Efficient transcription through an intron requires the binding of an Sm-type U1 snRNP with intact stem loop II to the splice donor.
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.
Alexander
Marina R
MR
Department of Microbiology and Immunology, University of Melbourne, Melbourne 3010, Australia.
Wheatley
Adam K
AK
Center
Rob J
RJ
Purcell
Damian F J
DF
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
D022821:RNA Splice Sites; D017412:Ribonucleoprotein, U1 Small Nuclear; D054299:env Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus