Cooperative Binding of SRSF3 to Structured 3’ss-α Exon RNA during α Exon Inclusion in the ZO-1 mRNA
Round 1
Reviewer 1 Report
1. line 55, iCLIP was 1st time introduced in the paper, stands for?
2. line 155, authors stated they focused on two SRSF3 binding sites, but the paragraph only mentioned the intronic one.
2. line 174, a 20 KDa band was observed, is there any other evidence (like Mass Spec) confirm it's SRSF3?
3. line 190, results are base on which figure?
4. line 194, should be Fig. 3A, not Fig. 3, correct?
5. Fig, 3A, author states two distinct bands were observed on the EMSA, but it seems only one smear band formed with increasing 3'I-E. I'm not fully convinced by the Fig 3A data.
6. line 214, should be Fig 4B, not 3B. Same problem in line 220.
Author Response
1: line 55, iCLIP was 1st time introduced in the paper, stands for?
A: iCLIP (Illumina´s individual nucleotide resolution CLIP software; https://emea.illumina.com/science/sequencing-method-explorer/kits-and-arrays/iclip.html; last accessed December 22, 2022)
- line 155, authors stated they focused on two SRSF3 binding sites, but the paragraph only mentioned the intronic one.
A: the intronic site CUUCA at positions -8 to -4 from the 3’ss which structural analyses show that it could bind to the second site — the exonic site CUCAUC at positions +12 to +17 within the α exon (Figure 1A) —, suggesting a possible SRSF3 interaction with an RNA hairpin structure (Figure 1B).
- line 174, a 20 KDa band was observed, is there any other evidence (like Mass Spec) confirm it's SRSF3?
A: Since there is no Mass Spec evidence for this 20 kDa band, we rephrased our findings:
To further explore the direct interaction of SRSF3 with the transcripts, UV cross-linking experiments were carried out. In agreement with the super-shift experiments, a twenty kDa protein from MDCK nuclear extract was cross-linked to the 3’I-E and 3’I probes but not to the 3’Δ (Fig. 2C), although further evidence is needed to confirm the identity of the 20 kDa protein observed.
- line 190, results are base on which figure?
A: Figure 3A show a discernible complex that migrates less in the gel (with no intermediate bands) as the concentration of the recombinant SRF3 increased. As previously described (Black et al., 1998), such migration pattern suggests cooperative interaction between two SRSF3 molecules and the SRSF3 binding sites.
- line 194, should be Fig. 3A, not Fig. 3, correct?
A: The sigmoid kinetics of the SRSF3/RNA interaction (Figs. 3A and B) and the Hill plot intersect and slope (m = 1.7) (Fig. 3C) were also indicative of cooperative binding between recombinant SRSF3 and the intronic and exonic SRSF3 target sites.
- Fig, 3A, author states two distinct bands were observed on the EMSA, but it seems only one smear band formed with increasing 3'I-E. I'm not fully convinced by the Fig 3A data.
A: We have rephrased the arguments of the previous points that respond to this issue. In addition, this point is addressed in the reference by Black et al., 1998 which was included.
- line 214, should be Fig 4B, not 3B. Same problem in line 220.
A: Thank you for indicating these typos. The manuscript was corrected.
Author Response File: 
Author Response.docx
Reviewer 2 Report
This manuscript describes the interaction between the splicing factor SRSF3 and the 3’ splice site (3’aa) of alpha exon. It is demonstrated by EMSA that SRSF3 (SRp20) binds to the wild type 3’aa (3’I-E) but not to the mutant 3’mI-E. The conformational change of 3’ss and other mutants upon binding of SRSF3 was also analyzed by enzymatic probing, and the effect of SRSF3 on the exon inclusion was also examined. The presented results are interesting. However, the interpretation may not be enough especially for Fig. 4.
The authors stated that “a hairpin RNA structure is formed (abstract)” without any direct evidence. Does the RNase V1 digestion pattern in Fig. 4 match to the predicted secondary structure? It is also stated that “the presence of SRSF3 decreased the intensity of several RNase V1 bands (lines 212-213). This reviewer cannot identify the decreased bands in Fig. 4B. How the authors define the SRSF3 footprint (bar to the right in Fig. 4B)? Please map (i) RNase V1 sensitive residues without SRSF3 and (ii) the decreased bands on the predicted secondary structures.
Author Response
The authors stated that “a hairpin RNA structure is formed (abstract)” without any direct evidence. Does the RNase V1 digestion pattern in Fig. 4 match to the predicted secondary structure? It is also stated that “the presence of SRSF3 decreased the intensity of several RNase V1 bands (lines 212-213). This reviewer cannot identify the decreased bands in Fig. 4B. How the authors define the SRSF3 footprint (bar to the right in Fig. 4B)? Please map (i) RNase V1 sensitive residues without SRSF3 and (ii) the decreased bands on the predicted secondary structures.
A: The introduction was modified as follows:
Using V1 RNAse we probed the possible formation of a hairpin RNA structure between the intronic and the proximal exonic SRSF3 binding sites. The hairpin sufficed for complex formation in EMSA. The interaction of SRSF3 with the intronic site promoted the cooperative binding of SRSF3 to the exonic site. Finally, SRSF3 restored α exon activation in SRSF3 knockdown transfectants.
The results section was modified as follows:
As expected, the presence of SRSF3 decreased the intensity of several RNase V1 bands (between residues 62-67) in the wild-type 3´I-E probe (Fig. 4B, lanes 1-3), but not in the intronic mutant 3’mI-E (lanes 4-5), suggesting that SRSF3 binding to the intronic site stabilized double-stranded RNA interactions, probably exposing other nearby single-stranded sites. To delineate the relevance of the loop structure, we analyzed the structural mutant 3’I-mE that does not disturb the SRSF3 binding sites but altered the hairpin arrangement of such sequences. Interestingly, the effect was similar to that observed for intronic mutants (Fig. 4B, lines 6-7), suggesting a structural dependence for SRSF3 interaction.
In addition, Figure 4 was replaced with a newer version and its caption now states:
The bar to the right indicates the SRSF3 footprint; residues G63 - G66 are indicated by arrowheads; in agreement with the predicted lowest energy secondary structure, black arrowheads at positions 49-52, 41-46, 31-36, 18-21, 14-16, and 7-9 show the hypersensitive sites resulting from SRSF3-RNA interaction.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
The authors have addressed all my comments.
Reviewer 2 Report
The authors add “in agreement with the predicted lowest energy secondary structure” without additional interpretation of the Fig. 4B. This may be removed.
