Abstract
Most human genes contain multiple introns, necessitating mechanisms to effectively define exons and ensure their proper connection by spliceosomes. Human spliceosome assembly involves both cross-intron and cross-exon interactions, but how these work together is unclear. We examined in human nuclear extracts dynamic interactions of single pre-mRNA molecules with individual fluorescently tagged spliceosomal subcomplexes to investigate how cross-intron and cross-exon processes jointly promote pre-spliceosome assembly. U1 subcomplex bound to the 5' splice site of an intron acts jointly with U1 bound to the 5' splice site of the next intron to dramatically increase the rate and efficiency by which U2 subcomplex is recruited to the branch site/3' splice site of the upstream intron. The flanking 5' splice sites have greater than additive effects implying distinct mechanisms facilitating U2 recruitment. This synergy of 5' splice sites across introns and exons is likely important in promoting correct and efficient splicing of multi-intron pre-mRNAs.
Highlights
Spliceosomes consist of the U1, U2, and U4/U6.U5 small nuclear ribonucleoproteins and multiprotein Prp19-complex as major building blocks, plus many transiently interacting splicing factors (Wahl et al, 2009)
In S. cerevisiae, where the cross-intron pathway predominates, single-molecule approaches have proven invaluable for elucidating the kinetic pathways and subcomplex dynamics involved in spliceosome assembly (Semlow et al, 2016; Warnasooriya and Rueda, 2014)
No spliced products were observed in the absence of ATP, which is required for spliceosome assembly
Summary
Spliceosomes consist of the U1, U2, and U4/U6.U5 small nuclear ribonucleoproteins (snRNPs) and multiprotein Prp19-complex as major building blocks, plus many transiently interacting splicing factors (Wahl et al, 2009). This machinery recognizes and assembles stepwise at splice sites (SS) (U1 at 5’SS and U2 at the 3’SS/branch site) to form pre-spliceosomes, which are subsequently remodeled into catalytically active spliceosomes. We developed the tools necessary to implement colocalization single-molecule spectroscopy (CoSMoS) in human cell extracts and used this system to investigate the dynamic mechanism of cross-intron and cross-exon cooperation in human pre-spliceosome assembly
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