Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public Review):
Summary:
This manuscript builds upon the authors' previous work on the cross-talk between transcription initiation and post-transcriptional events in yeast gene expression. These prior studies identified an mRNA 'imprinting' phenomenon linked to genes activated by the Rap1 transcription factor (TF), a surprising role for the Sfp1 TF in promoting RNA polymerase II (RNAPII) backtracking, and a role for the non-essential RNAPII subunits Rpb4/7 in the regulation of mRNA decay and translation. Here the authors aimed to extend these observations to provide a more coherent picture of the role of Sfp1 in transcription initiation and subsequent steps in gene expression. They provide evidence for (1) a physical interaction between Sfp1 and Rpb4, (2) Sfp1 binding and stabilization of mRNAs derived from genes whose promoters are bound by both Rap1 and Sfp1 and (3) an effect of Sfp1 on Rpb4 binding or conformation during transcription elongation.
Strengths:
This study provides evidence that a TF (yeast Sfp1), in addition to stimulating transcription initiation, can at some target genes interact with their mRNA transcripts and promote their stability. Sfp1 thus has a positive effect on two distinct regulatory steps. Furthermore, evidence is presented indicating that strong Sfp1 mRNA association requires both Rap1 and Sfp1 promoter binding and is increased at a sequence motif near the polyA track of many target mRNAs. Finally, they provide compelling evidence that Sfp1-bound mRNAs have higher levels of RNAPII backtracking and altered Rpb4 association or conformation compared to those not bound by Sfp1.
Weaknesses:
The Sfp1-Rpb4 association is supported only by a two-hybrid assay that is poorly described and lacks an important control. Furthermore, there is no evidence that this interaction is direct, nor are the interaction domains on either protein identified (or mutated to address function).
Indeed, our two hybrid, immunoprecipitation and imaging results do not allow us to conclusively discern whether the interaction between Rpb4 and Sfp1 is direct or indirect. While the interaction holds significance, we consider the direct versus indirect distinction to be of secondary importance in the context of this paper. In the current text we indicated that 'our two hybrid, immunoprecipitation and imaging results do not differentiate between a direct or indirect interactions' (see page 6, sentences highlighted in blue)
The contention that Sfp1 nuclear export to the cytoplasm is transcription-dependent is not well supported by the experiments shown, which are not properly described in the text and are not accompanied by any primary data.
This section has been re-written for better clarity (see page 7). We note that this assay was originally developed and published by Lee, M. S., M. Henry, and P. A. Silver in their 1996 paper in G&D and has since been reported in numerous subsequent studies. Reassuringly, our conclusion is bolstered by the observation that Sfp1 binds to Pol II transcripts co-transcriptionally, suggesting that Sfp1 is exported in the context of the mRNA.
The presence of Sfp1 in P-bodies is of unclear relevance and the authors do not ask whether Sfp1-bound mRNAs are also present in these condensates.
P-bodies consist of both RNA and proteins (reviewed in doi: 10.1021/acs.biochem.7b01162). The significance of this experiment lies in its contribution to further confirming the co-localization of Sfp1 with mRNAs and Rpb4. This observation could also yield valuable insights for future investigations into the role of Sfp1.
Further analysis of Sfp1-bound mRNAs would be of interest, particularly to address the question of whether those from ribosomal protein genes and other growth-related genes that are known to display Sfp1 binding in their promoters are regulated (either stabilized or destabilized) by Sfp1.
Fig. 4A, C and D show that RP mRNAs become destabilized in sfp1Δ cells.
The authors need to discuss, and ideally address, the apparent paradox that their previous findings showed that Rap1 acts to destabilize its downstream transcripts, i.e. that it has the opposite effect of Sfp1 shown here.
We would like to thank Reviewer 1 for this valuable comment. In the revised paper, we delved into our hypothesis suggesting that Rap1 is likely responsible for regulating the imprinting of other proteins, that, in turn, lead to the destabilization of mRNAs, such as Rpb4. See blue paragraph in page 20.
Finally, recent studies indicate that the drugs used here to measure mRNA stability induce a strong stress response accompanied by rapid and complex effects on transcription. Their relevance to mRNA stability in unstressed cells is questionable.
Half-lives were determined mainly by the GRO analysis of optimally proliferating cells. This method does not requires any drug or stressful treatment. The results obtained by this method were consistent with those obtained after thiolutin addition. Using both methods, we discovered that disruption of Sfp1 results in substantial mRNA destabilization. Nevertheless, in our revised manuscript, we show results obtained by subjecting cells to a temperature shift to 42°C, a natural method to inhibit transcription. This approach to determine half-lives has been previously reported in our publications, such as Lotan et al. (2005, 2007) and Goler Baron et al. (2008). This may rule out effects of the drug on half-lives. Indeed, this assay clearly determine HL under heat stress. Thus it can clearly demonstrate that, at least during heat shock, Sfp1 stabilizes mRNAs. Since the results are similar to those obtained by the GRO method at 30oC, we concluded that Sfp1 stabilizes mRNA under optimal and hot conditions.
Reviewer #2 (Public Review):
Summary:
The manuscript by Kelbert et al. presents results on the involvement of the yeast transcription factor Sfp1 in the stabilisation of transcripts whose synthesis it stimulates. Sfp1 is known to affect the synthesis of a number of important cellular transcripts, such as many of those that code for ribosomal proteins. The hypothesis that a transcription factor can remain bound to the nascent transcript and affect its cytoplasmic half-life is attractive, but the methods used to demonstrate the half-life effects and the association of Sfp1 with cytoplasmic transcripts remain to be fully validated, as explained in my comments on the results below:
Comments on methodology and results:
(1) A two-hybrid-based assay for protein-protein interactions identified Sfp1, a transcription factor known for its effects on ribosomal protein gene expression, as interacting with Rpb4, a subunit of RNA polymerase II. Classical two-hybrid experiments depend on the presence of the tested proteins in the nucleus of yeast cells, suggesting that the observed interaction occurs in the nucleus. Unfortunately, the two-hybrid method cannot determine whether the interaction is direct or mediated by nucleic acids.
Indeed, our two hybrid, immunoprecipitation and imaging results do not allow us to conclusively discern whether the interaction between Rpb4 and Sfp1 is direct or indirect. While the interaction holds significance, we consider the direct versus indirect distinction to be of secondary importance in the context of this paper. In the current text we indicated that 'our two hybrid, immunoprecipitation and imaging results do not differentiate between a direct or indirect interactions' (see page 6)
(2) Inactivation of nup49, a component of the nuclear pore complex, resulted in the redistribution of GFP-Sfp1 into the cytoplasm at the temperature non-permissive for the nup49-313 strain, suggesting that GFP-Sfp1 is a nucleo-cytoplasmic shuttling protein. This observation confirmed the dynamic nature of the nucleo-cytoplasmic distribution of Sfp1. For example, a similar redistribution to the cytoplasm was previously reported following rapamycin treatment and under starvation (Marion et al., PNAS 2004). In conjunction with the observation of an interaction with Rpb4, the authors observed slower nuclear import kinetics for GFP-Sfp1 in the absence of Rpb4 when cells were transferred to a glucose-containing medium after a period of starvation. Since the redistribution of GFP-Sfp1 was abolished in an rpb1-1/nup49-313 double mutant, the authors concluded that Sfp1 localisation to the cytoplasm depends on transcription. The double mutant yeast cells may show a variety of non-specific effects at the restrictive temperature, and whether transcription is required for Sfp1 cytoplasmic localisation remains incompletely demonstrated.
We agree with Reviewer 2 that any heat inactivation of a temperature-sensitive (ts) protein can lead to non-specific effects. It is evident that nup49-313 does not prevent Sfp1 export to the cytoplasm. In the case of rpb1-1, these non-specific effects are expected due to transcriptional arrest, which can eventually result in a reduction in protein content. However, this process takes some time, while the impact on export is more rapid. It is worth noting that this assay was developed and previously published by Pam Silver (Henry and Silver G&D 1996) and has been reported in many subsequent papers. Importantly, our conclusion is supported by the observation that Sfp1 binds both nascent RNA (co-transcriptionally) and mature mRNA (cytoplasmic). These observations, along with the reduced mRNA export upon transcription blocking, are consistent with our proposal that Sfp1 is exported in association with mRNA.
(3) Under starvation conditions, which led to the presence of Sfp1 in the cytoplasm and have previously been correlated with a decrease in the transcription of Sfp1 target genes, the authors observed that a plasmid-based expressed GFP-Sfp1 accumulated in cytoplasmic foci. These foci were also labelled by P-body markers such as Dcp2 and Lsm1. The quality of the microscopic images provided does not allow to determine whether Rpb4-RFP colocalises with GFP-Sfp1.
The submitted PDF figure is of low quality. We believe that high quality figure of the final submission is convincing.
(4) To understand to which RNA Sfp1 might bind, the authors used an N-terminally tagged fusion protein in a cross-linking and purification experiment. This method identified 264 transcripts for which the CRAC signal was considered positive and which mostly correspond to abundant mRNAs, including 74 ribosomal protein mRNAs or metabolic enzyme-abundant mRNAs such as PGK1. The authors did not provide evidence for the specificity of the observed CRAC signal, in particular, what would be the background of a similar experiment performed without UV cross-linking. In a validation experiment, the presence of several mRNAs in a purified SFP1 fraction was measured at levels that reflect the relative levels of RNA in a total RNA extract. Negative controls showing that abundant mRNAs not found in the CRAC experiment were clearly depleted from the purified fraction with Sfp1 would be crucial to assessing the specificity of the observed protein-RNA interactions. The NON-CRAC+ selected mRNAs were enriched for genes whose expression was previously shown to be upregulated upon Sfp1 overexpression (Albert et al., 2019). The presence of unspliced RPL30 pre-mRNA in the Sfp1 purification was interpreted as a sign of co-transcriptional assembly of Sfp1 into mRNA, but in the absence of valid negative controls, this hypothesis would require further experimental validation.
We would like to thank Reviewer 2 for bringing this issue up, as it helped us to clarify it in the revised paper.
First, we emphasized in the Discussion that many CRAC+ genes do not fall into the category of highly transcribed genes. Please see more detailed discussion below.
Secondly, we examined various features of the 264 genes - classified as CRAC+ - to estimate their specificity and biological significance. Our various experiments revealed that the CRAC+ genes represent a distinct group with many unique features.
The biological significance of the 264 CRAC+ mRNAs was demonstrated by various experiments; all are inconsistent with technical flaws. In fact, all the experiments and analyses that we have pursued indicate the unique nature of the CRAC+ genes. Some examples are:
(1) Fig. 2a and B show that most reads of CRAC+ mRNA were mapped to specific location – close the pA sites.
(2) Fig. 2C shows that most reads of CRAC+ mRNA were mapped to specific RNA motif located near the 3’ ends of the mRNAs.
(3) Most RiBi CRAC+ promoter contain Rap1 binding sites (p= 1.9x10-22), whiles the vast majority of RiBi non-CRAC+ promoters do not. (Fig. 3C).
(4) Fig. 4A shows that RiBi CRAC+ mRNAs become destabilized due to Sfp1 deletion, whereas RiBi non-CRAC+ mRNAs do not. Fig. 4B shows similar results due to Sfp1 depletion.
(5) Fig. 6B shows that the impact of Sfp1 on backtracking is substantially higher for CRAC+ than for non-CRAC+ genes. This is most clearly visible in RiBi genes.
(6) Fig. 7A shows that the Sfp1-dependent changes along the transcription units is substantially more rigorous for CRAC+ than for non-CRAC+.
(7) In Fig. S4B, the chromatin binding profile of Sfp1 is shown to be different for CRAC+ and non-CRAC+ genes.
Taken together, the many unique features, in fact, any feature that we examined, indicate the specificity and significance of this group, demonstrating that our CRAC results are biologically significant.
Most importantly, these genes do not all fall into the category of highly transcribed genes. On the contrary, as depicted in Figure 6A (green dots), it is evident that CRAC+ genes exhibit a diverse range of Rpb3 ChIP and GRO signals. Furthermore, as illustrated in Figure 7A, when comparing CRAC+ to Q1 (the most highly transcribed genes), it becomes evident that the Rpb4/Rpb3 profile of CRAC+ genes behaves differently from the Q1 group. Evidently, despite the heterogeneous transcription of CRAC+ genes (as mentioned above), the Rpb4/Rpb3 profile decreases more substantially than that of the highly transcribed genes (Q1). Moreover, despite similar expression levels among all RiBi mRNAs, only a portion of them binds Sfp1.
Thus, all our results indicate that CRAC+ genes represent biologically significant group, irrespective of the expression of it members. In response to this comment, we included a new paragraph discussing the validity of our conclusions. See page 18, blue paragraph.
(5) To address the important question of whether co-transcriptional assembly of Spf1 with transcripts could alter their stability, the authors first used a reporter system in which the RPL30 transcription unit is transferred to vectors under different transcriptional contexts, as previously described by the Choder laboratory (Bregman et al. 2011). While RPL30 expressed under an ACT1 promoter was barely detectable, the highest levels of RNA were observed in the context of the native upstream RPL30 sequence when Rap1 binding sites were also present. Sfp1 showed better association with reporter mRNAs containing Rap1 binding sites in the promoter region. However, removal of the Rap1 binding sites from the reporter vector also led to a drastic decrease in reporter mRNA levels. Whether the fraction of co-purified RNA is nuclear and co-transcriptional or not cannot be inferred from these results.
The proposed co-transcriptional binding of Sfp1 is based on the findings presented in Figure 5C and Figure S2D, as well as the observed binding of Sfp1 to transcripts containing introns, as shown in Figures 2D and 3B. The results of Fig. 3 led us to the assertion that the "RNA-binding capacity of Sfp1 is regulated by Rap1-binding sites located at the promoter." We maintain our stance on this conclusion. Indeed, the Rap1 binding site does impact mRNA levels, as highlighted by Reviewer 2. However, "construct E," which possesses a promoter with a Rap1 binding site, exhibits lower transcript levels compared to "construct F," which lacks such a binding site in its promoter. Despite this difference in transcript levels, Sfp1 was able to pull down the former transcript but not the latter, even though expression of the former gene is relatively low. Thus, the results appear to be more reliant on the specific capacity of Sfp1 to interact with the transcript rather than on the transcript's expression level.
(6) To complement the biochemical data presented in the first part of the manuscript, the authors turned to the deletion or rapid depletion of SFP1 and used labelling experiments to assess changes in the rate of synthesis, abundance, and decay of mRNAs under these conditions. An important observation was that in the absence of Sfp1, mRNAs encoding ribosomal protein genes not only had a reduced synthesis rate but also an increased degradation rate. This important observation needs careful validation, as genomic run-on experiments were used to measure half-lives, and this particular method was found to give results that correlated poorly with other measures of half-life in yeast (e.g. Chappelboim et al., 2022 for a comparison). Similarly, the use of thiolutin to block transcription as a method of assessing mRNA half-life has been reported to be problematic, as thiolutin can specifically inhibit the degradation of ribosomal protein mRNA (Pelechano & Perez-Ortin, 2008). Specific repressible reporters, such as those used by Baudrimont et al. (2017), would need to be tested to validate the effect of Sfp1 on the half-life of specific mRNAs. Also, it would be very difficult to infer from the images presented whether the rate of deadenylation is altered by Sfp1.
Various methods exist for assessing mRNA half-lives (HLs), and each of them carries its own set of challenges and biases. Consequently, it becomes problematic to directly compare HL values of a specific mRNA when different methods are employed. The superiority of one particular method over others remains unclear (in my opinion). However, they exhibit a high degree of reliability when it comes to comparing different strains under the identical conditions using a single method.
Estimating HLs through the GRO approach is a non-invasive method, applied on optimally proliferating cells, which has been employed in numerous publications. While no method is without its limitations, our experience along the years reassured approach to be among the most dependable. Our HL determination using thiolutin to block transcription provided results that were consistent with the values obtained by the GRO approach.
Nevertheless, in our revised manuscript, we supplemented the HL data, obtain by thiolutin, with results obtained by subjecting cells to a temperature shift to 42°C, a natural method to block transcription in wild-type (WT) cells. This approach to determine HLs has been previously reported in our publications, such as Lotan et al. (2005, 2007) and Goler Baron et al. (2008). The new results are shown in Fig. S3B. They are consistent with our conclusion that Sfp1 stabilizes mRNAs.
Using a repressible promoter to determine mRNA HL is, unfortunately, not suitable in this paper because the promoter itself is involved in HL regulation. This observation is supported by Bregman et al. (2011) and depicted in Fig. 3, which illustrates that the promoter is critical for mRNA imprinting, consequently regulating HL.
(7) The effects of SFP1 on transcription were investigated by chromatin purification with Rpb3, a subunit of RNA polymerase, and the results were compared with synthesis rates determined by genomic run-on experiments. The decrease in polII presence on transcripts in the absence of SFP1 was not accompanied by a marked decrease in transcript output, suggesting an effect of Sfp1 in ensuring robust transcription and avoiding RNA polymerase backtracking. To further investigate the phenotypes associated with the depletion or absence of Sfp1, the authors examined the presence of Rpb4 along transcription units compared to Rpb3. One effect of spf1 deficiency was that this ratio, which decreased from the start of transcription towards the end of transcripts, increased slightly. The results presented are largely correlative and could arise from the focus on very specific types of mRNAs, such as those of ribosomal protein genes, which are sensitive to stress and are targeted by very active RNA degradation mechanisms activated, for example, under heat stress (Bresson et al., 2020).
Figure 7A illustrates a significant reduction in Rpb4/Rpb3 ratios along the transcription unit in WT cells. This reduction is notably more pronounced in CRAC+ genes compared to the highly transcribed quartile (Q1), which includes all ribosomal protein (RP) genes, and it is completely absent in sfp1∆ cells. Furthermore, it's important to highlight that the CRAC+ gene group displays a wide range of transcription rates, as measured by either Rpb3 ChIP or GRO (Figure 6A). Given these observations, we do not think that heightened sensitivity of RP mRNA degradation in response to stress is responsible for the pronounced difference in the configuration of the Pol II elongation complex that is detected in CRAC+ genes, mainly because this experiment was performed under standard (non-stress) culture conditions.
Correlative studies are particularly informative when a gene mutation eliminates a correlation, and this is precisely the type of study depicted in Figure 7B-C. The correlations shown in these panels are dependent on Sfp1. Indeed, RP genes are sensitive to stress. However, we used non-stressed conditions. Furthermore, CRAC+ genes did not display any apparent unusual destabilization but rather exhibited higher (not lower) mRNA stability compared to non-CRAC+ genes (Figure 7C).
