EM of individual spliceosomes assembled on Ftz-M3 pre-mRNA. Size bars are indicated at the bottom. The 3-cm bar is equal to 45 nm.

Strategy for MBP-affinity purification of the spliceosome. Spliceosomes are isolated by gel filtration followed by affinity purification (see text for details).

Visualization of purified functional spliceosomes by electron microscopy. (A) Three different fields of purified spliceosomes assembled on AdML-M3 pre-mRNA. (B) Fields of control purification. (C) Fields of spliceosomes after treatment with Proteinase K. (D) Fields of spliceosomes after treatment with RNase A. The scale bar represents 360 nm.

Analysis of snRNAs in purified spliceosomes. Spliceosomes were purified using AdML-M3, AdML, Ftz-M3, or Ftz pre-mRNAs as substrates (AdML or Ftz pre-mRNAs lacking hairpins were used for negative controls). Total RNAs extracted from the nuclear extract (NE), AdML, AdML-M3, Ftz, or Ftz-M3 elutions were ³²P-end-labeled (+) or not labeled (−) and fractionated on an 8% polyacrylamide gel. Note that the snRNAs are 3′-end-labeled differentially with RNA ligase (15).

Immunogold labeling of spliceosomes. Spliceosomes assembled on AdML-M3 pre-mRNA were incubated with antitrimethyl cap antibody followed by a secondary antibody conjugated with 10 nm gold (A) or with anti-U5–40kD antibody followed by a secondary antibody conjugated with 6 nm gold (B). Arrows indicate the positions of gold labels. The 3-cm bars are equal to 180 nm (A) and 110 nm (B), respectively.

Optimization of MBP-affinity purification. (a) Insertion of MS2 hairpins at the 3′ end of exon 2 does not affect splicing efficiency. AdML pre-mRNA containing 0, 1, 2, or 3 hairpins are spliced in nuclear extract for 40 min, and total RNAs were analyzed on a 16% denaturing polyacrylamide gel. (b) Sequence of the cassette containing three MS2 RNA binding sites (hairpins). The secondary structure and the bulged adenosine characteristic of the hairpins are shown. The restriction enzyme sites of the corresponding DNA sequence are indicated. (c) Schematic of MS2-MBP fusion protein. The mutations that prevent MS2 oligomerization are indicated. (d) The MS2-MBP protein specifically binds to AdML-M3 pre-mRNA in a dose-dependent manner. AdML (lane 1) or AdML-M3 (lanes 2–8) pre-mRNA was incubated with increasing amounts of MS2-MBP for 30 min. The RNA-protein complexes were separated on a 1.5% agarose gel. (e) Binding of MS2-MBP to AdML-M3 does not affect splicing efficiency. Increasing amounts of MS2-MBP protein were incubated with AdML-M3 pre-mRNA for 30 min, followed by incubation under splicing conditions in nuclear extract for 40 min. Total RNAs were analyzed on a 16% denaturing polyacrylamide gel.