I am responding to the peer reviewer's comments an...
Created on: March 31, 2025
Question
I am responding to the peer reviewer's comments and this is his question: Is there any biological significance to the 10% of genes that are co-regulated by FY, CPSF30, and FIP1 (line 144). The same for the 27% of genes that have increased readthrough in both fip1 and pap1?
I have written a draft response; please polish these sentences up. Please rephrase it for clarity, coherence and conciseness, ensuring each paragraph flows into the next. Please rephrase it for clarity, coherence and conciseness, ensuring each paragraph flows into the next. Remove jargon.除此之外我现在觉得提到他们识别polya signal的差异是在给自己找事，是不是删掉更合适，如果你觉得删掉不提更好，就删掉 My response: We thank the reviewer for this insightful question. The 10% overlap of genes co-regulated by FY, CPSF30, and FIP1, along with the 27% overlap of genes with increased readthrough in both fip1 and pap1 mutants, indeed suggests potential biological significance. However, as the reviewer pointed out, overlaps alone cannot confirm causality, and further investigation of their functional relevance is necessary in the future.
In our study, we observed that hundreds of protein-coding genes exhibited significant increases in transcriptional readthrough in fy, cpsf30, and fip1 mutants (Fig. 1e), highlighting their involvement in transcription termination regulation. The 10% overlap of co-regulated genes by FY, CPSF30, and FIP1 (Fig. S3a, Fig. S3b) could suggest a specific set of genes that are highly sensitive to CPSF complex regulation, and the relatively low overlap may reflect the modular of the CPSF complex. Unlike the tightly coordinated CstF complex, which regulates genes through a more conserved mechanism (the 75% overlap in cstf64 and cstf77 mutants), the CPSF components may depend on broader polyadenylation signals (PAS), indicating a more complex regulatory role. This modularity in CPSF may contribute to its regulation of distinct gene sets, as the components—such as FY and CPSF30, which recognize canonical PAS motifs, and FIP1, which binds U-rich sequences downstream—each play specific roles in mRNA 3' end processing. It is likely that genes co-regulated by FY, CPSF30, and FIP1 are particularly dependent on the assembly of the CPSF complex for transcription termination. However, the relatively small proportion of genes in this subset (10%) suggests that these factors may be involved in the regulation of a specific subset of genes, rather than influencing all genes.
The 27% overlap of genes showing increased readthrough in both fip1 and paps1 mutants (Fig. S3c) further suggests a complex interaction between these two factors. While the overlap hints at a potential regulatory relationship, the distinct gene subsets regulated by each factor point to independent, or at least partially independent, roles for FIP1 and PAPS1 in transcription termination. FIP1, known for its role in bridging CPSF and poly(A) polymerases, recruits PAPS1 to the polyadenylation site, but the lower overlap between FIP1 and PAPS1 genes suggests that PAPS1 may also function in conjunction with other factors, possibly in a way that is independent of FIP to regulate termination. This could imply that PAPS1's role in termination is not entirely dependent on FIP1 in Arabidopsis, underscoring the complexity of termination regulation.
In summary, the CstF complex operates more as a tightly coordinated unit, whereas the CPSF complex, due to its functional modularity, interacts with broader PAS contexts and regulatory factors. The overlap observed between the genes regulated by FIP1 and PAPS1 further supports the idea that these factors, while interacting, also function through distinct regulatory mechanisms. These observations highlight the specialized transcription termination regulation in Arabidopsis, where different factors contribute to finely tuned control over gene expression.
We have revised the Discussion section to better clarify these points and the potential implications of the observed overlaps:
Thought
Answer
Below is a revised version that is more concise, clear, and cohesive. Because you suggested potentially removing the explanation about different polyadenylation signals, I have omitted that section to keep the focus on the main points:
We thank the reviewer for this insightful question. The 10% overlap of genes co-regulated by FY, CPSF30, and FIP1, as well as the 27% overlap of genes with increased readthrough in both fip1 and paps1, does hint at potential biological significance. However, overlaps alone do not demonstrate a causal relationship, and further experiments will be needed to understand their functional relevance.
Our data show that hundreds of protein-coding genes exhibit increased transcriptional readthrough in fy, cpsf30, and fip1 mutants (Fig. 1e), underscoring the importance of these factors in transcription termination. The modest (10%) overlap suggests that a specific subset of genes may depend heavily on the assembly and activity of the CPSF complex for proper termination, while most other genes are regulated by different mechanisms or complexes. In contrast, the CstF complex appears to act more as a single, tightly coordinated unit, evidenced by the higher degree of overlap (75%) in cstf64 and cstf77 mutants.
The 27% overlap of genes showing increased readthrough in both fip1 and paps1 mutants (Fig. S3c) further underscores the complexity of termination control. While FIP1 recruits PAPS1 to the polyadenylation site, the relatively low overlap indicates that PAPS1 also works with other factors and may regulate additional genes independently of FIP1. Thus, FIP1 and PAPS1 likely cooperate in some contexts but also perform separate functions.
In summary, whereas CstF components seem to function in a tightly integrated manner, the CPSF complex demonstrates a modular strategy, in which each component (FY, CPSF30, and FIP1) contributes to the termination of distinct gene subsets. Similarly, FIP1 and PAPS1 overlap in some targets but also carry out independent roles. These findings highlight the specialized nature of transcription termination in Arabidopsis and the diverse regulatory mechanisms that control gene expression. We have revised the Discussion section to incorporate these points and clarify the potential implications of these overlaps.