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Molecular and Cellular Biology, December 2004, p. 10390-10396, Vol. 24, No. 23
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.23.10390-10396.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

3'-End Polishing of the Kinetoplastid Spliced Leader RNA Is Performed by SNIP, a 3'->5' Exonuclease with a Motley Assortment of Small RNA Substrates{dagger}

Gusti M. Zeiner, Robert A. Hitchcock, Nancy R. Sturm,* and David A. Campbell

Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California

Received 3 June 2004/ Returned for modification 29 July 2004/ Accepted 1 September 2004

In all trypanosomatids, trans splicing of the spliced leader (SL) RNA is a required step in the maturation of all nucleus-derived mRNAs. The SL RNA is transcribed with an oligo-U 3' extension that is removed prior to trans splicing. Here we report the identification and characterization of a nonexosomal, 3'->5' exonuclease required for SL RNA 3'-end formation in Trypanosoma brucei. We named this enzyme SNIP (for snRNA incomplete 3' processing). The central 158-amino-acid domain of SNIP is related to the exonuclease III (ExoIII) domain of the 3'->5' proofreading {varepsilon} subunit of Escherichia coli DNA polymerase III holoenzyme. SNIP had a preference for oligo(U) 3' extensions in vitro. RNA interference-mediated knockdown of SNIP resulted in a growth defect and correlated with the accumulation of one- to two- nucleotide 3' extensions of SL RNA, U2 and U4 snRNAs, a five-nucleotide extension of 5S rRNA, and the destabilization of U3 snoRNA and U2 snRNA. SNIP-green fluorescent protein localized to the nucleoplasm, and substrate SL RNA derived from SNIP knockdown cells showed wild-type cap 4 modification, indicating that SNIP acts on SL RNA after cytosolic trafficking. Since the primary SL RNA transcript was not the accumulating species in SNIP knockdown cells, SL RNA 3'-end formation is a multistep process in which SNIP provides the ultimate 3'-end polishing. We speculate that SNIP is part of an organized nucleoplasmic machinery responsible for processing of SL RNA.

* Corresponding author. Mailing address: Department of Microbiology, Immunology, and Molecular Genetics, University of California at Los Angeles, 609 Charles E. Young Dr. East, Los Angeles, CA 90095-1489. Phone: (310) 206-5556. Fax: (310) 206-5231. E-mail: nsturm{at}ucla.edu.

{dagger} This work is dedicated to the memory of Pauline "Pesh" Katz (1923-2004).

Molecular and Cellular Biology, December 2004, p. 10390-10396, Vol. 24, No. 23
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.23.10390-10396.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Zamudio, J. R., Mittra, B., Chattopadhyay, A., Wohlschlegel, J. A., Sturm, N. R., Campbell, D. A. (2009). Trypanosoma brucei Spliced Leader RNA Maturation by the Cap 1 2'-O-Ribose Methyltransferase and SLA1 H/ACA snoRNA Pseudouridine Synthase Complex. Mol. Cell. Biol. 29: 1202-1211 [Abstract] [Full Text]  
  • Zamudio, J. R., Mittra, B., Foldynova-Trantirkova, S., Zeiner, G. M., Lukes, J., Bujnicki, J. M., Sturm, N. R., Campbell, D. A. (2007). The 2'-O-Ribose Methyltransferase for Cap 1 of Spliced Leader RNA and U1 Small Nuclear RNA in Trypanosoma brucei. Mol. Cell. Biol. 27: 6084-6092 [Abstract] [Full Text]  
  • Zamudio, J. R., Mittra, B., Zeiner, G. M., Feder, M., Bujnicki, J. M., Sturm, N. R., Campbell, D. A. (2006). Complete Cap 4 Formation Is Not Required for Viability in Trypanosoma brucei. Eukaryot Cell 5: 905-915 [Abstract] [Full Text]  


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