aCPSF1 cooperates with terminator U-tract to dictate archaeal transcription termination efficacy
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Evaluation Summary:
This study presents new evidence that support a model of aCPSF1-dependent transcription termination in Archaea. Archaeal transcription termination is shown to rely on both poly-U tract terminator signals and the endoribonuclease aCPSF1 of the β-CASP family. This mechanism resembles the eukaryal RNAP II termination process. These new insights fill a gap in our understanding of the mechanism of transcription termination in Archaea and they are of general importance for the RNA biology community.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Abstract
Recently, aCPSF1 was reported to function as the long-sought global transcription termination factor of archaea; however, the working mechanism remains elusive. This work, through analyzing transcript-3′end-sequencing data of Methanococcus maripaludis , found genome-wide positive correlations of both the terminator uridine(U)-tract and aCPSF1 with hierarchical transcription termination efficacies (TTEs). In vitro assays determined that aCPSF1 specifically binds to the terminator U-tract with U-tract number-related binding affinity, and in vivo assays demonstrated the two elements are indispensable in dictating high TTEs, revealing that aCPSF1 and the terminator U-tract cooperatively determine high TTEs. The N-terminal KH domains equip aCPSF1 with specific-binding capacity to terminator U-tract and the aCPSF1-terminator U-tract cooperation; while the nuclease activity of aCPSF1 was also required for TTEs. aCPSF1 also guarantees the terminations of transcripts with weak intrinsic terminator signals. aCPSF1 orthologs from Lokiarchaeota and Thaumarchaeota exhibited similar U-tract cooperation in dictating TTEs. Therefore, aCPSF1 and the intrinsic U-rich terminator could work in a noteworthy two-in-one termination mode in archaea, which may be widely employed by archaeal phyla; using one trans-action factor to recognize U-rich terminator signal and cleave transcript 3′-end, the archaeal aCPSF1-dependent transcription termination may represent a simplified archetypal mode of the eukaryotic RNA polymerase II termination machinery.
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Author Response:
Reviewer #1 (Public Review):
The authors conducted an extensive analysis of transcription termination in Methanococcus maripaludis, which is an archael species. Using a combination of functional genomics, statistical analyses, in vitro/biochemical approaches, and reporters; the authors explore mechanistic aspects of termination. The authors determine that the archael CPSF protein binds an upstream uridine tract using a KH domain. The RNA binding activity of aCPSF is not present in eukaryotes and is shown to be important for termination in a uridine tract dependent manner.
Overall, the work is well-conceived and, in general, the conclusions by the authors are supported by the data. Investigation of archael species is not mainstream but still has significant potential to impact the field of transcription as the …
Author Response:
Reviewer #1 (Public Review):
The authors conducted an extensive analysis of transcription termination in Methanococcus maripaludis, which is an archael species. Using a combination of functional genomics, statistical analyses, in vitro/biochemical approaches, and reporters; the authors explore mechanistic aspects of termination. The authors determine that the archael CPSF protein binds an upstream uridine tract using a KH domain. The RNA binding activity of aCPSF is not present in eukaryotes and is shown to be important for termination in a uridine tract dependent manner.
Overall, the work is well-conceived and, in general, the conclusions by the authors are supported by the data. Investigation of archael species is not mainstream but still has significant potential to impact the field of transcription as the process of termination is still be unraveled. Moreover, several aspects of the methodology would benefit other researchers - notably, the development of Term-seq. The readers would benefit from consulting early structural studies of aCPSF to more fully gain a perspective on the interesting aspects of KH domain presence in this homology of CPSF.
Immensely thank you for the positive comments.
Reviewer #2 (Public Review):
Transcription termination defines accurate transcript 3'-ends and ensures programmed transcriptomes, making it critical to gene expression regulation. Our understanding of archaeal transcription termination mechanisms is still limited. Li et al present new evidences that support their model of aCPSF1-dependent transcription termination in Archaea (Yue et al NAR 2020). Importantly, they show that aCSPF1 recognize U-rich terminator signals with its KH-domain, using the methanogen Methanococcus maripaludis, as study model. The reported results of the manuscript are the continuity of their work published in Nucleic Acid Research journal in 2020 (Yue et al NAR 2020). Previously, the authors demonstrated the importance of the absolutely conserved ribonuclease aCPSF1 to terminate transcription by cleaving the transcripts at their 3' ends. They proposed a model for transcription termination in Archaea in which they defined aCPSF1 as general transcription termination factor. In here, by reinvestigating Term-seq data and by using RNA-protein binding assays (EMSA & SPR) and genetics experiments, Li et al argue that (i) PolyU-tracts are signals for transcription termination in M. maripaludis, (ii) aCPSF1 binds to PolyU-tract signals in transcript 3'-ends through its KH domain, and (iii) transcription termination is more effective in presence of aCPSF1. In general, the experiments and analyses that are shown are well-conducted. A major criticism is the lack (i) of quantification of RNA-protein binging assays which will allow going deeper in the understanding of how aCPSF1 specifically recognized PolyU-tract signals and (ii) of data on the oligomerisation status of aCPSF1. It is important to decipher if aCPSF1 is acting as a monomer or a dimer. Both will be helpful for proposing a more precise model for transcription termination mechanism in Archaea.
Immensely thank you for the positive comments and the valuable suggestions. According to your comments and suggestions, we have supplemented the following experimental data in the revision as (i) the aCPSF1-U rich RNA binding association contents (Kd) have been quantified for the binding assays; (ii) truncation of the C-terminal 13 residues (C13) that are essential for aCPSF1 dimerization leads to the loss of the in vivo termination function of aCPSF1, demonstrating that the dimerization of aCPSF1 is essential to its routine function; (iii) Footprint assay also determined that aCPSF1 binds to the U-tract region of the tested terminator. All the related results and discussions have been supplemented in the revised manuscript.
Reviewer #3 (Public Review):
In this manuscript by Li et al. examine U-rich motifs enriched for the transcript end sites in archaea M. marpaludis and analyze the role of aCPSF1 and its KH domains in binding these U-rich motifs. Their data indicate aCPSF1 binding to U-rich motifs is necessary for efficient 3' end definition of transcripts. Overall this work is well carried out, but there are also several key issues that should be addressed in order to more fully support the conclusions of the authors.
Immensely thank you for the positive comments.
Major:
- The conclusion that aCPSF1 functions as a back-up termination is not supported by their data. As far as I can tell, back-up termination should happen at non-primary sites, which have a lower frequency of U quadruplex. It is not clear how aCPSF1 functions at those sites.
Combination of the comments of you and the reviewer 1, this related inappropriate conclusion has been removed in the revised manuscript.
- The authors appear to indicate that aCPSF1 is the sole factor for 3' end cleavage of archaeal transcripts. But this is not supported by the data. Their data indicate both U-rich motif and aCPSF1 are necessary for cleavage. No data are shown to indicate that these two alone are sufficient for 3' end cleavage.
The obscure related description has been revised as “the only trans-action factor”. (Line 36 in the Abstract and Lines 476-477).
- The U-rich motif (U quadruplex) was recently reported in their NAR paper (Yue et al.). There appears to be limited additional information on motifs in this work. It would be useful to readers to know if U-rich motifs are the only type for 3' end cleavage. The authors may want to examine motifs beyond single nucleotide models (which is what they are doing in this work). For example, are there any k-mer enrichments besides U quadruplex?
According to your advice, we searched RNA sequences proximal to TTSs; however, no other motif in addition the U-rich sequence is found.
Minor:
- Strictly speaking, they are measuring transcript ending sites, not polymerase termination sites. The Term-seq data do not map the polymerase termination site. The authors should make this distinction in their writing. Likewise, it is better to rename transcription termination efficiency (TTE) to transcript termination frequency, because they are measuring steady state RNAs which embody both 3' end cleavage efficiency and RNA stability. The efficiency implies termination kinetics, which is not what they are measuring.
Thank you for the advice. To deliver the information more clearly, description of Term-seq method has been added as “Recently, Term-seq, an approach that enables accurate mapping of all exposed RNA 3′-ends in prokaryotes and determines the transcription termination sites (TTSs) at the genome-wide level in representative bacteria and archaea (Dar et al., 2016b; Porrua et al., 2016; Yue et al., 2020), has been developed. ” (Lines 82-85). In addition, “transcription termination efficiency (TTE)” has been revised as “Transcription termination efficacy” throughout the manuscript according to the suggestion.
- Figure 3 needs better quantification. Binding curves showing binding constant are needed to make quantitative conclusions.
Equilibrium dissociation constants (Kd) were calculated based on the binding curves, which is generated by quantifying the unbound and bound substrates in Figures 3, 4, and 6A, and the Kd values are indicated in respective figures.
- Figure 7. the eukaryotic model seems based on budding yeast. This should be noted. 3' end motifs in other eukaryotes are quite different than those in the budding yeast.
Thank you for the point. Budding yeast has been indicated in Figure 7 legend (Lines 1200).
- There are numerous grammatical errors, which the authors should address.
English language and grammar have revised by a professional English language editing company. We hope the language and grammar have been improved.
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Evaluation Summary:
This study presents new evidence that support a model of aCPSF1-dependent transcription termination in Archaea. Archaeal transcription termination is shown to rely on both poly-U tract terminator signals and the endoribonuclease aCPSF1 of the β-CASP family. This mechanism resembles the eukaryal RNAP II termination process. These new insights fill a gap in our understanding of the mechanism of transcription termination in Archaea and they are of general importance for the RNA biology community.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
The authors conducted an extensive analysis of transcription termination in Methanococcus maripaludis, which is an archael species. Using a combination of functional genomics, statistical analyses, in vitro/biochemical approaches, and reporters; the authors explore mechanistic aspects of termination. The authors determine that the archael CPSF protein binds an upstream uridine tract using a KH domain. The RNA binding activity of aCPSF is not present in eukaryotes and is shown to be important for termination in a uridine tract dependent manner.
Overall, the work is well-conceived and, in general, the conclusions by the authors are supported by the data. Investigation of archael species is not mainstream but still has significant potential to impact the field of transcription as the process of termination is …
Reviewer #1 (Public Review):
The authors conducted an extensive analysis of transcription termination in Methanococcus maripaludis, which is an archael species. Using a combination of functional genomics, statistical analyses, in vitro/biochemical approaches, and reporters; the authors explore mechanistic aspects of termination. The authors determine that the archael CPSF protein binds an upstream uridine tract using a KH domain. The RNA binding activity of aCPSF is not present in eukaryotes and is shown to be important for termination in a uridine tract dependent manner.
Overall, the work is well-conceived and, in general, the conclusions by the authors are supported by the data. Investigation of archael species is not mainstream but still has significant potential to impact the field of transcription as the process of termination is still be unraveled. Moreover, several aspects of the methodology would benefit other researchers - notably, the development of Term-seq. The readers would benefit from consulting early structural studies of aCPSF to more fully gain a perspective on the interesting aspects of KH domain presence in this homology of CPSF.
-
Reviewer #2 (Public Review):
Transcription termination defines accurate transcript 3'-ends and ensures programmed transcriptomes, making it critical to gene expression regulation. Our understanding of archaeal transcription termination mechanisms is still limited. Li et al present new evidences that support their model of aCPSF1-dependent transcription termination in Archaea (Yue et al NAR 2020). Importantly, they show that aCSPF1 recognize U-rich terminator signals with its KH-domain, using the methanogen Methanococcus maripaludis, as study model.
The reported results of the manuscript are the continuity of their work published in Nucleic Acid Research journal in 2020 (Yue et al NAR 2020). Previously, the authors demonstrated the importance of the absolutely conserved ribonuclease aCPSF1 to terminate transcription by cleaving the …
Reviewer #2 (Public Review):
Transcription termination defines accurate transcript 3'-ends and ensures programmed transcriptomes, making it critical to gene expression regulation. Our understanding of archaeal transcription termination mechanisms is still limited. Li et al present new evidences that support their model of aCPSF1-dependent transcription termination in Archaea (Yue et al NAR 2020). Importantly, they show that aCSPF1 recognize U-rich terminator signals with its KH-domain, using the methanogen Methanococcus maripaludis, as study model.
The reported results of the manuscript are the continuity of their work published in Nucleic Acid Research journal in 2020 (Yue et al NAR 2020). Previously, the authors demonstrated the importance of the absolutely conserved ribonuclease aCPSF1 to terminate transcription by cleaving the transcripts at their 3' ends. They proposed a model for transcription termination in Archaea in which they defined aCPSF1 as general transcription termination factor. In here, by reinvestigating Term-seq data and by using RNA-protein binding assays (EMSA & SPR) and genetics experiments, Li et al argue that (i) PolyU-tracts are signals for transcription termination in M. maripaludis, (ii) aCPSF1 binds to PolyU-tract signals in transcript 3'-ends through its KH domain, and (iii) transcription termination is more effective in presence of aCPSF1.
In general, the experiments and analyses that are shown are well-conducted. A major criticism is the lack (i) of quantification of RNA-protein binging assays which will allow going deeper in the understanding of how aCPSF1 specifically recognized PolyU-tract signals and (ii) of data on the oligomerisation status of aCPSF1. It is important to decipher if aCPSF1 is acting as a monomer or a dimer. Both will be helpful for proposing a more precise model for transcription termination mechanism in Archaea.
-
Reviewer #3 (Public Review):
In this manuscript by Li et al. examine U-rich motifs enriched for the transcript end sites in archaea M. marpaludis and analyze the role of aCPSF1 and its KH domains in binding these U-rich motifs. Their data indicate aCPSF1 binding to U-rich motifs is necessary for efficient 3' end definition of transcripts. Overall this work is well carried out, but there are also several key issues that should be addressed in order to more fully support the conclusions of the authors.
Major:
1. The conclusion that aCPSF1 functions as a back-up termination is not supported by their data. As far as I can tell, back-up termination should happen at non-primary sites, which have a lower frequency of U quadruplex. It is not clear how aCPSF1 functions at those sites.
2. The authors appear to indicate that aCPSF1 is the sole …
Reviewer #3 (Public Review):
In this manuscript by Li et al. examine U-rich motifs enriched for the transcript end sites in archaea M. marpaludis and analyze the role of aCPSF1 and its KH domains in binding these U-rich motifs. Their data indicate aCPSF1 binding to U-rich motifs is necessary for efficient 3' end definition of transcripts. Overall this work is well carried out, but there are also several key issues that should be addressed in order to more fully support the conclusions of the authors.
Major:
1. The conclusion that aCPSF1 functions as a back-up termination is not supported by their data. As far as I can tell, back-up termination should happen at non-primary sites, which have a lower frequency of U quadruplex. It is not clear how aCPSF1 functions at those sites.
2. The authors appear to indicate that aCPSF1 is the sole factor for 3' end cleavage of archaeal transcripts. But this is not supported by the data. Their data indicate both U-rich motif and aCPSF1 are necessary for cleavage. No data are shown to indicate that these two alone are sufficient for 3' end cleavage.
3. The U-rich motif (U quadruplex) was recently reported in their NAR paper (Yue et al.). There appears to be limited additional information on motifs in this work. It would be useful to readers to know if U-rich motifs are the only type for 3' end cleavage. The authors may want to examine motifs beyond single nucleotide models (which is what they are doing in this work). For example, are there any k-mer enrichments besides U quadruplex?
Minor:
1. Strictly speaking, they are measuring transcript ending sites, not polymerase termination sites. The Term-seq data do not map the polymerase termination site. The authors should make this distinction in their writing. Likewise, it is better to rename transcription termination efficiency (TTE) to transcript termination frequency, because they are measuring steady state RNAs which embody both 3' end cleavage efficiency and RNA stability. The efficiency implies termination kinetics, which is not what they are measuring.
2. Figure 3 needs better quantification. Binding curves showing binding constant are needed to make quantitative conclusions.
3. Figure 7. the eukaryotic model seems based on budding yeast. This should be noted. 3' end motifs in other eukaryotes are quite different than those in the budding yeast.
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