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.
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 epsilon 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.
Zeiner
Gusti M
GM
Department of Microbiology, Immunology, and Molecular Genetics, University of California at Los Angeles, 609 Charles E. Young Dr. East, Los Angeles, CA 90095-1489, USA.
Hitchcock
Robert A
RA
Sturm
Nancy R
NR
Campbell
David A
DA
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D018105:DNA, Kinetoplast / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005092:Exonucleases / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D034622:RNA Interference; D012322:RNA Precursors; D012326:RNA Splicing; D012341:RNA, Ribosomal, 5S / Q000378:metabolism; D020038:RNA, Spliced Leader / Q000235:genetics*; D013997:Time Factors; D014162:Transfection; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000378:metabolism*
D018105:DNA, Kinetoplast; D012322:RNA Precursors; D012341:RNA, Ribosomal, 5S; D020038:RNA, Spliced Leader; D049452:Green Fluorescent Proteins; D012313:RNA; D005092:Exonucleases