FIG. 5.

MS2 affinity selection of in vitro spliced and unspliced mRNPs. (A) Schematic representation of the Ftz-M3 pre-mRNA and FtzΔI-M3 mRNA. PY tract, polypyrimidine tract; nts, nucleotides. (B) FtzΔI-M3 or Ftz-M3 RNA was incubated for 180 min under splicing conditions in Kc cell nuclear extract, followed by oligonucleotide-directed cleavage by RNase H. Splicing substrates, intermediates, and products are indicated on the right. (C) Splicing reaction mixtures containing Ftz-M3 or FtzΔI-M3 were loaded onto a linear 10-to-30% (vol/vol) glycerol gradient containing G150 buffer. The radioactivity contained in each fraction was determined by Cherenkov counting. The profiles show the percentage of radioactivity in each fraction. Sedimentation coefficients (indicated by arrowheads) were determined by analyzing the UV absorbance of fractions of a reference gradient containing prokaryotic ribosomal subunits. (D) Gradient fractions 10 to 12 were pooled and subjected to MS2 affinity selection. Bound complexes were eluted with maltose, and RNA was recovered, separated by 12% denaturing PAGE, and visualized by silver staining (upper panel) or by autoradiography to detect radioactive species (lower panel). Lanes 1 and 4, 2.5% of the input (I) pooled gradient fractions. Lanes 2 and 5, 2.5% of the flowthrough (FT) of the amylose column. Lanes 3 and 6, 10% of the eluted (E) mRNPs. The positions of snRNAs, unspliced pre-mRNA, and splicing products are indicated on the right.