Detailed analysis of protein distribution and tri-snRNP composition upon

To simplify the comparison of the shifts in protein distribution upon changing of salt conditions we converted the numbers from table: 3.3 and 3.4 into a percentage distribution throughout the gradient for each protein, the peptide count over all fractions set as 100 %.

First, we look at the situation for the Sad1-TAP tri-snRNP purified and gradient-fractionated under 75 mM conditions (Fig: 3.13). When analysed with a percentage profile, three protein groups become immediately discernible, each with a distinct distribution profile. First, there is the

group comprising the U5 proteins (except Prp28, Lin1 and Dib1) and U4/U6 proteins Prp31, Prp3 and Prp4, with a strong presence in the tri-snRNP position and a rather low baseline in the top of the gradient (Fig. 3.13). A second group with a common profile comprises the U5 protein Prp28p, the U4/U6 protein Snu13p and tri-snRNP-specific proteins Snu66p and Sad1p (Fig.

3.13, B). Compared with the previous group, the profile is similar but the baseline is somewhat higher and the protein presence in the tri-snRNP position slightly reduced. Most dramatic is the situation in the third group; here we see a strict confinement of three tri-snRNP-specific proteins, Spp381p, Prp38p and Snu23p, to the tri-snRNP position with only minute amounts in the gradient. The two U5 proteins Lin1p and Dib1p defy easy assignment to any of the three groups:

the non-essential U5 component Lin1p shows two peaks outside of the tri-snRNP position, while the essential U5 component Dib1p peaks well in the tri-snRNP peak with a relatively strong presence in the middle of the gradient (Fig.3.13, D). Taken together, again the picture of a compact, highly integer tri-snRNP emerges.

Figure 3.13 Percentage distribution of protein and tri-snRNP composition under 75 mM K+

conditions. For each gel fraction peptide counts from Tab. 3.3 were calculated as percentage of total counts for a particular protein. Vertical axis shows percent value, horizontal axis the fraction number.

Panel A Distribution of U5 and U4/U6 proteins (see insert and text). Panel B Prp28p/Snu13p/Snu66p/Sad1p group (see text). Panel C Spp381/ Snu23 group (see text). Panel D Distribution of Lin1p and Dib1p. The bars in A – C indicate the min/max percent value for the protein group in a particular fraction of the gradient. The tri-snRNP position is indicated in yellow.

Prp8%

Figure 3.14 Percentage distribution of protein and tri-snRNP composition under 150 mM K+

conditions. For each gel fraction peptide counts from Tab. 3.5 were calculated as percentage of total counts for a particular protein. Vertical axis shows percent value, horizontal axis the fraction number.

Panel A Distribution of U5 and U4/U6 proteins (see insert and text). Panel B Prp28p/Snu13p/Snu66p/Sad1p group (see text). Panel C Spp381/Snu23 group (see text). Panel D Distribution of Lin1p and Dib1p. The bars in A – C indicate the min/max percent value for the protein group in a particular fraction of the gradient. The tri-snRNP position is indicated in yellow.

Prp8%

If we now subject the Sad1-TAP tri-snRNP to 150 mM K⁺ during purification and gradient, remarkable changes occur with severe consequences for the tri-snRNP (Fig. 3.14). The group of U5 proteins (except Prp28p, Lin1p and Dib1p) and U4/U6 proteins Prp31p and Prp4 is still strongly present in the tri-snRNP (Fig. 3.12, A). Prp3p instead starts bleeding from the tri-snRNP and appears in top of the gradient, grouping now rather with the second set of proteins (U5 protein Prp28p, the U4/U6 protein Snu13p and tri-snRNP-specific proteins Snu66p and Sad1p).

Compared to 75 mM K⁺ this second group now shows a much stronger presence outside of the tri-snRNP position in the gradient (Fig. 3.14, B). Under 150 mM K⁺, proteins become destabilized from the tri-snRNP (Fig. 3.14). Sad1p and Snu66p are still present in the Sad1-TAP tri-snRNP, but start to dissociate. The Spp381p, Prp38p and Snu23p group of tri-snRNP-specific proteins is virtually absent from the tri-snRNP and even from the top fractions of the gradient (Fig. 3.14, C).

In any case, it is evident that the integrity of a yeast tri-snRNP purified under 150 mM K⁺ is severely compromised due to bleeding or complete loss of proteins. It was recently shown with reconstitution assays that the human orthologs of yeast Spp381p, Prp38p, and Snu23p, namely MFAP1, hPRP38 and hSNU23 could form a stable subcomplex (Ulrich and Wahl, 2017). This human subcomplex depends on the same, evolutionarily highly conserved, interactions as observed between Spp381p, Prp38p, and Snu23p (Ulrich and Wahl, 2017). Most remarkably, the human MFAP1 protein is a B complex-specific protein and the hSNU23 – hPRP38 – MFAP1 complex indeed enters the human spliceosome at the level of the B-complex and independent of the human tri-snRNP. It has been suggested that in yeast the Snu23p – Prp38p – Spp381p subcomplex is part of the tri-snRNP and enters the spliceosome with the tri-snRNP (Jia and Sun, 2018), however, it is also important to note that so far, purified yeast tri-snRNPs are shown to be devoid of loosely associated factors such as, Sad1 (Häcker et al., 2008), and Prp28 (Nguyen et al., 2015; Wan et al., 2016). This may hint towards the mutually exclusive presence of Sad1/Prp28 and Prp38 – Snu23 –Spp381 in the yeast snRNP (as observed in the case of Brr2-TAP snRNPs as well, table 3.1- column: B). Interaction behaviour of Dib1p and Lin1p with the tri-snRNP particles at 150 mM K⁺ appears similar to as under 75 mM K⁺, with Lin1p outside of the tri-snRNP peak and Dib1p inside and outside (Fig. 3.14, D).

Based on the conclusions of the biochemical experiments and comprehensive MS analyses, it is apparent that the Sad1-TAP tri-snRNP, purified at 75 mM K+ concentration, not only is remarkably resistant to dissociative conditions in the presence of ATP but also shows a complete and strong set of all the constitutive parts of this snRNP. These results adequately support the interesting possibility that such a Sad1-TAP tri-snRNP may have a different conformational state when compared to tri-snRNPs obtained from previous purifications used for structure determinations. Therefore, I set out to investigate the structural features of the Sad1-TAP tri-snRNPs using cryo-electron microscopy.