Novel features of the yeast tri-snRNP structure

As mentioned in the previous section, highly flexible and vigorously moving structural elements are difficult to resolve by EM analysis. In contrast, stable and fixed regions of the structure are relatively easy to resolve. While we were able to reconstruct the highly flexible head domain only

A

D

C yeast&Prp6&structure&&

U4/U6&snRNA&

yeast&Prp6&placed&in&EM&density&

B

U5&snRNA&

U4/U6&snRNA&fi;ed&in&EM&density&

in our 6.7 Å Cryo -EM density map, comparatively stable and fixed structural features (body, foot, and linker domain) were relatively quickly resolved in our 5.8 Å 3D cryo-EM density map.

Nevertheless, our structure revealed some novel features of the Sad1-TAP tri-snRNP structure.

In order to compare our 3D cryo-EM model of Sad1-TAP tri-snRNP with the published yeast tri-snRNP model (Nguyen et al., 2015, Wan et al., 2016; Nguyen et al., 2016), we superimposed our cryo-EM electron density map (low-resolution unmasked model in this case) on the published yeast tri-snRNP model (Nguyen et al., 2016; PDB - 5gan) taking the well-resolved lower region as fixed reference region (fig: 3.27) (i.e., relative to Snu114 and the Prp8 NTD1 domain).

A comparison of the two models revealed some structural features that, for the first time, were exclusively observed in the Sad1-TAP tri-snRNP structure. While comparing with the published structure of the yeast tri-snRNP (Nguyen et al., 2016), we noted that the Sad1-TAP tri-snRNP has a missing density in one of its regions. The missing density region of Sad1-TAP tri-snRNP is located at the base of the head domain – a density region that has been annotated in the published structure (PDB-5gan) as that of the yeast Snu66 protein (fig: 3.27, C; represented by a blue dotted circle). Notably, in a recent study, my colleagues (Bertram et al., 2017) compared the molecular architecture of the yeast tri-snRNP reported by Wan et al. (2016) and Nguyen et al.

(2016) respectively, with the human spliceosomal B complex as determined by cryo-EM (Bertram et al., 2017; PDB-5O9Z). The aforementioned study not only showed that the structural organisation of Prp8, Brr2, Prp6 and the U4/U6 proteins in the reported yeast tri-snRNP is analogous to the organisation in the human B complex, but also, that the density which was tentatively assigned to helical regions of yeast Snu66 by Nguyen et al. (2016) more likely comprises the yeast Prp38 complex (Prp38/ Snu23/ Spp381 protein complex), as this position is equivalent to the hPrp38 complex in the human B complex and can even be superimposed (Bertram et al., 2017; PDB-5o9z). Later that year another study which was related to the yeast B complex structure (Plaschka et al., 2017), showed that, indeed, this density was comprised of the yeast Prp38/ Snu23/ Spp381 protein complex.

Secondly, we observed an extra density in the Sad1-TAP tri-snRNP when compared with the same published tri-snRNP structure (fig: 3.27, C; represented by a red dotted circle).

To identify the likely occupant of this extra density in the Sad1-TAP tri-snRNP, we compared the yeast Sad1-TAP tri-snRNP model with the available human tri-snRNP model (Agafonov et al., 2016a). In the case of the human tri-snRNP, the equivalent region was predicted to harbour the human Sad1 protein (PDB-3jcr). To test the possibility of having a similar situation in the yeast tri-snRNP, we used the X-ray crystal structure of the yeast Sad1 protein (PDB-4msx;

(Hadjivassiliou et al., 2014) and performed a rigid body placement in our Sad1-TAP tri-snRNP cryo-EM map. We found that the crystal structure of the Sad1 protein fits very nicely into the unique extra density of the Sad1-TAP tri-snRNP model (fig: 3.28).

Figure: 3.27 comparing the Sad1-TAP tri-snRNP’s 3D cryo-EM structure with the published cryo-EM structure of the yeast tri-snRNPs. The images show the structure comparison using superimposed features. A The low-resolution (8.8 Å, unmasked cryo-EM model of the Sad1-TAP tri-snRNP particle. B Published 3D cryo-EM model of the yeast tri-tri-snRNP (EMD-8012). C Superimposed view of the two structures (i.e., our low-resolution cryo-EM map with published tri-snRNP map) revealing the missing density (shown here in a blue dotted circle) and the electron density (shown by a red dotted circle) exclusively present in our cryo-EM model of the Sad1-TAP tri-snRNP.

Low resolution cryo-EM 3D map

Published Cryo-EM 3D map (EMD-8012)

A

B

C

Superimposed Low resolution cryo-EM map and Published cryo-EM model(EMD-8012)

Figure: 3.28 Sad1-TAP tri-snRNP’s 3D cryo-EM structure with ySad1 location. A: Image showing our cryo-EM model of Sad1-TAP tri-snRNP with the X-ray crystal structure of Sad1 protein placed in the “extra density”. B: Enlarged view of the crystal structure of published yeast Sad1 protein (PDB- 4msx; Hadjivassiliou et al., 2014) fitted in the extra density.

Though an extra density appeared in our 3D cryo-EM model, the current level of resolution did not allow us to place the Sad1p crystal structure on the basis of the secondary structure elements.

Therefore, we have performed protein-protein crosslinking experiments (see section: 3.3) to independently verify our tentative positioning of the Sad1p in this extra density. Crosslinking data clearly supported the stable presence of Sad1 protein in the Sad1-TAP tri-snRNP and its placement in our cryo-EM map. These findings were also in-line with the previously presented biochemical experiments (section: 3.1.4.1) and MS data (table: 3.4 and table: 3.5) in earlier chapters, which showed that the Sad1-TAP tri-snRNP indeed retained Sad1p.