title: Splice-site mutations cause Rrp6-mediated nuclear retention of the unspliced RNAs and transcriptional down-regulation of the splicing-defective genes.
abstract: BACKGROUND
  Eukaryotic cells have developed surveillance mechanisms to prevent the expression of aberrant transcripts. An early surveillance checkpoint acts at the transcription site and prevents the release of mRNAs that carry processing defects. The exosome subunit Rrp6 is required for this checkpoint in Saccharomyces cerevisiae, but it is not known whether Rrp6 also plays a role in mRNA surveillance in higher eukaryotes.
  
  
  RESULTS
  We have developed an in vivo system to study nuclear mRNA surveillance in Drosophila melanogaster. We have produced S2 cells that express a human beta-globin gene with mutated splice sites in intron 2 (mut beta-globin). The transcripts encoded by the mut beta-globin gene are normally spliced at intron 1 but retain intron 2. The levels of the mut beta-globin transcripts are much lower than those of wild type (wt) ss-globin mRNAs transcribed from the same promoter. We have compared the expression of the mut and wt beta-globin genes to investigate the mechanisms that down-regulate the production of defective mRNAs. Both wt and mut beta-globin transcripts are processed at the 3', but the mut beta-globin transcripts are less efficiently cleaved than the wt transcripts. Moreover, the mut beta-globin transcripts are less efficiently released from the transcription site, as shown by FISH, and this defect is restored by depletion of Rrp6 by RNAi. Furthermore, transcription of the mut beta-globin gene is significantly impaired as revealed by ChIP experiments that measure the association of the RNA polymerase II with the transcribed genes. We have also shown that the mut beta-globin gene shows reduced levels of H3K4me3.
  
  
  CONCLUSIONS
  Our results show that there are at least two surveillance responses that operate cotranscriptionally in insect cells and probably in all metazoans. One response requires Rrp6 and results in the inefficient release of defective mRNAs from the transcription site. The other response acts at the transcription level and reduces the synthesis of the defective transcripts through a mechanism that involves histone modifications.
authors.affiliation: Department of Molecular Biology and Functional Genomics, Stockholm University, Stockholm, Sweden.
authors.forename: Andrea B
authors.initials: AB
authors.lastname: Eberle
authors.forename: Viktoria
authors.initials: V
authors.lastname: Hessle
authors.forename: Roger
authors.initials: R
authors.lastname: Helbig
authors.forename: Widad
authors.initials: W
authors.lastname: Dantoft
authors.forename: Niclas
authors.initials: N
authors.lastname: Gimber
authors.forename: Neus
authors.initials: N
authors.lastname: Visa
mesh_terms: D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster; D063326:Exosome Multienzyme Ribonuclease Complex; D005455:Fluorescent Antibody Technique; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D009154:Mutation; D034622:RNA Interference; D012326:RNA Splicing / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055544:beta-Globins / Q000235:genetics
chemical_list: D029721:Drosophila Proteins; D012333:RNA, Messenger; C545895:Rrp6 protein, Drosophila; D055544:beta-Globins; D063326:Exosome Multienzyme Ribonuclease Complex