title: Quantifying the ratio of spliceosome components assembled on pre-mRNA.
abstract: RNA processing by the splicing machinery removes intronic sequences from pre-mRNA to generate mature mRNA transcripts. Many splicing events occur co-transcriptionally when the pre-mRNA is still associated with the transcription machinery. This mechanism raises questions regarding the number of spliceosomes associated with the pre-mRNA at a given time. In this protocol, we present a quantitative FISH approach that measures the ratio of intensities between two different spliceosomal components associated on a nascent mRNA, and compares to the number of introns in the mRNA, thereby calculating the number of spliceosome complexes assembled with each transcript.
authors.affiliation: The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, Israel.
authors.forename: Noa
authors.initials: N
authors.lastname: Neufeld
authors.forename: Yehuda
authors.initials: Y
authors.lastname: Brody
authors.forename: Yaron
authors.initials: Y
authors.lastname: Shav-Tal
mesh_terms: D001483:Base Sequence; D006801:Humans; D007438:Introns; D008967:Molecular Biology / Q000379:methods*; D012322:RNA Precursors / Q000235:genetics*; D012326:RNA Splicing / Q000235:genetics; D012261:Ribonucleoproteins / Q000235:genetics; D017381:Spliceosomes / Q000235:genetics* / Q000648:ultrastructure; D014158:Transcription, Genetic
chemical_list: D012322:RNA Precursors; D012261:Ribonucleoproteins