DNA repair pathways, and potential applications for basic research and medicine. Specifically, it outlines ongoing clinical trials using CRISPR-Cas to treat genetic diseases.
Advanced Genome Engineering Services and Transgenic Model Generation
at MSU’s Transgenic and Genome Editing Facility
Huirong Xie, Elena Demireva, Nate Kauffman, Richard Neubig
This document provides an overview of CRISPR/Cas9 genome editing. It discusses the history and limitations of prior genome engineering techniques like recombinant DNA and zinc finger nucleases. It then explains how CRISPR/Cas9 works as a RNA-guided DNA endonuclease and how this allows it to efficiently and specifically edit genomes. The document outlines several applications of CRISPR/Cas9 like generating knockout animals and cell lines. It also notes some concerns about using the technique for human genome editing.
Genome editing is a method of making specific changes to the DNA of a cell or organism. An enzyme scissors the DNA at a specific sequence, and when this is repaired by the cell, a change or ‘edit’ is made to the sequence.
https://www.creative-biolabs.com/gene-therapy/approaches-to-genome-editing.htm
This document provides an overview of genome engineering techniques from older methods to newer CRISPR/Cas9 technology. It discusses how genes can be transferred between organisms using vectors like plasmids or viruses. Older techniques like ZFN and TALEN cut DNA at specific sites, while CRISPR/Cas9 uses a Cas9 enzyme guided by CRISPR RNA to make precise cuts. Delivery methods for Cas9 include plasmids, mRNA, and RNP complexes. Viral vectors like AAV are commonly used but have limits. Physical methods also deliver Cas9 via nanoparticles or peptides.
This document provides an overview of biotechnology concepts and applications. It discusses how recombinant DNA can be created in the laboratory using restriction enzymes, DNA ligase, and gel electrophoresis. This DNA can then be used to genetically transform cells and organisms using vectors like plasmids and viruses. Genes can be manipulated through techniques like gene knockout, antisense RNA, and microarrays. Finally, the document outlines wide applications of biotechnology in medicine, agriculture, and industry, including producing insulin, pharmaceuticals in transgenic animals/plants, and genetically modifying crops.
This document summarizes a study that investigated the complexes containing the RNA helicase DDX6, which is a key component of P-bodies involved in posttranscriptional regulation. The researchers identified DDX6 complexes using tandem affinity purification coupled with mass spectrometry. They found that DDX6 was present in three main complexes: the decapping complex, a CPEB-like complex, and an Ataxin2/Ataxin2L complex. Investigation of P-body assembly under various conditions identified three proteins required for assembly in all conditions: DDX6, 4E-T, and LSM14A. The results reveal that P-body assembly involves different pathways that nevertheless share these three key factors connecting
The 5' terminal uracil of let-7a is critical for the recruitment of mRNA to A...David W. Salzman
This document investigates the interaction between let-7a microRNA, Argonaute2 protein, and mRNA targets. It finds that recombinant Argonaute2 is sufficient to direct let-7a-guided cleavage of a fully complementary mRNA target in vitro. Additionally, it determines that the 5' terminal uracil of let-7a is critical for recruitment of the mRNA target to the let-7a-Argonaute2 complex. Mutation of this 5' uracil inhibits formation of the ternary let-7a-Argonaute2-mRNA complex, but does not affect formation of the binary let-7a-Argonaute2 complex. This suggests the 5' urac
Genome editing technologies allow genetic material to be added, removed or altered at specific locations in an organism's genome. Several approaches exist, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR/Cas9, and base editors. These tools create precise breaks in DNA that can be repaired through non-homologous end joining or homology-directed repair. They enable trait discovery and crop improvement by generating plants with high yield, stress resistance, or other desired properties. While powerful, challenges remain in fully editing complex genomes and reducing off-target mutations.
This document discusses genome editing using the CRISPR-Cas9 system. It begins by introducing three main genome editing technologies - zinc-finger nucleases, TALENs, and the CRISPR-Cas9 system. It then describes the key events in the discovery of CRISPR-Cas9, including its origins as a bacterial defense system. The document outlines the main components of the CRISPR-Cas9 system, including crRNA, tracrRNA, sgRNA, and Cas9. It also summarizes the two main steps in genome editing using CRISPR-Cas9 - knocking out genes and DNA repair. The document concludes by discussing opportunities for applying CRISPR-Cas9 technology across various
Genome editing is one of the most important tools which supports genetic engineering. It is based on the naturally occurring mechanism of DNA recombination which involves the initiation of breaks with the double stranded DNA followed by repair by the endogenous DNA polymerases.
Conventional techniques such as gene knockouts using P-elements and transposable genetic elements have been superseded by more accurate genome editing methods such as TALENs and CRISPR/Cas.
Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cut specific DNA sequences. TALENs are made by fusing a DNA-binding domain from TALE (transcription activator-like effector) proteins with a DNA cleavage domain from the FokI nuclease. This fusion allows TALENs to be targeted to specific DNA sequences and induce double-stranded breaks, making them useful for genome editing applications.
This document describes a study that used transposon mutagenesis and targeted gene deletions to identify genes involved in perchlorate reduction in the bacterium Azospira suillum PS. Transposon mutagenesis identified 18 mutants that were completely unable to grow using perchlorate as an electron acceptor (perchlorate null mutants). Most of these mutants had transposon insertions in genes located within the previously identified perchlorate reduction genomic island. Targeted deletions of each gene within this island identified 8 genes that were essential for perchlorate reduction, including those encoding the key enzymes chlorite dismutase and perchlorate reductase as well as genes for a putative regulatory system. This
This presentation provides an overview of genome editing. It defines genome editing as a technique used to modify DNA within a cell precisely and efficiently using engineered nucleases. The presentation discusses different tools for genome editing including meganucleases, zinc finger nucleases, TALENs, and CRISPR. It explains how these tools work by creating cuts in DNA at specific sequences, and how cells naturally repair this using homologous directed repair or non-homologous end joining. Applications of genome editing discussed include gene therapy, modifying crops and animals, disease treatment, and ecological control.
CRISPR is a novel genome editing tool using Cas9 nuclease guided by CRISPR RNA. The document discusses CRISPR's mechanism and applications in editing plant fungal pathogens. It provides examples where CRISPR was used to modify genes conferring disease resistance in rice, citrus, and viruses. The advantages of CRISPR include specificity, minimizing off-target effects, and applicability across species. Challenges include off-target effects and mosaicism. Highlights of studies editing fungi include creating mutants in Alternaria alternata and Ganoderma lucidum. CRISPR also suppressed pathogenicity in Sclerotinia sclerotiorum by editing oxalate genes.
Genome Editing Techniques by Kainat RamzanKainatRamzan3
Genome technology has revolutionized biological science through techniques of Gene Editing in order to edit any organism's genome.MegNs and zinc-finger nucleases are commonly understood to be used, as is the effector's transcriptional activator-like nucleases. In CRISPR/Cas9, genetic alterations, and gene functionality have become a well-known tool for understanding gene targeting.
This document discusses gene editing applications using CRISPR-Cas9, including in gametes and embryos. It provides background on the development of CRISPR-Cas9 as a gene editing tool. Genome editing has been applied to male and female germ cells in animal models and research embryos to correct genetic mutations. However, human embryo genome editing faces limitations such as mosaicism and off-target effects. While genome editing holds promise for treating genetic diseases, more research is needed to improve specificity and fidelity before clinical applications.
Have you considered that protein over-expression or inefficient mRNA knockdown may be masking physiological effects in your assays? Increasingly scientists are moving to endogenous gene-editing to characterise the function of their genes of interest.
Dr Chris Thorne from Cambridge Biotech Horizon Discovery discusses the ground breaking gene-editing technology CRISPR. The simplicity of experimental design has led to rapid adoption of the technology across the scientific community. However, challenges remain.
This Slidedeck focuses specifically on implementing CRISPR experiments, and explore a number of key considerations crucial to maximising chances of targeting success, whether your goal is to generate a knock-out or a knock-in. Chris also takes a look at some of the alternative uses of CRISPR, including sgRNA genome wide synthetic lethality screens.
The slides aim to support those researchers either planning to or already using CRISPR gene-editing in their lab. Horizon Discovery have also recently launched a program aimed specifically at academic cell biologists to promote the adoption of CRISPR by offering FREE CRISPR Reagents for knock-out cell line generation - more information available here. http://www.horizondiscovery.com/what-we-do/discovery-toolbox/genassist-crispr--raav-genome-editing-tools
TAPPING THE RNA WORLD FOR THERAPEUTICSHasnat Tariq
ASO drugs, Si-RNA, Delivery of si-RNA based drugs, CRISPR-Cas gene editing, mRNA based drugs, Aptamer based therapeutics, RNAI PATHWAY, Aptamer based delivery.
Genome editing is a technique used to precisely modify DNA within a cell. It involves using artificially engineered nucleases called "molecular scissors" to cut DNA at specific locations. This creates breaks that can then be repaired through natural cellular processes, allowing the genome to be altered. Early methods like homologous recombination were inefficient. New tools like zinc finger nucleases, TALENs, and the CRISPR/Cas9 system allow genome editing to be targeted to specific DNA sequences with greater accuracy and efficiency. These programmable nucleases make targeted cuts in the genome that can then be repaired through mechanisms like non-homologous end joining or homology-directed repair. CRISPR/Cas9 has become particularly
The document describes an experiment using CRISPR/Cas9 to introduce mutations into the PNPLA3 gene in HepG2 cells. Sequencing of genomic DNA and mRNA from the cells showed the presence of SNP1, which changes an amino acid, but not SNP2. The researcher will use this sequence as a template to generate four combinations of the SNPs in order to study their effects on fat accumulation when transfected into HepG2 cells. This could provide insight into how genetic variants influence susceptibility to NAFLD in different ethnic groups.
The next generation of crispr–cas technologies and Applicationsiqraakbar8
The document discusses recent advances in CRISPR-Cas gene editing tools, including Cas9, Cas12a, and Cas13a. It describes how these tools work, how they can be used to make various genomic alterations through DNA repair pathways, and potential applications for basic research and medicine. Specifically, it outlines ongoing clinical trials using CRISPR-Cas to treat genetic diseases.
Advanced Genome Engineering Services and Transgenic Model Generation
at MSU’s Transgenic and Genome Editing Facility
Huirong Xie, Elena Demireva, Nate Kauffman, Richard Neubig
This document provides an overview of CRISPR/Cas9 genome editing. It discusses the history and limitations of prior genome engineering techniques like recombinant DNA and zinc finger nucleases. It then explains how CRISPR/Cas9 works as a RNA-guided DNA endonuclease and how this allows it to efficiently and specifically edit genomes. The document outlines several applications of CRISPR/Cas9 like generating knockout animals and cell lines. It also notes some concerns about using the technique for human genome editing.
Genome editing is a method of making specific changes to the DNA of a cell or organism. An enzyme scissors the DNA at a specific sequence, and when this is repaired by the cell, a change or ‘edit’ is made to the sequence.
https://www.creative-biolabs.com/gene-therapy/approaches-to-genome-editing.htm
This document provides an overview of genome engineering techniques from older methods to newer CRISPR/Cas9 technology. It discusses how genes can be transferred between organisms using vectors like plasmids or viruses. Older techniques like ZFN and TALEN cut DNA at specific sites, while CRISPR/Cas9 uses a Cas9 enzyme guided by CRISPR RNA to make precise cuts. Delivery methods for Cas9 include plasmids, mRNA, and RNP complexes. Viral vectors like AAV are commonly used but have limits. Physical methods also deliver Cas9 via nanoparticles or peptides.
This document provides an overview of biotechnology concepts and applications. It discusses how recombinant DNA can be created in the laboratory using restriction enzymes, DNA ligase, and gel electrophoresis. This DNA can then be used to genetically transform cells and organisms using vectors like plasmids and viruses. Genes can be manipulated through techniques like gene knockout, antisense RNA, and microarrays. Finally, the document outlines wide applications of biotechnology in medicine, agriculture, and industry, including producing insulin, pharmaceuticals in transgenic animals/plants, and genetically modifying crops.
This document summarizes a study that investigated the complexes containing the RNA helicase DDX6, which is a key component of P-bodies involved in posttranscriptional regulation. The researchers identified DDX6 complexes using tandem affinity purification coupled with mass spectrometry. They found that DDX6 was present in three main complexes: the decapping complex, a CPEB-like complex, and an Ataxin2/Ataxin2L complex. Investigation of P-body assembly under various conditions identified three proteins required for assembly in all conditions: DDX6, 4E-T, and LSM14A. The results reveal that P-body assembly involves different pathways that nevertheless share these three key factors connecting
The 5' terminal uracil of let-7a is critical for the recruitment of mRNA to A...David W. Salzman
This document investigates the interaction between let-7a microRNA, Argonaute2 protein, and mRNA targets. It finds that recombinant Argonaute2 is sufficient to direct let-7a-guided cleavage of a fully complementary mRNA target in vitro. Additionally, it determines that the 5' terminal uracil of let-7a is critical for recruitment of the mRNA target to the let-7a-Argonaute2 complex. Mutation of this 5' uracil inhibits formation of the ternary let-7a-Argonaute2-mRNA complex, but does not affect formation of the binary let-7a-Argonaute2 complex. This suggests the 5' urac
This study examines the degradation pathway of the thiazide-sensitive NaCl cotransporter (NCC) in yeast and mammalian cells. The authors show that NCC is a substrate of endoplasmic reticulum-associated degradation (ERAD) in yeast. Using yeast strains with mutations in ERAD components, they identify the E3 ubiquitin ligase Hrd1 and the cytoplasmic Hsp70 chaperone Ssa1/Hsp70 as important for NCC ubiquitination and degradation. Expression of NCC in mammalian kidney cells reveals similar polyubiquitination and proteasome-dependent degradation. Cytoplasmic Hsp70 preferentially associates with immature glycosylated NCC, indicating its role
1. The document discusses lateral root development in Arabidopsis thaliana, including the initiation and stages of lateral root primordia formation. Key processes like cell division and emergence are described.
2. Lateral root development is regulated by auxin and other signals in a transcriptional cascade. Auxin binds to repressor proteins and activates transcription factors that regulate genes involved in cell wall remodeling and lateral root primordia development.
3. The document also covers root gravitropism, describing the three phases of gravity perception in columella cells, signal transduction through auxin transport, and the gravitropic response through differential cell elongation. Many genes involved in these processes remain unidentified.
1) Wnt3a protein rapidly increases the frequency of miniature excitatory synaptic currents in hippocampal neurons through a mechanism involving calcium influx and post-translational modifications enhancing vesicle exocytosis.
2) While previous studies suggested Wnt signaling modulates neurotransmission, this is the first to demonstrate a direct effect of a purified Wnt ligand, Wnt3a, on synaptic transmission.
3) The results identify Wnt3a and its receptor LRP6 as key molecules in neurotransmission modulation and suggest crosstalk between canonical and Wnt/calcium signaling in central neurons.
Regulation Of Atp7 A Gene Expression By The Grx1 As An Inducer In Menkes D...pranamees
This document summarizes an engineering approach to address Menkes disease by expressing an ATP7A-Glrx1 gene cassette. Menkes disease is caused by mutations in the ATP7A gene resulting in a truncated copper-transporting protein. The approach involves designing an expression vector containing the ATP7A gene and the Glrx1 gene, which interacts with ATP7A, to allow Glrx1 to potentially restore copper transport function despite ATP7A truncation upon transfection into intestinal cells. Validation would assess GFP expression and copper transport capability of the transfected truncated ATP7A protein with co-expressed Glrx1.
This document is a thesis presented by Archie Lovatt for the degree of Doctor of Philosophy at the University of Leicester in 1994. The thesis describes molecular analysis of a P-N-acetyl-hexosaminidase gene from the oral pathogen Porphyromonas gingivalis W83. Lovatt cloned the nahA gene encoding P-N-acetyl-hexosaminidase from P. gingivalis and characterized the gene and its protein product. Sequence analysis revealed that nahA is 2331 base pairs long and encodes a 111 amino acid protein with homology to hexosaminidases from other sources. The nahA gene appears to be conserved in P. gingivalis strains
DNA damage repair Neil3 gene Knockout in MOLT-4iosrjce
RNAi is superannuated cellular mechanism that protect organism against viruses that replicate
through double- stranded RNA. RNAi can be used to diminish gene expression from plasmid expressing and
inserted sequence repeat. A stable harpin would be expressed after the vector was integrated into the genome.
In this paper a shiRNA expressing vector for Neil3 was designed and developed which is capable of replication
in MOLT-4. This shiRNA vector had the ability to arose the RNAi pathway, and reduce the gene expression of
Neil3. This was assessed by using pSilence 4.1CMV as a vector, and Gapdh as positive control.
This document describes a method for performing RNA sequencing on single nuclei. Key points:
1) The authors demonstrate that double-stranded cDNA can be synthesized from single mouse neural progenitor cell nuclei and hippocampal tissue nuclei, allowing for whole transcriptome sequencing.
2) On average, sequencing of single nuclei detected over 16,000 of the approximately 24,000 mouse protein-coding genes.
3) Analysis of single nuclei avoids issues with dissociating intact cells from complex tissues, is applicable across eukaryotic species, and provides insight into nuclear gene regulation.
4) RNA sequencing of single nuclei is a powerful new method for investigating gene expression at the single cell level without disrupting cells.
Western Blotting Of Camkii Β And T 287Beth Salazar
1. Tomato production is affected by various bacterial, fungal and viral diseases which can cause considerable yield losses.
2. One of the most devastating diseases is tomato leaf curl disease (ToLCD), caused by geminiviruses, which is increasing worldwide and poses a major constraint to tomato production in India.
3. ToLCD causes serious yield losses according to studies from the 1940s and more recently. Effective management strategies are needed to control this and other diseases threatening tomato production.
Lydia Yeshitla, Research Scholar at the Neurobiology Section of UCSDLydia Yeshitla
1) The document describes an experiment cloning a pH-sensitive fluorescent protein (pHRed) onto the GLUA1 AMPA receptor subunit to track intracellular trafficking and degradation of AMPA receptors by lysosomes.
2) Restriction enzymes (AGE1 and BSRG1) were used to cut the DNA in order to ligate pHRed onto GLUA1 using PCR. This would allow detection of AMPA receptors in the acidic lysosome lumen.
3) Bacteria were transformed with the ligated pHRed-GluA1 DNA. Colonies were selected and the DNA was sequenced to validate that the cloning procedure was done correctly.
The document discusses several key topics in biology including different philosophies of scientific discovery, evolutionary theory and computational analysis bridging approaches, complexity across levels of biological organization, and challenges in the scientific process. It provides examples of protein domain analysis illuminating relationships across life and computational analysis of biological networks. It also discusses sociological challenges around scientific competition, publication, and peer review that can hamper effective transmission of discoveries.
This study examines the expression of nicotinic acetylcholine receptors (nAChRs) in neuroepithelial bodies (NEBs) of neonatal hamster lung. The key findings are:
1) NEB cells express mRNA for the β2 subunit of nAChRs and contain α4, α7, and β2 nAChR subunits, as shown by in situ hybridization and immunofluorescence.
2) Patch clamp recordings of NEB cells show that nicotine activates inward currents in a concentration-dependent manner, mediated by nAChRs.
3) The nicotine-induced currents have properties consistent with α3β2, α4β2, and α7
This study tested the hypothesis that a base-pairing interaction between nucleotide A79 in the Hepatitis Delta Virus (HDV) ribozyme and nucleotide U(-1) in its substrate is necessary for catalytic activity. Mutant ribozymes and substrates with variations at these positions were created and their kinetic activity analyzed. While mutation of A79 significantly reduced activity, further experiments found the hypothesis was incorrect. Additional nucleotides like A78 may interact with the substrate and warrant further investigation.
This document summarizes a study investigating the minimum requirements for reconstituting an RNA interference (RNAi) pathway in yeast. The key findings are:
1) RNAi can be reconstituted in yeast by introducing the Dicer and Argonaute proteins from another yeast species, Saccharomyces castellii, but not with human Dicer.
2) Both S. castellii and human Argonaute proteins require regulation by the heat shock protein Hsp90 to function in the yeast RNAi pathway, suggesting this regulatory mechanism has been conserved.
3) Unlike previous reports, the study found that human Dicer, TRBP2 and Argonaute2 were not sufficient to reconstit
Modulation of MMP and ADAM gene expression in human chondrocytes by IL-1 and OSMpjtkoshy
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P68 RNA helicase unwinds the human let-7 microRNA precursor duplex and is required for let-7-directed silencing of gene expression.
1. Shubert-Coleman and Henry Furneaux
David W. Salzman, Jonathan
Gene Expression
Required for let-7-directed Silencing of
let-7 MicroRNA Precursor Duplex and Is
P68 RNA Helicase Unwinds the Human
RNAs:
RNA-Mediated Regulation and Noncoding
doi: 10.1074/jbc.M705054200 originally published online August 27, 2007
2007, 282:32773-32779.J. Biol. Chem.
10.1074/jbc.M705054200Access the most updated version of this article at doi:
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at YALE UNIV | Kline Science Library on August 21, 2013http://www.jbc.org/Downloaded from
3. EXPERIMENTAL PROCEDURES
HeLa S3 cells were obtained from the National Cell Culture
Center (Minneapolis, MN). Synthetic RNAs and siRNAs were
obtained from Dharmacon Research Inc. (Lafayette, CO).
Luciferase reporter plasmids were provided by the David Bartel
laboratory. The His-P68 plasmid was provided by the Zhi-Ren
Liu laboratory. Anti-P68 monoclonal antibody (PAB204) was
obtained from Upstate Biochemicals, and monoclonal antibod-
ies against GAPD and Vimentin were obtained from Abcam.
Preparation of HeLa Cell Extract—HeLa S3 cells (National
Cell Culture Center) were resuspended in hypotonic buffer (50
mM Tris, pH 7.5, 10 mM KCl, 5 mM dithiothreitol). The swollen
cells were homogenized, and KCl, MgCl2, and glycerol were
added to final concentrations of 100 mM, 2 mM, and 10%,
respectively. The homogenate was centrifuged at 500 ϫ g for 10
min. The supernatant was removed,
and the pellet was resuspended in
buffer A (50 mM Tris, pH 7.5, 2 mM
MgCl2, 5 mM dithiothreitol, 10%
glycerol). KCl was added dropwise
to a final concentration of 400 mM.
The homogenate was centrifuged at
10,000 ϫ g for 10 min. The resultant
nuclear extract was stored at Ϫ80 °C
in aliquots.
RNA Affinity Chromatography—
0.5 ml of avidin A beads (Vector
Laboratories) were incubated with
36 mol of biotinylated let-7 pre-
cursor hairpin RNA in 0.6 ml vol-
ume with buffer A (50 mM Tris, pH
7.5, 0.01% Nonidet P-40, 10% glyc-
erol) at 4 °C for 8 h. Beads were
washed with buffer A containing 1 M
NaCl and then equilibrated with
buffer A containing 50 mM NaCl.
Nuclear extract was applied to the
column and washed with buffer A
containing 50 mM NaCl. The col-
umn was then eluted with buffer A
containing a 50 mM stepwise 0.05
M-0.8 M NaCl gradient.
Preparation of Recombinant His-
P68 RNA Helicase—Recombinant
His-P68 was prepared as described
previously (31). In short, His-P68
was induced in BL21 (DE3) cells
with 0.1 mM isopropyl-1-thio--D-
galactopyranoside at 37 °C for 6 h.
Bacterial pellets were resuspended
in lysis buffer (50 mM Tris, pH 8.0,
0.1 M NaCl, 0.5 mM EDTA, pH 8.0)
and were lysed by the addition of
lysozyme (0.2 mg/ml) and Triton
X-100 (1%). The resulting superna-
tant was applied to a nickel-nitrilo-
triacetic acid agarose column. The
column was first washed with lysis
buffer containing 20 mM imidazole followed by lysis buffer con-
taining 20 mM imidazole and 0.15 mM NaCl. Recombinant His-
P68 was then eluted with buffer containing 250 mM NaCl and
250 mM imidazole.
Preparation of Labeled MicroRNA Precursor Duplex—The
let-7 guide strand was labeled using T4 polynucleotide kinase
and [␥-32
P]ATP (Amersham Biosciences). After phenol-chlo-
roform extraction, it was annealed (65 °C for 5 min and then
37 °C for 25 min) to a 5-fold excess of let-7 passenger strand.
The duplex was then gel-purified and stored in 50 mM Tris, pH
7.5, and 0.2 M potassium acetate.
Precursor Duplex-unwinding Assay—Reaction mixtures
(0.02 ml) contained labeled let-7 precursor duplex (1 nM), 50
mM Tris, pH 7.5, 50 mM NaCl, 2 mM MgCl2, 5 mM ATP, and
nuclear extract or recombinant P68 RNA helicase as indicated.
FIGURE 1. Identification of a novel activity from human cells that unwinds the let-7 duplex. A, sequence
and structure of the Let-7 microRNA duplex substrate. The microRNA or guide strand is the bottom strand.
B,theduplexlet-7substrateanditssinglestrandedderivative(producedbyheatpenetration)asanalyzedbynative
gel electrophoresis. C, 32
P-labeled let-7 duplex was incubated with increasing amounts of crude extract made from
HeLacells(amountsindicated).Reactionsweredeproteinizedandanalyzedforthepresenceofsingle-strandedRNA
using native polyacrylamide gel electrophoresis. The asterisk indicates the addition of heat-inactivated extract.
D, crude HeLa cell extract was fractionated using a Sephadex S-200 column. Fractions (2 l) were assayed for
unwinding activity, using 32
P-labeled let-7 duplex. Molecular mass markers are: alcohol dehydrogenase (150 kDa),
bovineserumalbumin(68kDa),andcytochromec(12.4kDa).E,32
P-labeledlet-7duplexwasassayedforunwinding
activity,usingfraction20fromtheSephadexS-200column(2g)intheabsenceorpresenceofdifferentnucleoside
cofactors (as indicated). The asterisk indicates a reaction containing a heat-inactivated Sephadex fraction.
P68 RNA Helicase Is Required for Silencing of Gene Expression
32774 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282•NUMBER 45•NOVEMBER 9, 2007at YALE UNIV | Kline Science Library on August 21, 2013http://www.jbc.org/Downloaded from
4. After 30 min at 37 °C, the reaction was terminated by the addi-
tion of SDS-EDTA buffer (50% glycerol, 0.1 M Tris, pH 8.0, 0.5%
SDS, 20 mM EDTA, 0.1% Nonidet P-40, 0.1% each bromphenol
blue, and xylene cyanol) and analyzed by 15% native polyacryl-
amide gel electrophoresis.
Luciferase Assay of MicroRNA Function—HeLa cells were
transfected using Lipofectamine 2000 in a 12-well plate using
the indicated amounts of firefly luciferase reporter plasmid and
either -galactosidase or Renilla luciferase plasmid. Firefly and
either -galactosidase or Renilla luciferase activities were
assayed 36–48 h after transfection using the Dual Luciferase
assay (Promega). Firefly activity was normalized by cotransfec-
tion of either -galactosidase or Renilla plasmids to control for
transfection efficiency.
RESULTS
The Identification of an ATP-dependent Activity That Can
Unwind the let-7 MicroRNA Duplex—We elected to use the
human let-7 duplex (Fig. 1A) as a model since let-7 is absolutely
conserved between worms and humans and has been biochem-
ically studied in many systems (12, 33–39). This choice there-
fore permits the ready comparison of any identified human pro-
tein co-factors with those identified in other organisms.
However, one disadvantage of the human let-7 duplex is that a
discernible level of single strand is produced merely on incuba-
tion at 37 °C. Thus, in all experiments, we included a negative
comparison control (a heat-inactivated corresponding cellular
fraction) so that we could clearly
distinguish any cellular helicase
activity from the enzyme-indepen-
dent background. Fig. 1B shows that
the let-7 duplex was unwound on
incubation with nuclear extract
made from HeLa cells. The extent of
unwinding titrated with the amount
of extract added to the reaction. To
extend this observation and to
establish the native molecular
weight of the activity, we fraction-
ated the extract using a Sephadex
S-200 column. Fractions were col-
lected and assayed for helicase
activity. There was a minor high
molecular weight species; however,
the majority of the unwinding activ-
ity eluted in an inclusion volume
consistent with a native molecular
mass of 68 kDa (Fig. 1C).
Next, using this partially purified
material, we investigated whether
the unwinding activity required
ATP. Little unwinding was seen in
the absence of ATP, whereas the
addition of ATP markedly stimu-
lated the reaction and appeared to
saturate at 5 mM. In addition, no
unwinding activity was seen in reac-
tion mixtures containing AMP-
PNP, a non-hydrolyzable ATP analog (Fig. 1D). From these
observations,weconcludedthatwehaveidentifiedanovelATP-
dependent activity in HeLa cell extract that is capable of
unwinding the let-7 microRNA duplex and that this activity has
a native molecular mass of ϳ68 kDa.
P68 RNA Helicase Co-purifies with the MicroRNA Duplex-
unwinding Activity—Our initial observations suggested that
the activity that unwinds the let-7 duplex precursor might
correspond to P68 RNA helicase. P68 RNA helicase is an
ATP-dependent RNA-unwinding enzyme of 68 kDa that has
been found to be a subunit of the Drosha-processing com-
plex (8, 30–32, 40). Accordingly, we elected to affinity-purify
our unwinding activity and investigate whether it co-puri-
fied with P68 RNA helicase. We prepared an affinity column
by immobilizing a biotinylated hairpin sequence containing
the let-7 passenger and guide sequences (Fig. 2A) to avidin
beads.
HeLa cell extract was applied, the column was washed with
low salt, and bound protein was eluted using a step gradient
from 0.05 to 0.8 M NaCl (Fig. 2B). A significant portion of the
microRNA helicase activity was retained and eluted at 200–250
mM NaCl (Fig. 2C). Next, we assayed for the presence of P68
RNA helicase by Western blot and found that it was precisely
coincident with the unwinding activity (Fig. 2D). Thus, we con-
cluded that P68 was indeed a strong candidate for the
microRNA-unwinding activity.
FIGURE 2. Purification of the let-7 microRNA helicase activity yields P68 RNA helicase as a likely candidate.
A,thesequenceandlikelystructureofthelet-7hairpinRNAusedtopurifytheactivity.BiindicatestheBiotinmoiety.
B, HeLa cell extract was applied to a let-7 hairpin affinity column and eluted with a salt gradient. C, fractions (2 l)
wereassayedforunwindingactivity,using32
P-labeledlet-7duplex.D,fractionsfromthelet-7hairpinaffinitycolumn
(2 l) were analyzed via Western blot for P68 RNA helicase using PAb204. Molecular size markers are
indicated in kDa.
P68 RNA Helicase Is Required for Silencing of Gene Expression
NOVEMBER 9, 2007•VOLUME 282•NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 32775at YALE UNIV | Kline Science Library on August 21, 2013http://www.jbc.org/Downloaded from
5. Recombinant P68 RNA Helicase Is Sufficient to Unwind the
let-7 MicroRNA Duplex—Next, we investigated whether
recombinant P68 was sufficient to unwind the let-7
microRNA duplex, and if so, whether its properties resem-
bled those displayed by the native activity. First, we investi-
gated the structural features of the microRNA duplex that
are necessary for unwinding by the native activity. We syn-
thesized two mutant derivatives of the let-7 duplex. In the
first mutant (mutant 1), nucleotide substitutions were made
so that every nucleotide of the microRNA guide strand was
annealed to the passenger. This mutant is a “bad” siRNA in
which the 5Ј end of the guide strand is annealed to the pas-
senger strand. Previous studies
have shown that the guide strand
of such an siRNA will not be
readily incorporated into a silenc-
ing complex (21, 22, 26, 41). This
mutant was a very poor substrate;
virtually no unwinding activity
was noted even on incubation with
saturating amounts of affinity-pu-
rified unwinding activity (Fig. 3, A
and B).
Next, we synthesized a mutant
that lacked the internal bulges of
the microRNA duplex yet retains
the unpaired structure of the 5Ј
end of the guide strand. This
mutant (mutant 2) is analogous to
a “good” siRNA in which the guide
strand is readily incorporated into
the silencing complex (41). Impor-
tantly, this mutant was also a very
poor substrate and indicates that
the unwinding activity recognized
the internal bulges and therefore
can distinguish between an siRNA
duplex and a microRNA duplex.
Indeed, the importance of the
internal bulges was illustrated by
the observation that a third
mutant, in which the bulges were
retained and the 5Ј end of the
guide was annealed to the passen-
ger strand, exhibited significant
unwinding Fig. 3, A and B).
Importantly, recombinant P68
RNA helicase also displayed a
marked preference for a microRNA
duplex, and the critical role of the
internal bulges was similarly evi-
dent. Thus, we concluded that P68
RNA helicase is sufficient to unwind
the let-7 duplex, and like the affini-
ty-purified activity, it prefers a
microRNA duplex to an siRNA
duplex (Fig. 3, A and C).
P68 RNA Helicase Is Required
for let-7 MicroRNA Function in HeLa Cells—If P68 RNA
helicase was required for the unwinding of the let-7
microRNA duplex, one would predict that its down-regula-
tion would prevent loading of let-7 microRNA into the
silencing complex and that significant inhibition of
microRNA activity would result. To test this hypothesis, we
utilized a reporter plasmid (pIS-Lin41(s) (42)) that contains
a previously characterized let-7-response element found in
lin-41 mRNA. Indeed, transfection of this plasmid into HeLa
cells resulted in a 22-fold repression when compared with
normalized luciferase activity in comparison with that
expressed by the parental plasmid that lacks the response
FIGURE 3. Purified recombinant His-P68 RNA helicase is sufficient to unwind the let-7 microRNA duplex.
A, 32
P-labeled let-7 duplexes were analyzed for helicase activity using affinity-purified material (left panel,
amounts indicated) or recombinant His-P68 (middle panel, amounts indicated). I, indicates the input into each
reaction. ⌬T indicates heat-denatured input. The sequence and structure of each let-7 duplex are indicated
next to the companion assay. Mutations along the passenger strand are indicated in red. * and N indicate heat
denaturedandnativeprotein,respectively.B,quantificationoftheunwindingactivityexhibitedbytheaffinity-
purified material. C, quantification of the unwinding activity exhibited by recombinant His-P68.
P68 RNA Helicase Is Required for Silencing of Gene Expression
32776 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282•NUMBER 45•NOVEMBER 9, 2007at YALE UNIV | Kline Science Library on August 21, 2013http://www.jbc.org/Downloaded from
6. element (Fig. 4A). Evidence that this suppression was exerted
by let-7 was provided by the observation that this suppression
was alleviated by AntagomiRs against let-7 but not by
AntagomiRs against an irrelevant microRNA (Fig. 4A).
Moreover, the suppressive activ-
ity of the element was attenuated
by mutations that compromise the
annealing of let-7 (Fig. 4B). In the
following experiment, we have
used the comparison of the lucif-
erase activity between the wild
type (WT) and mutant (MUT) let-
7-response elements as a measure
of let-7 activity. siRNA-mediated
down-regulation of P68 RNA heli-
case, but not GAPDH, attenuated
the activity of let-7 microRNA
(Fig. 4B). Importantly, GAPDH
was successfully down-regulated
as shown by Western blot analysis
using Vimentin as a loading con-
trol (Fig. 4C). Thus, we conclude
that P68 RNA helicase is indeed
required for microRNA activity
and is likely required for the incor-
poration of the microRNA into the
Argonaute2 complex.
DISCUSSION
The single-stranded small RNA-
directed silencing of mRNA has
been reconstituted with recombi-
nant Argonaute2 (18). However, the
precursor microRNA duplex-di-
rected silencing of mRNA has not
yet been reconstituted with the can-
didate recombinant proteins. Thus,
it is likely that other factors remain
to be discovered. In these studies,
we have identified an ATP-depend-
ent unwinding activity that specifi-
cally unwinds the let-7 microRNA
duplex yet exhibits little activity
on a derived siRNA duplex. This
observation reinforces the current
perception that the guide strands
of siRNA and microRNA duplexes
arrive in Argonaute2 complexes
through different pathways (20,
22, 26). Indeed, it will be interest-
ing to see whether RNA helicase A,
which has been implicated in the
unwinding of the siRNA duplex
(27), is also capable of unwinding
some microRNA duplexes.
Our size fractionation analysis
and affinity purification studies sug-
gested that a principal component
of the unwinding activity corresponds to the P68 RNA helicase.
P68 was originally identified in human cells due to its coinci-
dental reactivity with a monoclonal antibody directed against
SV40 large T antigen (40).
FIGURE 4. P68 RNA helicase is required for let-7 microRNA function in HeLa cells. A, HeLa cells were
co-transfected with a -galactosidase plasmid (100 ng) and either the parental plasmid (pIS-0) or a let-7-
responsive reporter (lin41(s) WT (where WT indicates wild type)) (100 ng, as indicated), along with AntagomiRs
against let-7 or Mir-16 (amounts indicated). Cells were incubated for 36 h and subsequently analyzed for
luciferase activity. Results are normalized to cotransfected plasmid encoding -galactosidase. B, HeLa cells
wereco-transfectedwithaRenillaluciferasereporter(100ng)andthepIS-0,lin-41(s)WT,orlin-41(s)MUT(where
MUT indicates mutant) (42) luciferase reporter (100 ng, as indicated) along with siRNA directed against either
P68 RNA helicase or GAPDH (amounts indicated). Cells were incubated for 40 h and analyzed for luciferase
activity. Results are normalized to Renilla luciferase. C, left panel, HeLa cells treated with siRNA against P68 RNA
helicase or GAPDH (amounts indicated) were analyzed for protein via Western blot. Protein levels were nor-
malized to Vimentin.
P68 RNA Helicase Is Required for Silencing of Gene Expression
NOVEMBER 9, 2007•VOLUME 282•NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 32777at YALE UNIV | Kline Science Library on August 21, 2013http://www.jbc.org/Downloaded from
7. P68 was originally believed to be an RNA helicase due to its
homology to the DEAD box of eIF-4A, a well characterized
RNA helicase (43, 44). When P68 was assayed for unwinding
activity, it was found to be an ATP-dependent RNA helicase,
which can unwind RNA duplexes in both 3Ј to 5Ј and 5Ј to 3Ј
directions (30–32). The substrates unwound by P68 RNA heli-
case range in size from 22 to 175 nucleotides in length and
contain overhangs of varied lengths ranging from 6 to 185
nucleotides long (30–32). Thus, our observations are consist-
ent with the known properties of P68 RNA helicase.
P68 RNA helicase has been implicated in many cellular func-
tions. In some cases, for example, in its perceived role as a tran-
scriptional regulator, this function does not require helicase
activity (45). In most cases, however, its touted role in mRNA
splicing, rRNA processing, and mRNA decay requires the
integrity of the helicase domain (46–48). Our studies here lead
us to speculate that P68 RNA helicase might regulate the
expression of many microRNAs with a consequent pleiotropic
effect upon cellular function. It is possible that this function
might accommodate many of the previously ascribed functions
in RNA metabolism. In any event, the regulation of microRNA
activity would be consistent with its well described role in cel-
lular proliferation. However, it remains possible that P68 RNA
helicase may only unwind particular subclasses of microRNA
duplexes, and its role in a particular cell function may be pecu-
liar to the miRNAs that are expressed in a given cell type.
Our contention that P68 RNA helicase plays a role in the
unwinding of the let-7 microRNA duplex is strengthened by the
previous observation that it is a subunit of the affinity-purified
Drosha-processing complex (48). In addition, it has been
recently demonstrated that mouse embryonic fibroblasts that
lack P68 RNA helicase are compromised in their expression of
many microRNAs (48). Importantly, we show here, for the first
time, that a recombinant RNA helicase is sufficient to unwind a
microRNA precursor duplex. From this key observation, we
would propose that P68 RNA helicase might drive the selective
uptake of the let-7 guide strand into the silencing complex. So
far, however, our preliminary attempts to directly demonstrate
this using purified, recombinant proteins have not been suc-
cessful. However, the present studies have identified an essen-
tial co-factor that may ultimately facilitate the reconstitution of
this critical step.
REFERENCES
1. Calin, G. A., and Croce, C. M. (2006) Cancer Res. 66, 7390–7394
2. Ambros, V. (2001) Cell 107, 823–826
3. Zamore, P. D., and Haley, B. (2005) Science 309, 1519–1524
4. Meister, G., and Tuschl, T. (2004) Nature 431, 343–349
5. Thai, T. H., Calado, D. P., Casola, S., Ansel, K. M., Xiao, C., Xue, Y.,
Murphy, A., Frendewey, D., Valenzuela, D., Kutok, J. L., Schmidt-Sup-
prian, M., Rajewsky, N., Yancopoulos, G., Rao, A., and Rajewsky, K. (2007)
Science 316, 604–608
6. Lee, Y., Kim, M., Han, J., Yeom, K. H., Lee, S., Baek, S. H., and Kim, V. N.
(2004) EMBO J. 23, 4051–4060
7. Lee, Y., Ahn, C., Han, J., Choi, H., Kim, J., Yim, J., Lee, J., Provost, P.,
Radmark, O., Kim, S., and Kim, V. N. (2003) Nature 425, 415–419
8. Gregory, R. I., Yan, K. P., Amuthan, G., Chendrimada, T., Doratotaj, B.,
Cooch, N., and Shiekhattar, R. (2004) Nature 432, 235–240
9. Han, J., Lee, Y., Yeom, K. H., Kim, Y. K., Jin, H., and Kim, V. N. (2004)
Genes Dev. 18, 3016–3027
10. Han, J., Lee, Y., Yeom, K. H., Nam, J. W., Heo, I., Rhee, J. K., Sohn, S. Y.,
Cho, Y., Zhang, B. T., and Kim, V. N. (2006) Cell 125, 887–901
11. Provost, P., Dishart, D., Doucet, J., Frendewey, D., Samuelsson, B., and
Radmark, O. (2002) EMBO J. 21, 5864–5874
12. Hutvagner, G., McLachlan, J., Pasquinelli, A. E., Balint, E., Tuschl, T., and
Zamore, P. D. (2001) Science 293, 834–838
13. Macrae, I. J., Zhou, K., Li, F., Repic, A., Brooks, A. N., Cande, W. Z., Adams,
P. D., and Doudna, J. A. (2006) Science 311, 195–198
14. Lee, Y. S., Nakahara, K., Pham, J. W., Kim, K., He, Z., Sontheimer, E. J., and
Carthew, R. W. (2004) Cell 117, 69–81
15. Liu, J., Carmell, M. A., Rivas, F. V., Marsden, C. G., Thomson, J. M., Song,
J. J., Hammond, S. M., Joshua-Tor, L., and Hannon, G. J. (2004) Science
305, 1437–1441
16. Meister, G., Landthaler, M., Patkaniowska, A., Dorsett, Y., Teng, G., and
Tuschl, T. (2004) Mol. Cell 15, 185–197
17. Okamura, K., Ishizuka, A., Siomi, H., and Siomi, M. C. (2004) Genes Dev.
18, 1655–1666
18. Rivas, F. V., Tolia, N. H., Song, J. J., Aragon, J. P., Liu, J., Hannon, G. J., and
Joshua-Tor, L. (2005) Nat. Struct. Mol. Biol. 12, 340–349
19. Gregory, R. I., Chendrimada, T. P., Cooch, N., and Shiekhattar, R. (2005)
Cell 123, 631–640
20. Maniataki, E., and Mourelatos, Z. (2005) Genes Dev. 19, 2979–2990
21. Tomari, Y., Matranga, C., Haley, B., Martinez, N., and Zamore, P. D.
(2004) Science 306, 1377–1380
22. Liu, X., Jiang, F., Kalidas, S., Smith, D., and Liu, Q. (2006) RNA (Cold Spring
Harbor) 12, 1514–1520
23. Liu, Q., Rand, T. A., Kalidas, S., Du, F., Kim, H. E., Smith, D. P., and Wang,
X. (2003) Science 301, 1921–1925
24. Haase, A. D., Jaskiewicz, L., Zhang, H., Laine, S., Sack, R., Gatignol, A., and
Filipowicz, W. (2005) EMBO Rep. 6, 961–967
25. Leuschner, P. J., Ameres, S. L., Kueng, S., and Martinez, J. (2006) EMBO
Rep. 7, 314–320
26. Matranga, C., Tomari, Y., Shin, C., Bartel, D. P., and Zamore, P. D. (2005)
Cell 123, 607–620
27. Robb, G. B., and Rana, T. M. (2007) Mol. Cell 26, 523–537
28. Chendrimada, T. P., Gregory, R. I., Kumaraswamy, E., Norman, J., Cooch,
N., Nishikura, K., and Shiekhattar, R. (2005) Nature 436, 740–744
29. Giraldez, A. J., Cinalli, R. M., Glasner, M. E., Enright, A. J., Thomson, J. M.,
Baskerville, S., Hammond, S. M., Bartel, D. P., and Schier, A. F. (2005)
Science 308, 833–838
30. Iggo, R. D., and Lane, D. P. (1989) EMBO J. 8, 1827–1831
31. Huang, Y., and Liu, Z. R. (2002) J. Biol. Chem. 277, 12810–12815
32. Hirling, H., Scheffner, M., Restle, T., and Stahl, H. (1989) Nature 339,
562–564
33. Johnson, S. M., Grosshans, H., Shingara, J., Byrom, M., Jarvis, R., Cheng,
A., Labourier, E., Reinert, K. L., Brown, D., and Slack, F. J. (2005) Cell 120,
635–647
34. Mayr, C., Hemann, M. T., and Bartel, D. P. (2007) Science 315, 1576–1579
35. Pasquinelli, A. E., Reinhart, B. J., Slack, F., Martindale, M. Q., Kuroda, M. I.,
Maller, B., Hayward, D. C., Ball, E. E., Degnan, B., Muller, P., Spring, J.,
Srinivasan, A., Fishman, M., Finnerty, J., Corbo, J., Levine, M., Leahy, P.,
Davidson, E., and Ruvkun, G. (2000) Nature 408, 86–89
36. Pillai, R. S., Bhattacharyya, S. N., Artus, C. G., Zoller, T., Cougot, N.,
Basyuk, E., Bertrand, E., and Filipowicz, W. (2005) Science 309,
1573–1576
37. Reinhart, B. J., Slack, F. J., Basson, M., Pasquinelli, A. E., Bettinger, J. C.,
Rougvie, A. E., Horvitz, H. R., and Ruvkun, G. (2000) Nature 403,
901–906
38. Vella, M. C., Choi, E. Y., Lin, S. Y., Reinert, K., and Slack, F. J. (2004) Genes
Dev. 18, 132–137
39. Kloosterman, W. P., Wienholds, E., Ketting, R. F., and Plasterk, R. H.
(2004) Nucleic Acids Res. 32, 6284–6291
40. Lane, D. P., and Hoeffler, W. K. (1980) Nature 288, 167–170
41. Schwarz, D. S., Hutvagner, G., Du, T., Xu, Z., Aronin, N., and Zamore,
P. D. (2003) Cell 115, 199–208
42. Lewis, B. P., Shih, I. H., Jones-Rhoades, M. W., Bartel, D. P., and Burge,
C. B. (2003) Cell 115, 787–798
43. Lane, D. (1988) Nature 334, 478
P68 RNA Helicase Is Required for Silencing of Gene Expression
32778 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282•NUMBER 45•NOVEMBER 9, 2007at YALE UNIV | Kline Science Library on August 21, 2013http://www.jbc.org/Downloaded from
8. 44. Ray, B. K., Lawson, T. G., Kramer, J. C., Cladaras, M. H., Grifo, J. A.,
Abramson, R. D., Merrick, W. C., and Thach, R. E. (1985) J. Biol. Chem.
260, 7651–7658
45. Bates, G. J., Nicol, S. M., Wilson, B. J., Jacobs, A. M., Bourdon, J. C., War-
drop, J., Gregory, D. J., Lane, D. P., Perkins, N. D., and Fuller-Pace, F. V.
(2005) EMBO J. 24, 543–553
46. Lin, C., Yang, L., Yang, J. J., Huang, Y., and Liu, Z. R. (2005) Mol. Cell. Biol.
25, 7484–7493
47. Ishizuka, A., Siomi, M. C., and Siomi, H. (2002) Genes Dev. 16, 2497–2508
48. Fukuda, T., Yamagata, K., Fujiyama, S., Matsumoto, T., Koshida, I., Yo-
shimura, K., Mihara, M., Naitou, M., Endoh, H., Nakamura, T., Akimoto,
C., Yamamoto, Y., Katagiri, T., Foulds, C., Takezawa, S., Kitagawa, H.,
Takeyama, K., O’Malley, B. W., and Kato, S. (2007) Nat. Cell Biol. 9,
604–611
P68 RNA Helicase Is Required for Silencing of Gene Expression
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