title: Efficient transcription through an intron requires the binding of an Sm-type U1 snRNP with intact stem loop II to the splice donor.
abstract: The mechanism behind the positive action of introns upon transcription and the biological significance of this positive feedback remains unclear. Functional ablation of splice sites within an HIV-derived env cDNA significantly reduced transcription that was rescued by a U1 snRNA modified to bind to the mutated splice donor (SD). Using this model we further characterized both the U1 and pre-mRNA structural requirements for transcriptional enhancement. U1 snRNA rescued as a mature Sm-type snRNP with an intact stem loop II. Position and sequence context for U1-binding is crucial because a promoter proximal intron placed upstream of the mutated SD failed to rescue transcription. Furthermore, U1-rescue was independent of promoter and exon sequence and is partially replaced by the transcription elongation activator Tat, pointing to an intron-localized block in transcriptional elongation. Thus, transcriptional coupling of U1 snRNA binding to the SD may licence the polymerase for transcription through the intron.
authors.affiliation: Department of Microbiology and Immunology, University of Melbourne, Melbourne 3010, Australia.
authors.forename: Marina R
authors.initials: MR
authors.lastname: Alexander
authors.forename: Adam K
authors.initials: AK
authors.lastname: Wheatley
authors.forename: Rob J
authors.initials: RJ
authors.lastname: Center
authors.forename: Damian F J
authors.initials: DF
authors.lastname: Purcell
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D022821:RNA Splice Sites; D012326:RNA Splicing; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic; D054299:env Gene Products, Human Immunodeficiency Virus / Q000235:genetics; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000378:metabolism
chemical_list: D022821:RNA Splice Sites; D017412:Ribonucleoprotein, U1 Small Nuclear; D054299:env Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus