Synergistic assembly of human pre-spliceosomes across introns and exons

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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.

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All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided.

Article and author information

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Joerg E Braun

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-8309-6401
Larry J Friedman

Department of Biochemistry, Brandeis University, Waltham, United States

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0003-4946-8731
Jeff Gelles

Department of Biochemistry, Brandeis University, Waltham, United States

For correspondence
gelles@brandeis.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-7910-3421
Melissa J Moore

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States

For correspondence
melissa.moore@umassmed.edu

Competing interests
The authors declare that no competing interests exist.

Funding

National Institutes of Health (R01 GM053007)

Melissa J Moore

Human Frontier Science Program (LT000166/2013)

Joerg E Braun

European Molecular Biology Organization (ALTF 890-2012)

Joerg E Braun

Howard Hughes Medical Institute

Melissa J Moore

National Institutes of Health (R01 GM081648)

Jeff Gelles

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.