MNAB, a paralog of Roquin, is unable to downregulate ICOS

MNAB and Roquin are paralogs with a similar domain organization. Both proteins have amino acid identities ranging from 71.4% within the RING finger, 92.4% in the newly defined ROQ domain and only 18.9% in the zinc finger domain. However, this degree of similarity for the RING finger and ROQ domain indicates that both proteins could perform similar functions.

To address this question we compared protein levels of mouse Roquin and mouse MNAB. Since Roquin protein levels in mouse tissue are highest in the thymus (data not shown), we compared Roquin and MNAB protein levels in this organ. To be able to detect both proteins, an enrichment was performed by immunoprecipitation with an antibody (Q4-2) that recognizes an epitope from a peptide that is shared in both proteins. Endogenous MNAB migrates higher than Roquin, as expected (section 8.1.1). MNAB appears only in one band, whereas endogenous Roquin migrates in two isoforms. Interestingly, MNAB was much higher expressed than Roquin (Figure 8-11 a). Comparing mRNA expression levels of Roquin and MNAB in lymphoid organs, Roquin and MNAB mRNAs show a similar expression profile, with highest expression in the thymus, followed by slightly decreased expression in lymphnodes and a significant lower expression in spleen (Figure 8-11 b). It is not clear how Roquin and MNAB levels relate to each other on mRNA levels since the measurements were performed in different PCR runs. These cannot be compared reliably with each other due to technical variations. Summarizing, Roquin and MNAB show a similar expression profile, but MNAB might be higher expressed in general, at least on the protein level. It should be noted that the differential expression of MNAB and Roquin in thymus, lymhnodes and spleen can also be due to the different T and B cell-composition of these organs and that the data presented above probably reflect the differential expression of Roquin and MNAB mRNA in lymphocytic cells.

Figure 8-11: MNAB and Roquin protein and mRNA expression in lymphoid organs. (a) Immunoprecipitations of Roquin and MNAB from thymus were performed with an antibody recognizing MNAB and Roquin (Q4-2) (section 8.1.1). Extracts from equal cell numbers were used for immunoprecipitation and analysis by SDS-PAGE. Immunoblots were probed with the indicated antibodies. (b) Thymus, lymph nodes and spleen were isolated from BALB/c mice. Single cell suspensions were used for RNA isolation. Roquin and MNAB mRNA expression levels were determined

by quantitative PCR.

To investigate whether MNAB can also downregulate ICOS, ICOS surface levels of in vitro differentiated Th2 cells that overexpressed Roquin or its paralog MNAB were compared. Primary CD4 T cells from DO11.10 CAR∆1 mice (section 6.6) were used to introduce adenoviral expression vectors (section 6.3.2). After in vitro differentiation of primary CD4 T cells into Th2 cells, the cells were infected with bicistronic adenoviruses that encode MNAB, Roquin or Roquin M199R and in each case GFP from an IRES sequence. Cells overexpressing MNAB, and consequently also GFP, did not significantly decrease ICOS expression (Figure 8-12 a, c, page 64). In the same approach Roquin effectively reduced ICOS surface levels (Figure 8-12 a). Furthermore, the effect was exclusively observed in GFP positive cells, in cells that actually overexpressed Roquin. The exchange of amino acid

199 from methionine to arginine created a functional hypomorph of Roquin that downregulated ICOS with an attenuated dose response, similar to previously reported findings [1, 2]. This effect of Roquin was specific for ICOS as there was no change in the surface expression of another activation-induced protein CD44 (Figure 8-12 b, d).

Figure 8-12: Roquin has specialized to repress ICOS surface levels on T cells. (a-d) In vitro differentiated

Th2 cells from DO11.10 CAR∆1 mice were infected with the indicated adenoviral vectors at an MOI of 50 on day 4 and analyzed two days later by flow cytometry for ICOS (a) or CD44 (b). The boxes (a, b) indicate GFP positive cells, for which ICOS (c) or CD44 expression levels (d) were quantified.

To identify unique sequences in Roquin that are critical for ICOS downregulation chimeras of Roquin and MNAB were analyzed for ICOS repression. In these chimeras the conserved carboxy-termini of Roquin and MNAB were reciprocally exchanged (section 6.3). Expression of Roquin/MNAB chimeras in MEF cells revealed that sequences carboxy- terminal to the zinc finger in Roquin contribute to downregulate the co-expressed ICOS (Figure 8-13, page 65).

In fact, the amino-terminus of MNAB in the context of the carboxy-terminus of Roquin was able to support ICOS downregulation. In contrast, the carboxy-terminus of MNAB fused to the amino-terminus of Roquin produced a protein with strongly reduced activity (Figure 8-13, page 65).

Figure 8-13: The carboxy-terminus of Roquin has specialized for efficient ICOS repression. (a-b) MEF cells were transduced with retroviruses that encoded the human ICOS cDNA with 3’UTR and superinfected to co-express Roquin or MNAB or chimeras of both proteins from bicistronic Thy1.1 co- expressing retroviruses. Flow cytometric analysis showing contour plots of antibody-stained ICOS and Thy1.1 surface expression (a) or a bar diagram of the average ICOS expression from three independent experiments of the relative mean fluorescence intensity (MFI) of all cells normalized to the ICOS MFI in cells transduced with MNAB expressing constructs (b).

These data show that the amino-terminal sequences of MNAB can exert a similar function as those of Roquin, whereas the carboxy-terminus cannot. Retroviruses encoding both chimeras showed comparable mRNA expression as judged from Thy1.1 levels. Transfection of the chimera-encoding vectors led to similar protein expression in HEK293 cells (Figure 8-2). However, quantification of ICOS downregulation revealed that even the amino-terminus of Roquin in the context of the carboxy-terminus of MNAB was more potent in repressing ICOS than the corresponding sequences in MNAB (Figure 8-13). These findings demonstrate that, in contrast to MNAB, the entire Roquin protein, but especially its carboxy-terminus, has evolved for efficient downregulation of ICOS.