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

Abstract

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 ε 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.

We thank Ruslan Aphasizhev, Larry Simpson, and Wade Winkler for oligonucleotides and helpful suggestions with the in vitro exonuclease assay; Danielle Leuenberger and Carla Koehler for TOM70-His₆; C. C. Wang for 29-13 cells; Kent Hill for YTAT cells and for use of the Zeiss fluorescence microscope; Keith Matthews for the α/β-tubulin intragenic RNase protection probe; and Sean Thomas, Scott Westenberger, Jesse Zamudio, Bidyottam Mittra, and Carmen Zelaya for stimulating discussion and comments on the manuscript.

G.M.Z. was a predoctoral trainee on Microbial Pathogenesis Training Grant 2-T32-AI-07323. This study was supported by NIH grant AI 056034 to D.A.C. and N.R.S.