Knock-down of Tb927.10.14150 affects cell growth in the bloodstream

putative RNA-binding protein without any canonical RNA-binding domain that is conserved in Kinetoplastids and has also some orthologues in oomycetes and brown algae. Sequence identity in Kinetoplastida is equally spread through the protein sequence (Suppl. Figure 1).

However, sequence identity to the oomycetes and brown algae is not very high.

Tb927.10.14150 was an up-regulator in the tethering screen (Erben et al., 2014b) and the relative protein abundance of the protein is similar in both stages during differentiation (Dejung et al., 2016). Tb927.10.14150 protein displays in vivo mRNA binding (Lueong et al., 2016) although it lacks canonical RNA-binding domains. It has some similarities to yeast Bfr1p (Brefeldin A resistance protein), an ER- and polysome-associated protein (Lang et al., 2001; Weidner et al., 2014), which is not essential (Jackson and Kepes, 1994). Bfr1p interacts with RNA, although it lacks canonical RNA-binding domains. Bfr1p together with Scd160 inhibits P-body formation under normal growth conditions and thereby protects RNAs at ribosomes (Weidner et al., 2014). Since Tb927.10.14150 has some similarities to the yeast Bfr1 protein, even if the sequence identity is very low (Suppl. Figure 2.1), I will rename Tb927.1014150 as BFR1-like (BFR1L) protein. BFR1L consists of 479 amino acids.

It has a predicted molecular weight of 55.3 kDa and contains four low complexity regions, of which one is located in the N-terminal region and the other three are located in the C-terminal region (Figure 2.1 A). To investigate whether BFR1L is essential in bloodstream form trypanosomes (BF), cell growth after RNAi-mediated knock-down of BFR1L was analyzed. I could observe a growth defect upon RNAi (+ Tet) in comparison to the uninduced cells (- Tet), but it was not lethal (Figure 2.1 B). The decrease of the in situ V5-tagged protein after RNAi was monitored over time and analyzed by Western Blotting (Figure 2.1 C). The protein level decreased already after 1 day of Tetracycline induction and was not detectable at later points, which showed successful knock-down. The division

Figure 2.1: Knockdown of BFR1L expression in BF V5-BFR1L cells. A. Domains of BFR1L. LC:

Low complexity region. B. Growth curve for dsRNAi of BFR1L in BF V5-BFR1L cells of three independent clones. Error bars indicate standard deviation. +Tet (dashed lines) and -Tet (solid lines). C. 3x10⁶ cells were collected each day after counting and Western Blot of V5-BFR1L was performed. Aldolase was used as loading control. Numbers below the blot indicate quantification of the V5-BFR1L signal. Day 0 was set as 1. D. Division times (in h) of +/- Tet calculated from 3

time after knock-down (+Tet) was 11.6 ± 0.5 h, whereas the division time of the control cells (-Tet) was 7.9 ± 0.3 h (Figure 2.1 D).

To investigate whether BFR1L is essential in the bloodstream form, both ORFs of BFR1L were replaced with two different genes encoding for antibiotic resistances (Figure 2.2 A). At first, a single knockout (SKO) was created in which one ORF of BFR1L was replaced by the gene encoding for Blasticidin-S deaminase (BSD). To create a double knockout (DKO) the second ORF of BFR1L was replaced by the gene encoding for Puromycin N-acetyltransferase (PAC). To confirm that both genes of BFR1L were knocked out in this cell

Figure 2.2: Growth of bloodstream form cells without BFR1L. A. Schematic representation of the different fragments that were amplified by PCR. BSD: Blasticidin-S deaminase; PAC: Puromycin N-acetyltransferase. B. gDNA of wild type BF cells (WT) and BF BFR1L^-/- (DKO) was extracted and amplified using different primer pairs to amplify the different fragments described in A. C.

Cumulative growth curve of 3 independent experiments, which were done at different times but with the same clones, for BF (WT), BF BFR1L^+/- (SKO) and BF BFR1L^-/- (DKO). Error bars indicate standard deviation.

line, gDNA of the WT and DKO was extracted and different fragments were amplified by PCR, as shown in figure 2.2 A. The amplified PCR fragments can be seen in figure 2.2 B.

Fragment 1 or 2 could only be amplified using the DKO gDNA as template, but not the WT gDNA as template, which indicated that one allele of the BFR1L ORF was replaced by BSD gene. Otherwise, primers to amplify the ORF of BFR1L could only amplify fragment 3 by using the WT genomic DNA (gDNA) as template, but not by using the DKO gDNA as template. Replacement of the second BFR1L allele by PAC gene was investigated by amplification of fragment 4 and 5. In both cases, the fragment could be amplified by using the DKO gDNA as template, but not by using the WT gDNA. In addition, primers to amplify the PAC gene can only amplify fragment 6 by using the DKO gDNA as template, but not by using the WT gDNA as template. Finally, amplification of fragment 7 was investigated. This fragment has different sizes dependent on the sequence length of the different genes. It can be seen that fragment 7 had a size of 2200 bp, when WT gDNA was used as template.

When DKO gDNA was used as template two bands with sizes of 1100 bp and 1300 bp appeared indicating replacement of the BFR1L gene by the PAC and BSD gene. After confirmation of successful knock-out of BFR1L, the growth of DKO cells was compared with SKO cells and WT cells over seven days in 3 independent experiments (Figure 2.2 C). The SKO cells showed a negligible growth defect in comparison to the WT cells. I could observe a growth defect of the DKO cells in comparison to the WT and SKO cells, but it was not lethal. This reflects the data of BFR1L knock-down by dsRNAi.

Next, I investigated, whether the growth defect of the DKO bloodstream form cells could be complemented by overexpression of myc-tagged BFR1L. For that reason, double-knockout cells with inducible ectopically expressed N-terminally myc-BFR1L (BF BFR1L^{-/-, MYC}) were created, which is a conditional DKO (cDKO). The growth of these cells with and without induction of myc-tagged BFR1L expression was compared with the growth of WT BF cells and the BFR1L^-/- cells (DKO). As described before, I could observe a growth defect of the DKO cells in comparison to the WT cells. The BF BFR1L^{-/-, MYC} cells grew as the WT in both cases: with and without tetracycline induction (Figure 2.3 A). However, the overexpression plasmid is leaky as it can be seen by Western Blotting. Even in the uninduced cells (-Tet), a myc-BFR1L band could be observed. Surprisingly, BFR1L appeared as double band, which I could not observe on other WBs (see figure 2.1 C). This experiment showed that the growth defect of the DKO cells can be rescued by overexpression of myc-tagged BFR1L. However, when repeating this experiment, I could not observe a growth defect of the DKO. It is possible that cells have simply been adapted to loss of BFR1L during culturing. In procyclic cells (PC), I was not able to generate a gene knock-down of the protein neither by dsRNAi nor by stem-loop RNAi, despite several attempts (data not shown). In addition, I was also not able to generate a double knockout of BFR1L in PCs;

however, I could create a SKO of the protein in PCs. These cells were then transfected with the ectopic expression plasmid to create PC BFR1L^{-/+, MYC} cells (conditional SKO=cSKO).

In these cells, myc-tagged BFR1L was expressed by Tetracycline induction and then they were transfected with a plasmid that replaces the second ORF of BFR1L with the PAC gene to create a conditional DKO (PC BFR1L^{-/-, MYC}). Surprisingly, all different cell lines (SKO, cSKO, cDKO) cells grew as the WT in both cases: with and without tetracycline induction (Figure 2.3 B; performed by Pia Hartwig (PH)). As it can be seen by Western Blotting, the overexpression of myc-BFR1L is tightly regulated by tetracycline induction. In the uninduced cells no myc-BFR1L signal can be observed, which suggests that BFR1L is not essential in the procyclic form.

Taken together, knock-down or knock-out of BFR1L in BF trypanosomes leads to a slight growth defect, which can be rescued by ectopic expression of the protein, whereas I was neither able to generate a RNAi-mediated knock-down cell line of BFR1L nor to generate a DKO cell line in PC trypanosomes. However, I was able to generate a cDKO in PCs.

Surprisingly, growth was not restricted by removing of tetracycline from these cells and cells grew as wildtype even without ectopic expression of myc-tagged BFR1L.