我有一篇sci论文正在naature投稿，现在一个reviewer 问了一个棘手的问题：Is the...

我有一篇sci论文正在naature投稿，现在一个reviewer 问了一个棘手的问题：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?我在文章中仅通过In fy, cpsf30, and fip1 mutants, hundreds of protein-coding genes showed significant increases in TW length compared to WT (Fig. 1e). About 10% of these genes were co-regulated by FY, CPSF30, and FIP1 (Fig. S3a, Fig. S3b). 和There was a 27% overlap in genes with increased readthrough in both fip1 and paps1 mutants (Fig. S3c), suggesting a potential interaction between FIP1 and PAPS1 in promoting transcription termination.来描述我的数据，现在面对这个问题我已经写了一版response，请对其进行修改，使reviewer能够满意：
Reply: We appreciate 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 showing increased readthrough in both fip1 and pap1 mutants, suggests a potential biological significance. However, while our data highlight these relationships, it is important to note that overlaps alone cannot establish causality. This limited overlap may be attributed to the distinct functional roles of these components in recognizing different polyadenylation signals (PAS).

The 75% overlap between genes with increased readthrough in cstf64 and cstf77 mutants supports the idea that the CstF complex operates as a tightly coordinated unit, regulating a set of genes with substantial overlap. In contrast, the 10% overlap among CPSF-regulated genes for FY, CPSF30, and FIP1 highlights differences between these complexes. While the CstF complex functions as a tightly coupled unit, the CPSF components exhibit modular flexibility, adapting to a variety of PAS contexts. The lower overlap among CPSF components suggests functional modularity within the CPSF complex, which may reflect the recognition of different PAS sequences and the regulation of distinct gene sets. The more heterogeneous nature of the CPSF complex, compared to the more unified CstF complex, likely stems from its involvement in recognizing a wider range of PAS motifs.

For example, FY and CPSF30 recognize canonical PAS motifs (e.g., AAUAAA), while FIP1 binds U-rich sequences downstream of the PAS and recruits poly(A) polymerase (PAPS1) to facilitate efficient polyadenylation. It is possible that these genes rely on the efficient assembly of the CPSF complex to ensure optimal transcription termination and avoid readthrough, which could prevent transcriptional interference or the generation of aberrant transcripts. The 10% overlap of genes co-regulated by FY, CPSF30, and FIP1 suggests that these genes may be particularly sensitive to CPSF complex regulation. However, the relatively low overlap in the co-regulated gene set implies that these factors may work together to control specific gene subsets, rather than universally influencing all genes. In contrast, the CstF complex, with its more conserved recognition of PAS sequences downstream of the poly(A) site, likely plays a more consistent regulatory role across a broader set of genes. This highlights the specialized roles of the CPSF and CstF complexes in coordinating transcription termination for different gene sets.

The 27% overlap between the genes with increased readthrough in both fip1 and paps1 mutants further suggests a complex relationship between the factors that regulate transcription termination. The overlap is modest, which implies that PAPS1 and FIP1 likely regulate distinct subsets of genes. FIP1 is known to bridge CPSF and poly(A) polymerases in both yeast and humans, recruiting PAPS1 to polyadenylation. However, in Arabidopsis, the relatively low overlap between FIP1- and PAPS1-regulated genes indicates that PAPS1's role in termination is not entirely dependent on FIP1. This suggests that PAPS1 may act through additional pathways or interact with other factors involved in the termination process, possibly in a way that is independent of FIP to regulate termination.

In summary, the CstF complex operates in a more tightly coordinated manner, while the CPSF complex, with its diverse roles, likely interacts with a wider array of regulatory pathways, including those involving FIP1 and PAPS1. These observations highlight the complexity and specificity of transcription termination regulation in Arabidopsis. While there is some overlap in the regulation by different factors, each factor or complex may regulate distinct subsets of genes, underscoring the intricate and finely tuned nature of pre-mRNA processing.

We have revised the Discussion section to better clarify the potential implications:

For the 10% of genes co-regulated by FY, CPSF30, and FIP1, this limited overlap suggests that these factors may act together to control a specific subset of genes that are particularly sensitive to CPSF complex regulation. Their joint control may ensure optimal transcription termination and prevent readthrough transcription, which could otherwise lead to transcriptional interference or the generation of aberrant transcripts. The relatively small gene set may reflect the distinct roles these components play in recognizing different polyadenylation signals (PAS) and recruiting specific processing factors.

In contrast, the 27% overlap of genes showing increased readthrough in both fip1 and paps1 mutants indicates a more complex regulatory relationship. This modest overlap implies that while FIP1 and PAPS1 may influence some common targets, they largely regulate distinct gene subsets. In Arabidopsis, FIP1 is thought to bridge CPSF and poly(A) polymerases, whereas PAPS1 might also act through additional pathways or interact with other factors during termination. This suggests that PAPS1’s role in termination is not entirely dependent on FIP1 and may involve other regulatory mechanisms.

Overall, these observations highlight the complexity of transcription termination regulation in Arabidopsis. The differential overlap between gene sets regulated by various factors—such as those in the CPSF and CstF complexes—suggests that each factor or complex contributes uniquely to termination. Rather than acting redundantly, these factors appear to participate in multiple, partially overlapping pathways that fine-tune the regulation of gene expression.