2. Materials and Methods
3.5 Not all identified prey encoded proteins
Once sequence information was available, each prey construct could be categorised according to sequence type. Through BLAST (Basic Local Search Alignment Tool) searching, it was found that many of the prey-encoded sequences that did not correspond to any known protein (Table 3.3). These prey constructs were most commonly found to contain cDNA corresponding to the 3’-untranslated regions (UTRs) of proteins. This is most likely an artefact from the production of the cDNA library, which used a poly-dT primer to anneal to cellular mRNAs via the 3’-polyadenylation signal (Section 3.3.2). 3’-UTRs are commonly found between the stop codon of an open reading frame and the polyadenylation signal. 5’- degradation of the RNAs during the construction of the library could additionally explain why such a large proportion of identified prey constructs (up to 56% in a given screen) contained only 3’-UTR sequence without any protein-coding region. As these hits did not appear to represent novel binding partners for Isl1, they were not further investigated.
Table 3.3: Proportion of isolated prey encoding proteins across library screens.
Screen Ldb1LID/Isl1LIM
Isl1LIM screen A Isl1LIM screen B Isl1ΔLIM screen A Isl1ΔLIM screen B Prey isolated 74 180* 260* 35 200 Prey identified 74 105 258 31 177 Prey encoding proteins in frame 27 (36%) 73 (70%) 150 (58%) 5 (16%) 23 (12%) Prey encoding frameshifted proteins 8 (11%) 14 (13%) 33 (13%) 8 (25%) 57 (29%)
Prey not encoding
proteins 38 (51%) 18 (17%) 75 (29%) 18 (56%) 94 (47%) Non-redundant
in-frame proteins 11 25 46 4 21
* Screen grew >1000 colonies. Strongest interactions were isolated for identification.
3.5.1 Investigation of frameshifted proteins
A significant proportion of hits (30% overall) encoded protein-coding sequences that were out of frame from the upstream GAL4 protein sequence (Table 3.3). Documentation from Clontech indicated that yeast are tolerant to ribosomal frameshifts, meaning that during translation the mRNA can shift by one base in either direction with respect to the ribosome [211, 226]. This phenomenon would make it possible for these frameshifted sequences to be expressed correctly, even though they are encoded in a different reading frame to the upstream GAL4 sequence. However, it was important to establish whether this was the case for this set of sequences.
The pGADT7-RecAB plasmid encodes a hemagglutinin (HA) tag immediately downstream of the GAL4AD, upstream of the multiple cloning site (MCS) in which the prey protein coding
sequence is inserted, allowing detection of the expressed protein in an anti-HA Western blot. Several plasmids were chosen that contained protein-coding sequence that would result in a significant size difference of the expressed protein, depending on whether the protein sequence expressed was in frame with the GAL4 sequence or not (Table 3.4).
Table 3.4: Expected size of potentially frameshifted prey. Predicted size of the
protein product unique to each clone is given for each reading frame, in kDa. For each prey, the frame that corresponds to a known protein is underlined. Note that these sizes include the size of the upstream sequence, which includes the HA tag, GAL4AD, and SV40 NLS (simian
virus 40 nuclear localisation sequence) (~22 kDa).
Prey ID Frame 1 size (kDa) Frame 2 size (kDa) (GAL4 frame)
Frame 3 size (kDa)
E4 24.4 29.6 23.5 E7 22.9 24.6 44.9 M7 25.8 23.8 34.6 P10 23.5 28.2 33.3 T8 22 24.1 39.2 U5 22.5 29.2 27
The plasmids selected were isolated as part of the Isl1LIM screening process and showed
evidence of strong interactions with the Isl1LIM construct. The plasmids were co-transformed
into yeast along with IslLIM and grown in either media selecting for the presence of plasmids
(SD-L-W), or media selecting for a strong interaction between bait and prey (SD-L-W-H-A), to determine if inducing protein expression selected for protein produced in a particular frame. The protein was then extracted (Section 2.3.6) and subjected to anti-HA Western blot analysis, to observe the size of the expressed prey protein (Figure 3.5).
Figure 3.5: Anti-HA Western blots of protein extracts from yeast. Yeast were
co-transformed with pGBT9-Isl1LIM and a library pGADT7-RecAB (E4, E7, M7, P10, or
U5), and grown to saturation in selective media, before protein was extracted. Total protein extracts were subjected to anti-HA Western blot analysis. (A) Blot of extracts of yeast grown in SD-L-W media, selecting for the presence of plasmids. (B) Blot of extracts of yeast grown in media selecting for plasmid presence (SD-L-W; right lane for each sample) or for a strong interaction (SD-L-W-H-A; left lane for each sample).
Samples U5 and E7 did not show levels of expression detectable by Western blot, so conclusions were drawn from the other four samples. All extracts from media selecting for a strong interaction showed a more intense band, signifying increased levels of protein expression (Figure 3.5B). This may occur as the growth conditions would favour yeast that are able to produce more of the ADE2 gene product, potentially because they have higher levels of expression of the prey protein, inducing more expression of the ADE2 gene.
Comparing the sizes of the detected proteins between preys, it appears that E4 and P10 expressed proteins of very similar sizes (around 30 kDa), with the proteins being expressed in M7 and T8 being close in size, but slightly smaller (around 25 kDa). Of the three potential coding frames, only frame 2 fulfils all these criteria, with E4 and P10 being 1.4 kDa different in size (29.6 and 28.2 kDa respectively), M7 and T8 being 0.3 kDa different in size (23.8 and 24.1 kDa respectively), and the two pairs being ~5 kDa different in size (Table 3.4). Given that Frame 2 is expected, these data indicate that no ribosomal frameshifts are occurring. As all these protein sequences that interacted with Isl1LIM appear to represent nonsense peptides,
and not a protein-protein interaction that occurs in vivo, hits found to encode frameshifted proteins were not pursued further.