TM7SF3 affects alternative splicing both under basal conditions and under stress (A) U2-OS cells were transfected with TM7SF3-siRNA or with a non-targeting sequence. RNA was extracted, and splicing variants of the indicated genes were detected by PCR. (B) Bar graphs represent the mean expression of PIG3 exons that were determined by real-time PCR with primers targeting Ex1-2 junction and Ex3-5 junction, n = 3. Results are presented as means G SEM (**p<0.01). (C) HFF cells were transfected with TM7SF3-siRNA or with a non-targeting sequence. Splicing variants of the indicated genes were detected by PCR. (D and E) U2-OS were transfected with TM7SF3-siRNA or with a non-targeting sequence. Cells were then treated with H 2 O 2 (500 mM; 3 h) (D) or tunicamycin (5ug/mL; 8 h) (E). Splicing variants of MCL1 (D) or XBP-1 (E) were detected by PCR.

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... is a ubiquitous phenomenon that affects almost any gene, therefore the pre-mRNAs chosen for analysis were randomly selected. As shown in Figure 5A, silencing of TM7SF3 in U2OS cells selectively inhibited AS of a number of pre-mRNAs, including PIG3 and ATP5C1 but it did not affect AS of other pre-mRNAs (e.g. EZH2, SRSF7, XBP1, EIF6, VEDFB, and MRPL52). ...

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... certain instances, silencing of TM7SF3 affected both basal and alternative splicing (cf. MCL1, Figure 5A). The affected exons Total cell extracts of U2-OS (n = 3) or HFF cells were immunoprecipitated with pre-immune serum or anti-TM7SF3 antibodies. ...

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... iScience 25, 105270, November 18, 2022 iScience Article were Ex4 of PIG3 ( Figure 5B) and Ex2 of MCL-1 ( Figure 5C). In contrast, silencing of TM7SF3 in HFF cells promoted AS of EZH2 ( Figure 5C), suggesting that depending on the cell type, TM7SF3 can either promote or inhibit AS of a given pre-mRNA. ...

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... iScience 25, 105270, November 18, 2022 iScience Article were Ex4 of PIG3 ( Figure 5B) and Ex2 of MCL-1 ( Figure 5C). In contrast, silencing of TM7SF3 in HFF cells promoted AS of EZH2 ( Figure 5C), suggesting that depending on the cell type, TM7SF3 can either promote or inhibit AS of a given pre-mRNA. ...

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... iScience 25, 105270, November 18, 2022 iScience Article were Ex4 of PIG3 ( Figure 5B) and Ex2 of MCL-1 ( Figure 5C). In contrast, silencing of TM7SF3 in HFF cells promoted AS of EZH2 ( Figure 5C), suggesting that depending on the cell type, TM7SF3 can either promote or inhibit AS of a given pre-mRNA. Based on the above results, we wished to determine how general are the effects of TM7SF3 on alternative splicing and whether it affects specific splice sites. ...

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... of cellular stress alters both the structure of nuclear speckles (Figure 2) as well as the profile of AS ( Paronetto et al., 2016), therefore we checked whether TM7SF3 affects AS under stress. Treatment of U2OS cells with the stress inducers H 2 O 2 ( Figure 5D) or tunicamycin ( Figure 5E) markedly induced AS of MCL1 and XBP-1, respectively, whereas prior treatment of these cells with TM7SF3 siRNA significantly inhibited this process. These findings suggest that TM7SF3 regulates AS both under basal conditions and under stress. ...

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... of cellular stress alters both the structure of nuclear speckles (Figure 2) as well as the profile of AS ( Paronetto et al., 2016), therefore we checked whether TM7SF3 affects AS under stress. Treatment of U2OS cells with the stress inducers H 2 O 2 ( Figure 5D) or tunicamycin ( Figure 5E) markedly induced AS of MCL1 and XBP-1, respectively, whereas prior treatment of these cells with TM7SF3 siRNA significantly inhibited this process. These findings suggest that TM7SF3 regulates AS both under basal conditions and under stress. ...

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... gain a deeper mechanistic insight into the mode of action of TM7SF3, its expression was silenced independent of, or concomitantly with silencing of HNRNPK that forms stable complexes with TM7SF3 (Fig- ure 4G and, Table 1). HNRNPK binding motifs were selected by comparing previous findings (Selvanathan et al., 2015) with the LSVs data ( Figure S5). Only genes, whose global expression was not affected upon silencing of TM7SF3 ( Figure S5) were selected for further analysis. ...

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... binding motifs were selected by comparing previous findings (Selvanathan et al., 2015) with the LSVs data ( Figure S5). Only genes, whose global expression was not affected upon silencing of TM7SF3 ( Figure S5) were selected for further analysis. Furthermore, alternative-splicing events having functional consequences were selected for analysis. ...

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... 25, 105270, November 18, 2022iScience Article significant differential expression of 1465 genes between control cells and cells in which TM7SF3 has been silenced. Silencing upregulated the expression of 844 genes, whereas the expression of 621 genes was downregulated ( Figure S5). Gene ontology analysis revealed that silencing induced a number of pathways and biological processes, such as DNA damage responses (17 out of 120 genes) and telomere stress induced senescence (15 out of 80 genes) (Table S7). ...