Alternative Spliceosome Assembly Pathways Revealed by Single-Molecule Fluorescence Microscopy
Inna Shcherbakova,
Aaron A. Hoskins,
Larry J. Friedman,
Victor Serebrov,
Ivan R. Corrêa, Jr.,
Ming-Qun Xu,
Jeff Gelles,
Melissa J. Moore
Affiliations
Inna Shcherbakova
Department of Biochemistry and Molecular Pharmacology, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA
Aaron A. Hoskins
Department of Biochemistry and Molecular Pharmacology, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA
Larry J. Friedman
Department of Biochemistry, Brandeis University, Waltham, MA 02454, USA
Victor Serebrov
Department of Biochemistry and Molecular Pharmacology, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA
Ivan R. Corrêa, Jr.
New England Biolabs, Ipswich, MA 01938, USA
Ming-Qun Xu
New England Biolabs, Ipswich, MA 01938, USA
Jeff Gelles
Department of Biochemistry, Brandeis University, Waltham, MA 02454, USA
Melissa J. Moore
Department of Biochemistry and Molecular Pharmacology, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA
Removal of introns from nascent transcripts (pre-mRNAs) by the spliceosome is an essential step in eukaryotic gene expression. Previous studies have suggested that the earliest steps in spliceosome assembly in yeast are highly ordered and the stable recruitment of U1 small nuclear ribonucleoprotein particle (snRNP) to the 5′ splice site necessarily precedes recruitment of U2 snRNP to the branch site to form the “prespliceosome.” Here, using colocalization single-molecule spectroscopy to follow initial spliceosome assembly on eight different S. cerevisiae pre-mRNAs, we demonstrate that active yeast spliceosomes can form by both U1-first and U2-first pathways. Both assembly pathways yield prespliceosomes functionally equivalent for subsequent U5⋅U4/U6 tri-snRNP recruitment and for intron excision. Although fractional flux through the two pathways varies on different introns, both are operational on all introns studied. Thus, multiple pathways exist for assembling functional spliceosomes. These observations provide insight into the mechanisms of cross-intron coordination of initial spliceosome assembly.
