Extended Data Fig. 6. Translation of Split-ORF2, RNA-binding and oligomerization of SRSF7 contribute to the formation of SRSF7 bodies in vivo.

a, Intron 5 undergoes alternative 5’ splice site usage, which gives rise to four different SRSF7 isoforms lacking different portions of the four RSRSXSXR repeats (X – hydrophobic aa, highlighted in orange). b, Co-IP of purified SRSF7-GFP, SRSF7_Δ27aa-GFP and SRSF7_mutZn-GFP probed with α-SRSF7 to verify oligomerization with endogenous SRSF7. α-PABPN1 was used as control for RNase treatment. IgG – unspecific antibody control, In – Input. c, Quantification of (n=3) Co-IP experiments. Shown is the interaction of endogenous SRSF7 with SRSF7-GFP, SRSF7_Δ27aa or SRSF7_mutZn normalized to the respective baits. Two-sided T-test, **p-value < 0.01. Error bars s.d. d, Northern blot to compare the levels of intron-containing transcripts SRSF7-I3 (red asterisk) and SRSF7-I3+5 (purple asterisk) in SRSF7-GFP, SRSF7_Δ27aa or SRSF7_mutZn cells. e, RT-PCR of SRSF7 isoforms generated from reporter and endogenous SRSF7 genes in WT, SRSF7 OE, SRSF7_Δ27aa and SRSF7_mutZn P19 cells with (+) and without (-) CHX using the indicated primers. f, Same as in E), except that primers specific for both endogenous and reporter SRSF7 genes were used. g, RT-qPCR quantification of SRSF7-PCE 1/4 and SRSF7-PCE transcript level after CHX treatment in SRSF7-GFP, SRSF7_Δ27aa and SRSF7_mutZn cells normalized to the respective DMSO controls (n=4). Two-sided T-test, *p-value < 0.05. Error bars s.d. h, Line scans of example cells to assess co-localization of SRSF7 bodies with SRSF7-GFP, SRSF7_Δ27aa and SRSF7_mutZn. i, Autoradiograph of an iCLIP experiment using α-GFP antibodies to purify GFP-tagged SRSF7, SRSF7_Δ27aa and SRSF7_mutZn. Crosslinked RNA was labeled with ³²P. Non-crosslinked samples (-UV) served as controls. Cut bands are indicated in grey squares. j, Venn diagram displaying the overlap in RNA targets. K, Comparison of crosslink preferences of SRSF7-GFP and SRSF7_mutZn around 5’ splice sites. L, Mean signal intensities of SRSF7 bodies quantified from 100 cells. Box plot indicates median, first and third quartiles (box), whiskers show 1.5× interquartile range.