Premium
Single‐molecule imaging of pre‐mRNA splicing in situ (108.3)
Author(s) -
Sanjay Tyagi
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.108.3
Subject(s) - rna splicing , intron , exon , polypyrimidine tract , alternative splicing , polypyrimidine tract binding protein , precursor mrna , biology , gene , rna , splicing factor , messenger rna , transcription (linguistics) , locus (genetics) , microbiology and biotechnology , genetics , linguistics , philosophy
We have developed a single‐molecule RNA imaging method to study the intracellular sites of splicing. In this approach introns and exons in natural genes are probed in situ with distinctively labeled sets of about 50 oligonucleotide probes. The attachment of so many probes renders each target molecule so intensely fluorescent that it becomes visible as a bright diffraction‐limited spot. Spots that fluoresce in just one color are either free introns or spliced mRNAs, and those that are visible in both colors correspond to pre‐mRNA molecules. Our imaging studies confirm that constitutively spliced introns are removed at the gene locus during transcription. However, during alternative splicing events regulated by RNA binding proteins Sex lethal (in fruit flies) and polypyrimidine tract binding protein (in HeLa cells), which ensure that only one splice form is produced in a particular cell type, splicing gets uncoupled from transcription and occurs after the release of pre‐mRNA from the gene locus. Similar uncoupling occurrs when intronic polypyrimidine tracts are masked within hairpins. Live cell imaging of the post‐transcriptional splicing events suggests that they occur at the periphery of nuclear speckles where many splicing factors and poly adenylated transcripts are sequestered. Recent genome‐wide studies reveal that a fraction of alternative splicing events likely occur via this route.