Page 130 - 2022_03-Haematologica-web

V. Madan et al.
AB
C
Figure 4. Loss of ZRSR1 exacerbates retention of U12-type introns in ZRSR2-deficient murine hematopoietic cells. (A) Distribution of DMSI values for retention of U12-type introns in Lin−Kit+ bone marrow (BM) cells lacking either ZRSR1 (sh1 or sh10) or ZRSR2 or both compared to control cells. (B) Number of U12-type introns retained (P<0.05; Fisher's exact test) in various pair-wise comparisons of Lin−Kit+ BM cells deficient in either one or both ZRSR proteins. (C) Normalized expression of representative U12-type introns in Lin−Kit+ BM cells determined using quantitative real-time polymerase chain reaction. Expression of U12-type introns was meas- ured relative to expression of flanking exons. Data are from at least three replicates and represented as mean ± standard error of the mean. P-values for each group compared to the ‘ZRSR2 WT; con sh’ group are depicted in the plot. Statistical difference between the Zrsr2/Zrsr1-double deficient and Zrsr2 KO cells are shown below the graph. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; ns: not significant; MSI: mis-splicing index: Difference in MSI values (DMSI) was calculated as DMSI=MSIknockout−MSIwildtype.
deficient cells compared to control cells (Online Supplementary Figure S8F).
In order to verify that human ZRSR1 is a non-function- al copy of ZRSR2, we generated K562 cells lacking either ZRSR1 or ZRSR2 or both proteins together. DZRSR1 cells were first generated using CRISPR/Cas9 technique fol- lowed by silencing of ZRSR2 using shRNA. As expected, ZRSR2 deficiency alone impaired splicing of U12-type introns. However, loss of ZRSR1, either alone or when combined with ZRSR2, did not significantly alter the splicing of U12-type introns in K562 cells (Online Supplementary Figure S9). Taken together, our experiments highlight a functional role of murine ZRSR1 in splicing of the U12-type introns.
Mis-splicing of MAPK9 and MAPK14 impacts their protein expression in murine and human cells
Several crucial genes harbour U12-type introns includ- ing members of the mitogen-activated protein kinase
(MAPK) family of serine-threonine kinases. MAPK pro- teins play a crucial role in a wide range of biological processes including hematopoiesis.29 We focused on splic- ing of two members, MAPK9 (JNK2) and MAPK14 (p38α), as aberrant retention of their U12-type introns was verified in murine myeloid precursors deficient in both Zrsr1 and Zrsr2 (Figure 4C). Intron retention of both MAPK9 and MAPK14 resulted in the generation of aber- rant transcripts containing a premature stop codon. In order to investigate if the retention of U12-type introns in these two genes affects their protein expression, western blotting was performed with anti-MAPK9 and anti- MAPK14 antibodies. Protein levels of both MAPK9 and MAPK14 were reduced, albeit modestly, in murine myeloid precursors, and MAPK14 levels were also reduced in 32D cells lacking ZRSR2 and ZRSR1 (Online Supplementary Figure S10A and B). Retention of U12-type introns of MAPK9 and MAPK14 was also observed in ZRSR2 knockdown K562 and TF1 cells (Figure 5A and B).
686
haematologica | 2022; 107(3)