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Fig. 4 | Genome Biology

Fig. 4

From: SRSF3 and SRSF7 modulate 3′UTR length through suppression or activation of proximal polyadenylation sites and regulation of CFIm levels

Fig. 4

Two SRSF7-specific protein features promote its interaction with CPA factors. a Alignment (Clustal Omega) of SRSF3 (amino acids [aa] 1–164) and SRSF7 proteins (aa 1–238). Protein domains are highlighted in dark gray (RRM), light gray (linker domain), olive (CCHC-type Zn knuckle), and light brown (RS domain). The 27 aa stretch is highlighted in bold with hydrophobic residues marked in red. Cysteine residues mutated to alanine in Zn knuckle in SRSF7-mutZn are marked with asterisks. b Scheme of GFP-tagged SRSF7 mutants stably expressed in P19 cells. c Co-IPs using GFP-tagged SRSF7/SRSF7-Δ27aa/SRSF7-mutZn cells with RNase A treatment: Proteins were pulled-down by α-GFP antibodies and probed for CPA factors using specific antibodies. PABPN1 served as a control for RNA degradation. IP, immunoprecipitation; IgG, unspecific antibody control. d Scheme of GFP-tagged SRSF3 chimeras harboring Zn knuckle, 27 aa stretch of SRSF7, or both. e Co-IPs using GFP-tagged SRSF3 chimeras: Proteins were pulled-down by α-GFP antibodies and probed for CPA factors using specific antibodies. Lysates were treated with (+RNase) or without (−RNase) RNase A prior to IPs. PABPN1 served as a control for RNA degradation. Inp, input. Samples without RNase A treatment are shown in Fig. S7A. f Quantification of 3′RACE-PCRs (n = 3) of Hspa4, Pphln1, and Tnpo3 transcripts in Ctrl and Srsf7 knockdown P19 cells transiently overexpressing SRSF3-27aa and SRSF3-27aa + ZnF chimeric proteins. Data are represented as mean ± standard deviation of mean. Student’s t test, *P value < 0.05

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