Preparation of fluorescent pre-mRNA substrates for an smFRET study of pre-mRNA splicing in yeast.
The spliceosome is a complex small nuclear (sn)RNA-protein machine that removes introns from pre-mRNAs via two successive phosphoryl transfer reactions. For each splicing event, the spliceosome is assembled de novo on a pre-mRNA substrate and a complex series of assembly steps leads to the active conformation. To comprehensively monitor pre-mRNA conformational dynamics during spliceosome assembly, we developed a strategy for single-molecule FRET (smFRET) that utilizes a small, efficiently spliced yeast pre-mRNA, Ubc4, in which donor and acceptor fluorophores are placed in the exons adjacent to the 5' and 3' splice sites. In this chapter, we describe the identification of Ubc4 pre-mRNA that is efficiently spliced in vitro and the methods we have developed for the chemical synthesis of fluorescent Ubc4 pre-mRNA for smFRET.
Abelson
John
J
Department of Biochemistry and Biophysics, University of California, San Francisco, California, USA.
Hadjivassiliou
Haralambos
H
Guthrie
Christine
C
D001483:Base Sequence; D005091:Exons; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D007438:Introns; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D005456:Fluorescent Dyes; D012322:RNA Precursors; D029701:Saccharomyces cerevisiae Proteins; C501306:Ubc4 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes