Export of discarded splicing intermediates requires mRNA export factors and the nuclear basket

Reviewer #1 (Public Review):

Summary:

In this manuscript, Zeng and Staley provide a valuable analysis of the molecular requirements for the export of a reporter mRNA that contains a lariat structure at its 5' end in the budding yeast S. cerevisiae. The authors provide evidence that this is regulated by the main mRNA export machinery (Yra1, Mex67, Nab2, Npl3, Tom1, and Mlp1). Of note, Mlp1 has been mainly implicated in the nuclear retention of unspliced pre-mRNA (i.e. quality control), and relatively little has been done to investigate its role in mRNA export in budding yeast.

Strengths:

There is relatively little information in the current literature about the nuclear export of splicing intermediates. This paper provides one of the first analyses of this process and dissects the molecular components that promote this form of RNA export. Overall, the strength of the data presented in the manuscript is solid. The paper is well written and the message is clear and of general interest to the mRNA community.

Weaknesses:

There are three problems with the paper, although these are not major and likely would not affect the final model as most aspects of the molecular details are confirmed by multiple complementary assays.

(1) The brG reporter produces both unspliced pre-mRNA and a lariat-containing intermediate RNA. Based on the primer extension assay the authors claim that only 33% of the final product is in pre-mRNA form and that this "is insufficient to account for the magnitude of the cytoplasmic signal from the brG reporter (83%)". Nevertheless, it is possible that primer extension is incomplete or that the lariat-containing RNA is inaccessible for smFISH. The authors could easily perform a dual smFISH experiment (similar to Adivarahan et l., Molecular Cell 2018) where exon 1 is labelled with probes of one color, and the region that overlaps the lariat-containing intermediate is labelled with probes of a second color. If the authors are correct, then one-third of the smFISH foci should have both labels and the rest would have only the second label. This would also confirm that the latter (i.e. the lariat-containing RNAs) are exported to the cytoplasm. Using this approach, the authors could then show that MLP1-depletion (or depletion of any of the other factors) affect(s) one pool of RNAs (i.e. those that are lariat-containing) but not the other (i.e. pre-mRNA). Including these experiments would make the evidence for their model more convincing.

(2) In some cases, the number of smFISH foci appears to change drastically depending on the genetic background. This could either be due to the stochastic nature of mRNA expression between cells or reflect real differences between the genetic backgrounds that could alter the interpretation of the other observations.

(3) The authors state in the discussion that "the general mRNA export pathway transports discarded lariat intermediates into the cytoplasm". Although this appears to be the case for the reporters that are investigated in this paper, I don't think that the authors should make such a broad sweeping claim. It may be that some discarded lariat intermediates are exported to the cytoplasm while others are targeted for nuclear retention and/or decay.

Reviewer #2 (Public Review):

In this report, Zeng and Staley have used an elegant combination of RNA imaging approaches (single molecule FISH), RNA co-immunoprecipitations, and translation reporters to characterize the factors and pathways involved in the nuclear export of splicing intermediates in budding yeast. Their study notably involves the use of specific reporter genes, which lead to the accumulation of pre-mRNA and lariat species, in a battery of mutants impacting mRNA export and quality control.

The authors convincingly demonstrate that mRNA species expressed from such reporters are exported to the cytoplasm in a manner depending on the canonical mRNA export machinery (Mex67 and its adaptors) and the nuclear pore complex (NPC) basket (Mlp1). Interestingly, they provide evidence that the export of splicing intermediates requires docking and subsequent undocking at the nuclear basket, a step possibly more critical than for regular mRNAs.

However, their assays do not always allow us to define whether the impacted mRNA species correspond to lariats and/or pre-mRNAs. This is all the more critical since their findings apparently contradict previous reports that supported a role for the nuclear basket in pre-mRNA quality control. These earlier studies, which were similarly based on the use of dedicated yet distinct reporters, had found that the nuclear basket subunit Mlp1, together with different cofactors, prevents the export of unspliced mRNA species. It would be important to clarify experimentally and discuss the possible reasons for these discrepancies.

Reviewer #3 (Public Review):

Summary:

Zeng and Stanley show that in yeast, intron-lariat intermediates that accumulated due to defects in pre-mRNA splicing, are transported to the cytoplasm using the canonical mRNA export pathway. Moreover, they demonstrate that export requires the nuclear basket, a sub-structure of the nuclear pore complex previously implicated with the retention of immature mRNAs. These observations are important as they put into question a longstanding model that the main role of the nuclear basket is to ensure nuclear retention of immature or faulty mRNAs.

Strengths:

The authors elegantly combine genetic, biochemical, and single-molecule resolution microscopy approaches to identify the cellular pathway that mediates the cytoplasmic accumulation of lariat intermediates. Cytoplasmic accumulation of such splicing intermediates had been observed in various previous studies but how these RNAs reach the cytoplasm had not yet been investigated. By using smFISH, the authors present compelling, and, for the first time, direct evidence that these intermediates accumulate in the cytoplasm and that this requires the canonical mRNA export pathway, including the RNA export receptor Mex67 as well as various RNA-binding proteins including Yra1, Npl3 and Nab2. Moreover, they show that the export of lariat intermediates, but not mRNAs, requires the nuclear basket (Mlp1) and basket-associated proteins previously linked to the mRNP rearrangements at the nuclear pore. This is a surprising and important observation with respect to a possible function of the nuclear basket in mRNA export and quality control, as it challenges a longstanding model that the role of the basket in mRNA export is primarily to act as a gatekeeper to ensure that immature mRNAs are not exported. As discussed by the authors, their finding suggests a role for the basket in promoting the export of certain types of RNAs rather than retention, a model also supported by more recent studies in mammalian cells. Moreover, their findings also collaborate with a recent paper showing that in yeast, not all nuclear pores contain a basket (PMID: 36220102), an observation that also questioned the gatekeeper model of the basket, as it is difficult to imagine how the basket can serve as a gatekeeper if not all nuclear pore contain such a structure.

Weaknesses:

One weakness of this study is that all their experiments rely on using synthetic splicing reporter containing a lacZ gene that produces a relatively long transcript compared to the average yeast mRNA.

The rationale for using a reporter containing the brG (G branch point) resulting in more stable lariat intermediates due to them being inefficient substrates for the debranching enzyme Dbr1 could be described earlier in the manuscript, as this otherwise only becomes clear towards the end, what is confusing.

Discussion of their observation in the context that, in yeast, not all pores contain a basket would be useful.

Author Response

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In this manuscript, Zeng and Staley provide a valuable analysis of the molecular requirements for the export of a reporter mRNA that contains a lariat structure at its 5' end in the budding yeast S. cerevisiae. The authors provide evidence that this is regulated by the main mRNA export machinery (Yra1, Mex67, Nab2, Npl3, Tom1, and Mlp1). Of note, Mlp1 has been mainly implicated in the nuclear retention of unspliced pre-mRNA (i.e. quality control), and relatively little has been done to investigate its role in mRNA export in budding yeast.

Strengths:

There is relatively little information in the current literature about the nuclear export of splicing intermediates. This paper provides one of the first analyses of this process and dissects the molecular components that promote this form of RNA export. Overall, the strength of the data presented in the manuscript is solid. The paper is well written and the message is clear and of general interest to the mRNA community.

We thank the reviewer for highlighting these strengths.

Weaknesses:

There are three problems with the paper, although these are not major and likely would not affect the final model as most aspects of the molecular details are confirmed by multiple complementary assays.

(1) The brG reporter produces both unspliced pre-mRNA and a lariat-containing intermediate RNA. Based on the primer extension assay the authors claim that only 33% of the final product is in pre-mRNA form and that this "is insufficient to account for the magnitude of the cytoplasmic signal from the brG reporter (83%)". Nevertheless, it is possible that primer extension is incomplete or that the lariat-containing RNA is inaccessible for smFISH. The authors could easily perform a dual smFISH experiment (similar to Adivarahan et l., Molecular Cell 2018) where exon 1 is labelled with probes of one color, and the region that overlaps the lariat-containing intermediate is labelled with probes of a second color. If the authors are correct, then one-third of the smFISH foci should have both labels and the rest would have only the second label. This would also confirm that the latter (i.e. the lariat-containing RNAs) are exported to the cytoplasm. Using this approach, the authors could then show that MLP1-depletion (or depletion of any of the other factors) affect(s) one pool of RNAs (i.e. those that are lariat-containing) but not the other (i.e. pre-mRNA). Including these experiments would make the evidence for their model more convincing.

We appreciate the reviewer’s comments and suggestions. Concerning the primer extension analysis, we are considering alternative assays to quantitate the pre-mRNA and lariat intermediate levels. Concerning the accessibility of the lariat intermediate in smRNA-FISH, in a dbr1∆ strain the only major species from the UAc reporter that is detected by primer extension is the lariat intermediate (Fig. S3), and this reporter is readily detected by smRNA-FISH, indicate that the lariat intermediate is accessible to smRNA-FISH. Concerning discriminating between pre-mRNA and lariat intermediate by smRNA-FISH, we agree with the reviewer that a dual smFISH experiment would directly distinguish between the signals of these species. The brG reporter we used in most smRNA-FISH experiments has a 5’ exon that is too short for smRNA-FISH probes, as is typical of most budding yeast 5’ exons. We have tried to replace the 5’ exon with a longer sequence (GFP) to allow for smRNA-FISH; however, this substitution inhibited splicing. Therefore, to distinguish signals from pre-mRNA versus lariat intermediate, we used additional reporters: G1c and brC reporters, which accumulate pre-mRNA essentially exclusively (Fig. S2A-C), and the UAc reporter, which accumulates lariat intermediate exclusively, in a dbr1∆ strain (Fig. S3). Whereas the mlp1 deletion did not change beta-galactosidase activities of the G1c and brC pre-mRNA-accumulating reporters (Fig. S2E), the mlp1 deletion in a dbr1∆ background did reduce the beta-galactosidase activities of the UAc lariat intermediate-accumulating reporter (Fig. 3D) and did increase smRNA-FISH signal of this reporter in the nucleus (Fig. 3E). These observations corroborate our interpretation based on the brG reporter that Mlp1p is required for efficient export of lariat intermediates but not pre-mRNAs.

(2) In some cases, the number of smFISH foci appears to change drastically depending on the genetic background. This could either be due to the stochastic nature of mRNA expression between cells or reflect real differences between the genetic backgrounds that could alter the interpretation of the other observations.

We thank the reviewer for raising this point. We will review our data to distinguish between these possibilities.

(3) The authors state in the discussion that "the general mRNA export pathway transports discarded lariat intermediates into the cytoplasm". Although this appears to be the case for the reporters that are investigated in this paper, I don't think that the authors should make such a broad sweeping claim. It may be that some discarded lariat intermediates are exported to the cytoplasm while others are targeted for nuclear retention and/or decay.

The reviewer’s point is well-taken. We will revise the wording accordingly.

Reviewer #2 (Public Review):

In this report, Zeng and Staley have used an elegant combination of RNA imaging approaches (single molecule FISH), RNA co-immunoprecipitations, and translation reporters to characterize the factors and pathways involved in the nuclear export of splicing intermediates in budding yeast. Their study notably involves the use of specific reporter genes, which lead to the accumulation of pre-mRNA and lariat species, in a battery of mutants impacting mRNA export and quality control.

The authors convincingly demonstrate that mRNA species expressed from such reporters are exported to the cytoplasm in a manner depending on the canonical mRNA export machinery (Mex67 and its adaptors) and the nuclear pore complex (NPC) basket (Mlp1). Interestingly, they provide evidence that the export of splicing intermediates requires docking and subsequent undocking at the nuclear basket, a step possibly more critical than for regular mRNAs.

We thank the reviewer for this overall positive assessment.

However, their assays do not always allow us to define whether the impacted mRNA species correspond to lariats and/or pre-mRNAs. This is all the more critical since their findings apparently contradict previous reports that supported a role for the nuclear basket in pre-mRNA quality control. These earlier studies, which were similarly based on the use of dedicated yet distinct reporters, had found that the nuclear basket subunit Mlp1, together with different cofactors, prevents the export of unspliced mRNA species. It would be important to clarify experimentally and discuss the possible reasons for these discrepancies.

It is true that we did not assess export of all reporters in all mutant strains by smFISH; however, we did validate the key conclusion that the export of lariat intermediates requires the nuclear basket gene MLP1: the export of both the brG reporter (mostly lariat intermediate) and the UAc reporter (exclusively lariat intermediate) showed a dependence on MLP1 (Fig. 3). Further, by beta-galactosidase activity, we tested in total five separate reporters – three that accumulated lariat intermediate and two that accumulated exclusively pre-mRNA; only the three reporters accumulating lariat intermediate showed a dependence of export on MLP1 (Fig. 4B,D; Fig S2D); the reporters accumulating pre-mRNA did not show a dependence on MLP1 (Fig. S2E), further validating our main conclusion. We are considering additional experiments to validate this key conclusion even further. Also, see response to comment 1 from reviewer 1.

We agree that the main conclusion from this manuscript differs from earlier studies. A key difference is that prior studies monitored exclusively pre-mRNA. In our study, we monitored pre-mRNA and lariat intermediate species and in doing so revealed a role for MLP1 in the export of lariat intermediates. This study, our previous study, as well as the previous studies of others have all provided evidence for efficient export of pre-mRNA; all of these studies are in conflict with the studies purporting a general role for the nuclear basked in retaining immature mRNA. Still, these past apparently conflicting studies can be re-interpreted in the context of our model that the export of such species requires docking at the nuclear basket, followed by undocking. In a revised manuscript, we will discuss the possibility that pre-mRNA apparently “retained” by the nuclear basket are stalled in export at the undocking stage.

Reviewer #3 (Public Review):

Summary:

Zeng and Stanley show that in yeast, intron-lariat intermediates that accumulated due to defects in pre-mRNA splicing, are transported to the cytoplasm using the canonical mRNA export pathway. Moreover, they demonstrate that export requires the nuclear basket, a sub-structure of the nuclear pore complex previously implicated with the retention of immature mRNAs. These observations are important as they put into question a longstanding model that the main role of the nuclear basket is to ensure nuclear retention of immature or faulty mRNAs.

Strengths:

The authors elegantly combine genetic, biochemical, and single-molecule resolution microscopy approaches to identify the cellular pathway that mediates the cytoplasmic accumulation of lariat intermediates. Cytoplasmic accumulation of such splicing intermediates had been observed in various previous studies but how these RNAs reach the cytoplasm had not yet been investigated. By using smFISH, the authors present compelling, and, for the first time, direct evidence that these intermediates accumulate in the cytoplasm and that this requires the canonical mRNA export pathway, including the RNA export receptor Mex67 as well as various RNA-binding proteins including Yra1, Npl3 and Nab2. Moreover, they show that the export of lariat intermediates, but not mRNAs, requires the nuclear basket (Mlp1) and basket-associated proteins previously linked to the mRNP rearrangements at the nuclear pore. This is a surprising and important observation with respect to a possible function of the nuclear basket in mRNA export and quality control, as it challenges a longstanding model that the role of the basket in mRNA export is primarily to act as a gatekeeper to ensure that immature mRNAs are not exported. As discussed by the authors, their finding suggests a role for the basket in promoting the export of certain types of RNAs rather than retention, a model also supported by more recent studies in mammalian cells. Moreover, their findings also collaborate with a recent paper showing that in yeast, not all nuclear pores contain a basket (PMID: 36220102), an observation that also questioned the gatekeeper model of the basket, as it is difficult to imagine how the basket can serve as a gatekeeper if not all nuclear pore contain such a structure.

We thank the reviewer for highlighting the importance and surprising nature of our findings.

Weaknesses:

One weakness of this study is that all their experiments rely on using synthetic splicing reporter containing a lacZ gene that produces a relatively long transcript compared to the average yeast mRNA.

We are considering repeating some of our experiments to monitor export of RNAs with more average lengths.

The rationale for using a reporter containing the brG (G branch point) resulting in more stable lariat intermediates due to them being inefficient substrates for the debranching enzyme Dbr1 could be described earlier in the manuscript, as this otherwise only becomes clear towards the end, what is confusing.

We thank the reviewer for this comment. We will revise the text to explain sooner the rationale for using the brG reporter to assess the export of lariat intermediates.

Discussion of their observation in the context that, in yeast, not all pores contain a basket would be useful.

Thanks for this suggestion. We will raise this point that a nuclear basket is not present on all nuclear pores and discuss the implications.