Protein composition and EM structure of the S. cerevisiae U6 snRNP. (A) Secondary structure of U6 snRNA in the U6 snRNP. The U6 snRNA is shown with the secondary structure as determined by Karaduman et al. (2006). Important structural features are labeled. Arrows indicate nucleotides that in previous work (Karaduman et al. 2006) were found to be UV-cross-linked to Prp24p (blue) or to an unidentified LSm protein (red). The blue and the red outlines indicate U6 snRNA regions that are covered and protected by Prp24p and the LSm proteins, respectively. U6 snRNA sequences involved in base pairing with U4 snRNA in the U4/U6 di-snRNP are indicated by dotted lines and are labeled U4/U6 stem I and stem II, respectively. (B) Protein composition of purified U6 snRNPs. Proteins from purified U6 snRNPs were separated by SDS-PAGE and stained with silver. (Lane 1) Proteins of the wild-type particle with a TAP-tagged version of Prp24p; (lanes 2–8) U6 snRNPs containing a single tagged LSm protein (as indicated above each lane). The positions of molecular weight markers are indicated on the right and protein identities on the left. The molecular weights of the tagged LSm proteins are increased by 30 kDa due to the yECitrine; note the absence of untagged protein in each case (this is seen unambiguously for LSm2p, LSm7p, and LSm4p, but is less clear for the others, owing to pairwise overlap of the unmodified proteins). (Lanes 3,4) The protein composition of U6 snRNPs analyzed directly after TAP purification, while particles analyzed in all other lanes had additionally undergone glycerol-gradient centrifugation. (C) A typical electron micrograph showing a field of U6 snRNPs. After purification on a glycerol gradient, U6 particles were directly adsorbed onto a carbon carrier film and negatively stained with uranyl formate. Scale bar, 20 nm. (D) Gallery of selected U6 images. U6 particles were oriented with the round LSm domain at the bottom and the angular Prp24p domain at the top. In the images of the far right-hand column, the LSm ring is probably seen from the side. Scale bar, 20 nm.

Structural details of the U6 snRNPs. To analyze structural details of the purified U6 snRNPs, image processing was performed for each domain separately. U6 snRNP images were classified according to the Prp24p domain structure after masking the LSm domain (A) or according to the LSm ring structure after masking the Prp24p domain (B). The class members were finally averaged without the mask, so that the whole particle image is incorporated in the averages shown. Small characteristic protrusions of the LSm ring (B) are indicated by arrows. Scale bars, 20 nm.

Electron microscopy of U6 snRNP with yECitrine-tagged LSm proteins. (A,C) Fields from typical micrographs with proteins LSm7 (A) or LSm5 (C) tagged. (B,D–G) Galleries of selected images of U6 snRNPs containing a tagged LSm7, LSm5, LSm6, LSm8, or LSm4 protein, as indicated below each gallery. In addition to the round LSm domain and the angular Prp24p domain (oriented upward in all galleries), a third small globular domain (the yECitrine tag) about 4 nm in diameter is visible (some are indicated by arrows). Note that some of the figures appear as mirror images of others, because the LSm ring can lie with either face in contact with the carbon film. For LSm4p (G), the tag at the C terminus is often seen at some distance from the LSm ring. A region of stain low density, linking the tag to the LSm ring can be recognized in several images (labeled with arrowheads). The distance between the yECitrine tag and the Prp24p domain was estimated visually for particles with a clearly visible LSm ring. By classifying these into rough distance categories we observed characteristic (relative) distances for each tag position. Scale bars, 20 nm.

Cross-linking of LSm2p to the U28–G30 region of the U6 snRNA in the U6 snRNP. Purified U6 snRNPs with one of the seven LSm proteins tagged with yECitrine (as indicated above) were UV irradiated to induce RNA–protein cross-links (“+UV” lanes). Particles were disrupted with 1.5% (w/v) SDS, and the tagged LSm proteins, including any cross-linked RNA, were precipitated with anti-yECitrine antibodies. As controls, non-UV-irradiated (“−UV” lanes) U6 snRNPs were also subjected to immunoprecipitation. To identify the nucleotides cross-linked to the precipitated protein, primer-extension analysis of U6 snRNA was performed as described previously (Karaduman et al. 2006). Nonimmune serum (NIS) was used as a negative control (lanes 15,16). Lanes C, U, A, and G correspond to sequencing ladders made with the same oligonucleotide (Lane 0, no ddNTP). Nucleotide positions of reverse transcriptase stops due to specific cross-linking between LSm2p and the U6 snRNA are indicated on the left with black dots. The asterisks indicate the background of naturally occurring or UV-induced stops due to RNA–RNA cross-linking.

Chemical cross-linking yields a more compact form of the U6 snRNP. (A) Electron microscopy of purified U6 snRNP stabilized by chemical cross-linking in solution with glutaraldehyde. Except for the fixation step with glutaraldehyde, all preparation steps were identical to those used to purify the particles shown in Figure 1. (Left) A typical field of glutaraldehyde-stabilized U6 snRNPs. Columns 1–4, class averages of gluteraldehyde-stabilized U6 snRNP particles. Each row shows particles in a similar orientation. In columns 1 and 2, the round features of the LSm ring can be seen (arrows); the images in columns 3 and 4 are more angular, indicative of the Prp24p domain. Scale bars, 20 nm. (B) Summary of EM analysis of yECitrine-labeled U6 snRNPs. Characteristic images of unfixed U6 snRNP particles with yECitrine tags on LSm8p, LSm4p, LSm7p, LSm5p, and LSm6p are shown. Some images are mirrored (see caption to Fig. 3) to illustrate the agreement with the proposed LSm circular sequence in the ring. Below the images, schematic diagrams are shown representing the U6 snRNP particle with yECitrine tags (red disks). (C) Schematic representation of the interaction between Prp24p and the LSm ring for the open (left) and closed (right) form of U6 snRNP. The identities of the LSm proteins in the LSm ring are shown (2–8). The Prp24p domain is represented as arbitrarily arranged protein modules (slightly elongated modules represent the four RRMs and the N- and C-terminal sequence stretches). Proteins are drawn approximately to scale on the basis of the known sizes of the LSm proteins and the Prp24p domains involved.