ALBERT

Description: Publication date: Available online 26 September 2013 Source: Cell Reports Author(s): Inna Shcherbakova , Aaron A. Hoskins , Larry J. Friedman , Victor Serebrov , Ivan R. Corrêa Jr. , Ming-Qun Xu , Jeff Gelles , Melissa J. Moore 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. Graphical abstract Teaser Intron excision by the spliceosome is an essential process in eukaryotic gene expression. Gelles, Moore, and colleagues use single-molecule colocalization to monitor early spliceosome assembly events in yeast whole-cell extract. They demonstrate that pre-mRNAs can initiate the formation of functional spliceosomes by first binding either U1 or U2 snRNP. This branched pathway has important implications for mechanisms of cross-intron coordination during early spliceosome assembly.