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Editosome

Editosome. Stefan Rittinghaus 05.08.07. What are Editosomes ?. Editosomes are a multiprotein complex, catalyzing the editing of mRNA`s . Editing is the posttranscriptional modification mRNA`s. Editing changes, adds or deletes nucleotids of the mRNA.

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Editosome

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  1. Editosome Stefan Rittinghaus 05.08.07

  2. What are Editosomes ? • Editosomes are a multiprotein complex, catalyzing the editing of mRNA`s. • Editing is the posttranscriptional modification mRNA`s. • Editing changes, adds or deletes nucleotids of the mRNA.

  3. The mechanism of editing in general consists of three steps • Cleavage of the pre-mRNA • Changing, addition or deletion of nucleotids • Ligation of the edited mRNA.

  4. Example for Editing A gene of Trypanosoma brucei for one of the subunits of cytochrome c oxidase

  5. gRNA • Guide RNA`s are tiny RNA`s (50-70 nt) that guide the Editosomes through base-pairing with the mRNA`s in editing regions. • They are complimentary to small segments of the mRNA and function as templates.

  6. Structure of the gRNA NgCYb-558 • Anchor region (G1-C13) • Stem-loop/ guiding region (template for editing) • Poly U-tail

  7. Cross-linking of the gRNA via ist 5`-end to the 3`-end of either CYbU or CYbPES3 (Partially edited through site 3). • To discover the structure single strand specific (T1, T2 and MBN) and double strand specific (V1) RNase`s were used. • T1 is G specific • T2 and MBN have a slight preference for A

  8. gRNA cascade

  9. Proteins in Editosomes

  10. Subcomplexes via tagged KREN1, KREPB2 or KREN2 • related proteins , which contain U1-like zinc fingers, RNase III motifs and double stranded RNA binding motifs

  11. All three complexes catalyze precleaved deletion and insertion editing (in vitro). They have exonuclease and deletion editing-associated RNA ligaseactivities as well as TUTase, and insertion editing-associated RNA ligase activities, consistent with their protein content. Untagged Editosomes with gRNA as positiv control. Untagged Editosomes without gRNA as negativ control. Unedited substrat

  12. The KREN1 complex cleaves the editing site of a pre-mRNA deletion substrat, but not the editing site of a pre-mRNA insertion substrat. • The KREN2 complex cleaves the editing site of a pre-mRNA insertion substrat, but not the editing site of a pre-mRNA deletion substrat. • No clevead product detected with either substrat using KREPB2.

  13. To support this data KREN1 and KREN2 were mutated and the results are, that the complexes with the mutated proteins catalyze precleaved deletion and insertion editing, but they do not cleave either a deletion or an insertion editing site.

  14. Are the Endosomes really heterogeneous? • Could the overexpression of the tagged RNase III protein destabilizis the association of the other two proteins with the complex and replace them? • Overexpression of an inactive mutant of one of the RNase proteins in vivo results in cell death, but you still find the editosomes with the other two RNase proteins in a concentration similar to the wild type. • The knockdown of one RNase protein reduces the amount but not the sedimentation of the editosomes. • Different composition of the Endosomes

  15. Function of the proteins

  16. Many questions are still not answered. • At the moment there is no functional protei-protein interaction map • There are sequences in a pre-mRNA with both insertion and deletion editing sites, specified by one single mRNA.

  17. Literature • Horton, T.L.and L.F. Landweber, Rewriting the information in DNA;: RNA editing in kinetoplastids and myxomycetes.Curr Opin Microbiol, 2002. • Mian, I.S.,E.A.Worthey, and R. Salvati, Taking U out, with two nucleases? BMC Bioinformatics, 2006. • Yu, L.E. and D.J. Koslowsky, Interactions of mRNAs and gRNAs involved in trypanosome mitochondrial RNA editing: structure probing of a gRNA bound to ist cognate mRNA. Rna, 2006. • Panigrahi, A.K., et al., Compositionally and functionally distinct editosomes in Trypanosoma brucei. Rna 2006

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