Guide RNA‐directed uridine insertion RNA editing in vitro.

Guide RNAs (gRNAs) have been proposed to mediate uridine (U) addition/deletion editing of mitochondrial mRNAs in kinetoplastid protozoa. The Us are proposed to be derived either from UTP by two successive cleavage‐ligations or transesterifications, or from the 3′ end of the gRNA by the same mechanisms. We have demonstrated gRNA‐dependent U insertions into a specific editing site of a pre‐edited mRNA which was incubated in a mitochondrial extract from Leishmania tarentolae. The predominant number of U insertions was determined by the number of guiding nucleotides in the added gRNA, and the formation of a gRNA‐mRNA anchor duplex was necessary for activity. UTP and alpha‐beta bond hydrolysis of ATP were required, and the activity was inhibited above 50–100 mM KCl. A gRNA‐independent insertion of up to approximately 13 Us occurred in the absence of the added cognate gRNA; the extent of this activity was affected by sequences upstream and downstream of the edited region. Heparin inhibited the gRNA‐independent U insertion activity and had no effect on the gRNA‐dependent activity. Blocking the 3′ OH of the gRNA had little effect on the gRNA‐dependent U insertion activity. The data are consistent with a cleavage‐ligation model in which the Us are derived directly from UTP.

[1]  T. Cech,et al.  RNA editing: World's smallest introns? , 1991, Cell.

[2]  L. Simpson,et al.  In vitro RNA editing‐like activity in a mitochondrial extract from Leishmania tarentolae. , 1995, The EMBO journal.

[3]  J. Shaw,et al.  Internal frameshifts within the mitochondrial genes for cytochrome oxidase subunit II and maxicircle unidentified reading frame 3 of Leishmania tarentolae are corrected by RNA editing: evidence for translation of the edited cytochrome oxidase subunit II mRNA. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[4]  L. Simpson,et al.  The Leishmania kinetoplast-mitochondrion contains terminal uridylyltransferase and RNA ligase activities. , 1989, The Journal of biological chemistry.

[5]  Larry Simpson,et al.  The polarity of editing within a multiple gRNA-mediated domain is due to formation of anchors for upstream gRNAs by downstream editing , 1992, Cell.

[6]  L. Simpson,et al.  Chimeric gRNA-mRNA molecules with oligo(U) tails covalently linked at sites of RNA editing suggest that U addition occurs by transesterification , 1991, Cell.

[7]  Larry Simpson,et al.  A model for RNA editing in kinetoplastid mitochondria: RNA molecules transcribed from maxicircle DNA provide the edited information , 1990, Cell.

[8]  L. Simpson,et al.  Sense from nonsense: RNA editing in mitochondria of kinetoplastid protozoa and slime molds , 1995, Cell.

[9]  L. Simpson,et al.  Kinetoplast DNA in trypanosomid flagellates. , 1986, International review of cytology.

[10]  R. Wagner,et al.  The effect of tRNA binding on the structure of 5 S RNA in Escherichia coli. A chemical modification study. , 1984, The Journal of biological chemistry.

[11]  K. Stuart,et al.  Editing of kinetoplastid mitochondrial mRNAs by uridine addition and deletion generates conserved amino acid sequences and AUG initiation codons , 1988, Cell.

[12]  O. Uhlenbeck,et al.  Synthesis of small RNAs using T7 RNA polymerase. , 1989, Methods in enzymology.

[13]  P. Myler,et al.  Trypanosoma brucei minicircles encode multiple guide RNAs which can direct editing of extensively overlapping sequences. , 1993, Nucleic acids research.

[14]  L. Simpson,et al.  Guide RNAs in kinetoplastid mitochondria have a nonencoded 3′ oligo(U) tail involved in recognition of the preedited region , 1990, Cell.

[15]  K. Stuart,et al.  RNA editing: transfer of genetic information from gRNA to precursor mRNA in vitro. , 1994, Science.

[16]  L. Simpson,et al.  Isolation of kinetoplast-mitochondrial complexes from Leishmania tarentolae. , 1974, The Journal of protozoology.

[17]  L. Simpson,et al.  The mitochondrial genome of kinetoplastid protozoa: genomic organization, transcription, replication, and evolution. , 1987, Annual review of microbiology.

[18]  L. Simpson,et al.  Synchronization of Leishmania tarentolae by hydroxyurea. , 1970, The Journal of protozoology.

[19]  J. Shaw,et al.  RNA editing and the mitochondrial cryptogenes of kinetoplastid protozoa , 1989, Cell.

[20]  L. Simpson,et al.  Guide RNA-independent and Guide RNA-dependent Uridine Insertion into Cytochrome b mRNA in a Mitochondrial Lysate from Leishmania tarentolae , 1997, The Journal of Biological Chemistry.

[21]  K. Stuart,et al.  Direct Visualization of Uridylate Deletion In Vitro Suggests a Mechanism for Kinetoplastid RNA Editing , 1996, Cell.

[22]  D. Maslov,et al.  Generation of unexpected editing patterns in Leishmania tarentolae mitochondrial mRNAs: Misediting produced by misguiding , 1992, Cell.

[23]  S. Hajduk,et al.  Organization of minicircle genes for guide RNAs in trypanosoma brucei , 1990, Cell.

[24]  J. Feagin.  RNA editing in kinetoplastid mitochondria. , 1990, The Journal of biological chemistry.

[25]  F. Bringaud,et al.  Characterization of two classes of ribonucleoprotein complexes possibly involved in RNA editing from Leishmania tarentolae mitochondria. , 1994, The EMBO journal.

[26]  Douglas H. Turner,et al.  17 Thermodynamic Considerations for Evolution by RNA , 1993 .

[27]  R. Benne,et al.  RNA Editing: The Alteration of Protein Coding Sequences of RNA , 1993 .