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A tale of two TUTases.

Ruslan Aphasizhev1, Inna Aphasizheva, Larry Simpson

  • 1Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA.

Proceedings of the National Academy of Sciences of the United States of America
|September 5, 2003
PubMed
Summary

RNA editing involves 3' terminal uridylyl transferase (TUTase) activity. RET2 is responsible for U-insertion editing, while RET1 matures guide RNAs (gRNAs) for trypanosome mitochondrial RNA editing.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • RNA editing in trypanosome mitochondria relies on 3' terminal uridylyl transferase (TUTase) activity.
  • TUTase activity is crucial for both mRNA editing and the synthesis of 3' oligo[U] tails on guide RNAs (gRNAs).
  • Two TUTases, RET1 (RNA editing TUTase 1) and RET2 (RNA editing TUTase 2), have been identified as components of distinct editing complexes.

Purpose of the Study:

  • To functionally characterize the roles of RET1 and RET2 in trypanosome RNA editing and gRNA maturation.
  • To confirm the TUTase activity of RET2 and its specific function in U-insertion.
  • To differentiate the roles of RET1 and RET2 in mitochondrial RNA processing.

Main Methods:

  • Expression and localization of tandem affinity purification-tagged Trypanosoma brucei RET2 (TbRET2) in Leishmania tarentolae.
  • Double-affinity isolation of TbRET2 and associated proteins.
  • In vitro biochemical assays to assess U-transferase activity.
  • RNA interference (RNAi) to down-regulate RET2 and RET1 expression in T. brucei.

Main Results:

  • Purified TbRET2 demonstrated specific 3'-terminal uridylylation of single-stranded RNA, confirming its TUTase activity.
  • RNAi-mediated knockdown of RET2 inhibited in vitro U-insertion editing but did not affect gRNA 3' oligo[U] tail length.
  • Down-regulation of RET1 showed a minor effect on U-insertion editing but reduced the average length of gRNA 3' oligo[U] tails.

Conclusions:

  • RET2 is the primary enzyme responsible for U-insertions during mitochondrial mRNA editing in trypanosomes.
  • RET1 plays a significant role in the maturation of 3' oligo[U] tails of gRNAs, essential for functional gRNAs.
  • The TUT-II complex, containing RET1, is functionally redefined as the guide RNA processing complex.

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