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  1. Article ; Online: T4 RNA Ligase 2 truncated active site mutants

    Zhuang Fanglei / Munafo Daniela B / Fuchs Ryan T / Viollet Sebastien / Robb Gregory B

    BMC Biotechnology, Vol 11, Iss 1, p

    improved tools for RNA analysis

    2011  Volume 72

    Abstract: Abstract Background T4 RNA ligases 1 and 2 are useful tools for RNA analysis. Their use upstream of RNA analyses such as high-throughput RNA sequencing and microarrays has recently increased their importance. The truncated form of T4 RNA ligase 2, ... ...

    Abstract Abstract Background T4 RNA ligases 1 and 2 are useful tools for RNA analysis. Their use upstream of RNA analyses such as high-throughput RNA sequencing and microarrays has recently increased their importance. The truncated form of T4 RNA ligase 2, comprising amino acids 1-249 (T4 Rnl2tr), is an attractive tool for attachment of adapters or labels to RNA 3'-ends. Compared to T4 RNA ligase 1, T4 Rnl2tr has a decreased ability to ligate 5'-PO 4 ends in single-stranded RNA ligations, and compared to the full-length T4 Rnl2, the T4 Rnl2tr has an increased activity for joining 5'-adenylated adapters to RNA 3'-ends. The combination of these properties allows adapter attachment to RNA 3'-ends with reduced circularization and concatemerization of substrate RNA. Results With the aim of further reducing unwanted side ligation products, we substituted active site residues, known to be important for adenylyltransferase steps of the ligation reaction, in the context of T4 Rnl2tr. We characterized the variant ligases for the formation of unwanted ligation side products and for activity in the strand-joining reaction. Conclusions Our data demonstrate that lysine 227 is a key residue facilitating adenylyl transfer from adenylated ligation donor substrates to the ligase. This reversal of the second step of the ligation reaction correlates with the formation of unwanted ligation products. Thus, T4 Rn2tr mutants containing the K227Q mutation are useful for reducing undesired ligation products. We furthermore report optimal conditions for the use of these improved T4 Rnl2tr variants.
    Keywords Biotechnology ; TP248.13-248.65 ; Chemical technology ; TP1-1185 ; Technology ; T ; DOAJ:Biotechnology ; DOAJ:Life Sciences ; DOAJ:Biology and Life Sciences
    Subject code 540
    Language English
    Publishing date 2011-07-01T00:00:00Z
    Publisher BioMed Central
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article ; Online: T4 RNA ligase 2 truncated active site mutants: improved tools for RNA analysis.

    Viollet, Sebastien / Fuchs, Ryan T / Munafo, Daniela B / Zhuang, Fanglei / Robb, Gregory B

    BMC biotechnology

    2011  Volume 11, Page(s) 72

    Abstract: Background: T4 RNA ligases 1 and 2 are useful tools for RNA analysis. Their use upstream of RNA analyses such as high-throughput RNA sequencing and microarrays has recently increased their importance. The truncated form of T4 RNA ligase 2, comprising ... ...

    Abstract Background: T4 RNA ligases 1 and 2 are useful tools for RNA analysis. Their use upstream of RNA analyses such as high-throughput RNA sequencing and microarrays has recently increased their importance. The truncated form of T4 RNA ligase 2, comprising amino acids 1-249 (T4 Rnl2tr), is an attractive tool for attachment of adapters or labels to RNA 3'-ends. Compared to T4 RNA ligase 1, T4 Rnl2tr has a decreased ability to ligate 5'-PO4 ends in single-stranded RNA ligations, and compared to the full-length T4 Rnl2, the T4 Rnl2tr has an increased activity for joining 5'-adenylated adapters to RNA 3'-ends. The combination of these properties allows adapter attachment to RNA 3'-ends with reduced circularization and concatemerization of substrate RNA.
    Results: With the aim of further reducing unwanted side ligation products, we substituted active site residues, known to be important for adenylyltransferase steps of the ligation reaction, in the context of T4 Rnl2tr. We characterized the variant ligases for the formation of unwanted ligation side products and for activity in the strand-joining reaction.
    Conclusions: Our data demonstrate that lysine 227 is a key residue facilitating adenylyl transfer from adenylated ligation donor substrates to the ligase. This reversal of the second step of the ligation reaction correlates with the formation of unwanted ligation products. Thus, T4 Rn2tr mutants containing the K227Q mutation are useful for reducing undesired ligation products. We furthermore report optimal conditions for the use of these improved T4 Rnl2tr variants.
    MeSH term(s) Adenosine Monophosphate/chemistry ; Adenosine Monophosphate/metabolism ; Analysis of Variance ; Catalytic Domain ; Electrophoresis, Polyacrylamide Gel ; High-Throughput Screening Assays/methods ; Hydrogen-Ion Concentration ; Mutation ; Polyethylene Glycols/chemistry ; RNA/analysis ; RNA/genetics ; RNA/metabolism ; RNA Ligase (ATP)/chemistry ; RNA Ligase (ATP)/genetics ; RNA Ligase (ATP)/isolation & purification ; RNA Ligase (ATP)/metabolism ; Viral Proteins/chemistry ; Viral Proteins/genetics ; Viral Proteins/isolation & purification ; Viral Proteins/metabolism
    Chemical Substances Viral Proteins ; Polyethylene Glycols (3WJQ0SDW1A) ; Adenosine Monophosphate (415SHH325A) ; RNA (63231-63-0) ; RNA Ligase (ATP) (EC 6.5.1.3) ; bacteriophage T4 RNA ligase 2 (EC 6.5.1.3)
    Language English
    Publishing date 2011-07-01
    Publishing country England
    Document type Journal Article
    ISSN 1472-6750
    ISSN (online) 1472-6750
    DOI 10.1186/1472-6750-11-72
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: RNAi triggered by specialized machinery silences developmental genes and retrotransposons.

    Yamanaka, Soichiro / Mehta, Sameet / Reyes-Turcu, Francisca E / Zhuang, Fanglei / Fuchs, Ryan T / Rong, Yikang / Robb, Gregory B / Grewal, Shiv I S

    Nature

    2012  Volume 493, Issue 7433, Page(s) 557–560

    Abstract: RNA interference (RNAi) is a conserved mechanism in which small interfering RNAs (siRNAs) guide the degradation of cognate RNAs, but also promote heterochromatin assembly at repetitive DNA elements such as centromeric repeats. However, the full extent of ...

    Abstract RNA interference (RNAi) is a conserved mechanism in which small interfering RNAs (siRNAs) guide the degradation of cognate RNAs, but also promote heterochromatin assembly at repetitive DNA elements such as centromeric repeats. However, the full extent of RNAi functions and its endogenous targets have not been explored. Here we show that, in the fission yeast Schizosaccharomyces pombe, RNAi and heterochromatin factors cooperate to silence diverse loci, including sexual differentiation genes, genes encoding transmembrane proteins, and retrotransposons that are also targeted by the exosome RNA degradation machinery. In the absence of the exosome, transcripts are processed preferentially by the RNAi machinery, revealing siRNA clusters and a corresponding increase in heterochromatin modifications across large domains containing genes and retrotransposons. We show that the generation of siRNAs and heterochromatin assembly by RNAi is triggered by a mechanism involving the canonical poly(A) polymerase Pla1 and an associated RNA surveillance factor Red1, which also activate the exosome. Notably, siRNA production and heterochromatin modifications at these target loci are regulated by environmental growth conditions, and by developmental signals that induce gene expression during sexual differentiation. Our analyses uncover an interaction between RNAi and the exosome that is conserved in Drosophila, and show that differentiation signals modulate RNAi silencing to regulate developmental genes.
    MeSH term(s) Animals ; Drosophila melanogaster/genetics ; Exome/genetics ; Gene Expression Regulation, Fungal/genetics ; Genes, Fungal/genetics ; Heterochromatin/genetics ; Multigene Family/genetics ; Polynucleotide Adenylyltransferase/genetics ; RNA Interference ; RNA Stability/genetics ; RNA, Fungal/genetics ; RNA, Small Interfering/genetics ; Retroelements/genetics ; Schizosaccharomyces/cytology ; Schizosaccharomyces/enzymology ; Schizosaccharomyces/genetics ; Schizosaccharomyces/growth & development ; Schizosaccharomyces pombe Proteins/genetics ; Schizosaccharomyces pombe Proteins/metabolism ; Sex Differentiation/genetics
    Chemical Substances Heterochromatin ; RNA, Fungal ; RNA, Small Interfering ; Retroelements ; Schizosaccharomyces pombe Proteins ; Polynucleotide Adenylyltransferase (EC 2.7.7.19)
    Language English
    Publishing date 2012-11-14
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Intramural
    ZDB-ID 120714-3
    ISSN 1476-4687 ; 0028-0836
    ISSN (online) 1476-4687
    ISSN 0028-0836
    DOI 10.1038/nature11716
    Database MEDical Literature Analysis and Retrieval System OnLINE

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