To identify cap-independently translated transcripts genome wide, we performed LC-MS/MS analyses in three 4E-BP1(4Ala) expressing mESC clones after 24 h of induction. The proteomic analysis detected ~6,400 proteins and 13 out of them were significantly (p <0.05) enriched by more than 4-fold (log2(wt/mut)≤-2) in at least one individual 4E-BP1(4Ala) expressing clone when
Fig. 5: Inducible expression of dominant negative 4E-BP1 and ASCL1 promotes cap-independent translation and neuronal differentiation. (A) Lysates of untreated or 24 h induced monoclonal mESC lines A6, B4 and C6 were analyzed for 4E-BP1(4Ala) protein expression by Western blotting using anti-V5 (C-terminally attached to 4E-BP1(4Ala)) and anti-α-tubulin antibodies. (B) Marker gene expression of wt mESCs and monoclonal lines A6 and C6 was analyzed by quantitative real-time PCR in undifferentiated cells and induced neurons after seven days of induction. Expression levels were calculated by the comparative ΔΔCT method. Actin expression was used for normalization of Oct4, Sox1, V5 and Tubb3. V5-tag is attached to ASCL1 in wt cells and to 4E-BP1(4Ala) in clones A6, C6. Error bars represent standard deviation of two technical replicates. (C) Schematic representation of bicistronic reporter structure. Firefly luciferase (FLuc) open reading frame (ORF) is located upstream of Renilla luciferase (RLuc) ORF on bicistronic reporter mRNAs. ORFs are separated by an intermediate sequence of interest which is tested for its ability to mediate internal translation initiation. (D) Dual luciferase reporter assays of wt mESCs and 4E-BP1(4Ala) expressing clones A6, B4 and C6 after transfection with bicistronic EMCV IRES and functionally inactive EMCV* IRES reporters. Luciferase levels were determined 48 h after transfection including 24 h of doxycycline (Dox) induction when indicated. IRES mediated RLuc translation was normalized to cap-dependent FLuc translation. Error bars represent standard error of the mean of three biological replicates.
152
compared to wt stem cells (Fig. 6, Fig. 7). The clones displayed some differences in protein expression levels with the result that some of the most significantly upregulated genes in one clone were not significantly enriched in another. Such phenotypic heterogeneity within clonal cell populations can emerge from a combination of intrinsic and extrinsic factors and is often observed in mammalian cellular in vitro systems [855], [856]. In mESCs heterogenous gene expression can be caused by inherent expression noise, stochastic switching between two correlated gene expression states (Nanog-high and Nanog-low state) as well as stochastic expression bursts [857]. Epigenetic regulation, as well as the supplementation of media with “2i”, two commonly used inhibitors in stem cell culture targeting MEK and GSK3β, greatly influence heterogenous gene expression [857], [858]. Throughout the 24 h of 4E-BP1(4Ala) induction, cells were cultured in the absence of 2i, which likely enhanced the cell heterogeneity prior to MS analysis.
Besides, it is possible that transgenic clones are not genetically identical due to individual accumulation of numerical and/or structural chromosomal aberrations during passaging. It was already reported earlier that the E14TG2 cell line, which is a parental cell line of A17 used in this study, shows certain chromosomal instability and is aneuploid at passage 29 (p29) with a modal karyotype of 42 chromosomes that can easily increase to 52 chromosomes within four passages (p33) [859]. These cells are specifically prone to acquire trisomy 8 and partial or total duplication of chromosome 11 which are associated with higher proliferation rates constituting a selective advantage of aneuploid cells over euploid cells under long-term culture conditions [859]–[861].
Fig. 6: Upregulate genes after dominant negative 4E-BP1 induction in mESCs were selected as candidates for putative cap-independent translation. Differentially expressed proteins between wt mESCs and 4E-BP1(4Ala) expressing mESCs were analyzed individually for each clone (A6, B4 and C6) and were examined in a pooled analysis in which data of all clones was combined.
153
The first subset of candidate genes was selected based on either significant (p ≤ 0.05) enrichment of ≥ 4-fold (log2≤-2) in individual or pooled clones’ analyses (17 candidate genes). A second subset of candidate genes was selected based on persistent enrichment of ≥ 2.8-fold (log2≤-1.5) in all clones (nine candidate genes).
However, taking all clones together as a whole and comparing the combined clonal cell population against wt, variations of single clones are equalized and significantly upregulated genes in individual clones are indeed found to be upregulated in the combined cell population. In addition, the combined analysis discovered four more candidate genes which were significantly (p ≤ 0.05) enriched by more than 4-fold that were not found to be significantly enriched when looking at single clones only (Fig. 6, Fig. 7). According to these findings, we draw our attention to proteins which were consistently enriched in all the three clonal cell lines to minimize unspecific effects and clone-specific alterations in the candidate selection. In this manner, we could identify nine more candidate genes which were reliably enriched more than 2.8-fold in all 4E-BP1(4Ala) exrepssing cells (Fig. 7).
In total, the MS analysis identified a set of 26 proteins which were more than 2.8-fold upregulated after 24 h of 4E-BP1(4Ala) overexpression (Tab. S1). These 26 candidate genes of cap-independent/ 4E-BP1-resistant translation initiation were selected for further analysis.
154 Figure continues on the next page
155
Fig. 7: Proteomic analysis identified upregulated genes after dominant negative 4E-BP1 induction in mESCs. Differentially expressed proteins between wt mESCs and 4E-BP1(4Ala) expressing mESC clones A6, B4 and C6 were determined 24 h after induction by LC-MS/MS. Analysis was performed in three biological replicates and 6,409 proteins were detected. Volcano plots depict Log2(LFQ value wt/LFQ value clone) on the x-axis and –log10(p-value) on the y-axis (two sample t-test). Upregulated candidate genes are listed and highlighted according to different selection criteria. See candidate legend and color code panels for details.
LC-MS/MS performed by Guido Mastrobuoni
156
3.3 Dominant negative 4E-BP1 resistant genes are linked to DNA repair and neuron projection