Multiplexed Assays of Human Disease-relevant Mutations Reveal UTR Dinucleotide Composition as a Major Determinant of RNA Stability

Reviewer #1 (Public Review):

In the manuscript by Su et al., the authors present a massively parallel reporter assay (MPRA) measuring the stability of in vitro transcribed mRNAs carrying wild-type or mutant 5' or 3' UTRs transfected into two different human cell lines. The goal presented at the beginning of the manuscript was to screen for effects of disease-associated point mutations on the stability of the reporter RNAs carrying partial human 5' or 3' UTRs. However, the majority of the manuscript is dedicated to identifying sequence components underlying the differential stability of reporter constructs. This shows that TA dinucleotides are the most predictive feature of RNA stability in both cell lines and both UTRs.
The effect of AU rich elements (AREs) on RNA stability is well established in multiple systems, and the present study confirms this general trend but points out variability in the consequence of seemingly similar motifs on RNA stability. For example, the authors report that a long stretch of Us has extreme opposite effects on RNA stability depending on whether it is preceded by an A (strongly destabilizing) or followed by an A (strongly stabilizing). While the authors interpretation of a context-dependence of the effect is certainly well-founded, it seems counterintuitive that the preceding or following A would be the (only) determining factor. This points to a generally reductionist approach taken by the authors in the analysis of the data and in their attempt to dissect the contribution of "AU rich sequences" to RNA stability, with a general tendency to reduce the size and complexity of the features (e.g. to dinucleotides). While this certainly increases the statistical power of the analysis due to the number of occurrences of these motifs, it limits the interpretability of the results. How do TA dinucleotides per se contribute to destabilizing the RNA, both in 5' and 3' UTRs, but (according to limited data presented) not in coding sequences? What is the mechanism? RBPs binding to TA dinucleotide containing sequences are suggested to "mask" the destabilizing effect, thereby leading to a more stable RNA. Gain of TA dinucleotides is reported to have a destabilizing effect, but again no hypothesis is provided as to the underlying molecular mechanism. In addition to reducing the motif length to dinucleotides, the notion of "context dependence" is used in a very narrow sense; especially when focusing on simple and short motifs, a more extensive analysis of the interdependence of these features (beyond the existing analysis of the relationship between TA-diNTs and GC content) could potentially reveal more of the context dependence underlying the seemingly opposite behavior of very similar motifs.

The present MPRAs measures the effect of UTR sequences in one specific reporter context and using one experimental approach (following the decay of in vitro transcribed and transfected RNAs). While this approach certainly has its merits compared to other approaches, it also comes with some caveats: RNA is delivered naked, without bound RBPs and no nuclear history, e.g. of splicing (no EJCs), editing and modifications. One way to assess the generalizability of the results as well as the context dependence of the effects is to perform the same analysis on existing datasets of RNA stability measurements obtained through other methods (e.g. transcription inhibition). Are TA dinucleotides universally the most predictive feature of RNA half-lives?

The authors conclude their study with a meta-analysis of genes with increased TA dinucleotides in 5' and 3'UTRs, showing that specific functional groups are overrepresented among these genes. In addition, they provide evidence for an effect of disease-associated UTR mutations on endogenous RNA stability. While these elements link back to the original motivation of the study (screening for effects of point mutations in 5' and 3' UTRs), they provide only a limited amount of additional insights.

In summary, this manuscript presents an interesting addition to the long-standing attempts at dissecting the sequence basis of RNA stability in human cells. The analysis is in general very comprehensive and sound; however, at times the goal of the authors to find novelty and specificity in the data overshadows some analyses. One example is the case where the authors try to show that TA-dinucleotides and GC content are decoupled and not merely two sides of the same coin. They claim that the effect of TA dinucleotides is different between high- and low-GC content contexts but do not control for the fact that low GC-content regions naturally will contain more TA dinucleotides and therefore the effect sizes and the resulting correlation between TA-diNT rate and stability will be stronger (Fig. 5A). A more thorough analysis and greater caution in some of the claims could further improve the credibility of the conclusions.

Reviewer #2 (Public Review):

Summary of goals:

Untranslated regions are key cis-regulatory elements that control mRNA stability, translation, and translocation. Through interactions with small RNAs and RNA binding proteins, UTRs form complex transcriptional circuitry that allows cells to fine-tune gene expression. Functional annotation of UTR variants has been very limited, and improvements could offer insights into disease relevant regulatory mechanisms. The goals were to advance our understanding of the determinants of UTR regulatory elements and characterize the effects of a set of "disease-relevant" UTR variants.

Strengths:

The use of a massively parallel reporter assay allowed for analysis of a substantial set (6,555 pairs) of 5' and 3' UTR fragments compiled from known disease associated variants. Two cell types were used.

The findings confirm previous work about the importance of AREs, which helps show validity and adds some detailed comparisons of specific AU-rich motif effects in these two cell types.

Using a Lasso regression, TA-dinucleotide content is identified as a strong regulator of RNA stability in a context dependent manner based on GC content and presence of RNA binding protein binding motifs. The findings have potential importance, drawing attention to a UTR feature that is not well characterized.

The use of complementary datasets, including from half-life analyses of RNAs and from random sequence library MRPA's, is a useful addition and supports several important findings. The finding the TA dinucleotides have explanatory power separate from (and in some cases interacting with) GC content is valuable.

The functional enrichment analysis suggests some new ideas about how UTRs may contribute to regulation of certain classes of genes.

Weaknesses:

It is difficult to understand how the calculations for half-life were performed. The sequencing approach measures the relative frequency of each sequence at each time point (less stable sequences become relatively less frequent after time 0, whereas more stable sequences become relatively more frequent after time 0). Since there is no discussion of whether the abundance of the transfected RNA population is referenced to some external standard (e.g., housekeeping RNAs), it is not clear how absolute (rather than relative) half-lives were determined.

Fig. S1A and B are used to assess reproducibility. They show that read counts at a given time point correlate well across replicate experiments. However, this is not a good way to assess reproducibility or accuracy of the measurements of t1/2 are. (The major source of variability in read counts in these plots - especially at early time points - is likely the starting abundance of each RNA sequence, not stability.) This creates concerns about how well the method is measuring t1/2. Also creating concern is the observation that many RNAs are associated with half-lives that are much longer than the time points analyzed in the study. For example, based upon Figure S1 and Table S1 correctly, the median t1/2 for the 5' UTR library in HEK cells appears to be >700 minutes. Given that RNA was collected at 30, 75, and 120 minutes, accurate measurements of RNAs with such long half lives would seem to be very difficult.

There is no direct comparison of t1/2 between the two cell types studied for the full set of sequences studied. This would be helpful in understanding whether the regulatory effects of UTRs are generally similar across cell lines (as has been shown in some previous studies) or whether there are fundamental differences. The distribution of t1/2's is clearly quite different in the two cell lines, but it is important to know if this reflects generally slow RNA turnover in HEK cells or whether there are a large number of sequence-specific effects on stability between cell lines. A related issue is that it is not clear whether the relatively small number of significant variant effects detected in HEK cells versus SH-SY5Y cells is attributable to real biological differences between cell types or to technical issues (many fewer read counts and much longer half lives in HEK cells).

The general assertion is made in many places that TA dinucleotides are the most prominent destabilizing element in UTRs (e.g., in the title, the abstract, Fig. 4 legend, and on p. 12). This appears to be true for only one of the two cell lines tested based on Fig. 3.

Appraisal and impact:

The work adds to existing studies that previously identified sequence features, including AREs and other RNA binding protein motifs, that regulate stability and puts a new emphasis on the role of "TA" (better "UA") dinucleotides. It is not clear how potential problems with the RNA stability measurements discussed above might influence the overall conclusions, which may limit the impact unless these can be addressed.

It is difficult to understand whether the importance of TA dinucleotides is best explained by their occurrence in a related set of longer RBP binding motifs (see Fig 5J, these motifs may be encompassed by the "WWWWWW cluster") or whether some other explanation applies. Further discussion of this would be helpful. Does the LASSO method tend to collapse a more diverse set of longer motifs that are each relatively rare compared to the dinucleotide? It remains unclear whether TA dinucleotides are associated with less stability independent of the presence of the known larger WWWWWWW motif. As noted above, the importance of TA dinucleotides in the HEK experiments appears to be less than is implied in the text.

The inclusion of more than a single cell type is an acknowledgement of the importance of evaluating cell type-specific effects. The work suggests a number of cell type-specific differences, but due to technical issues (especially with the HEK data, as outlined above) and the use of only two cell lines, it is difficult to understand cell type effects from the work.

The inclusion of both 3' and 5' UTR sequences distinguishes this work from most prior studies in the field. Contrasting the effects of these regions on stability is of interest, although the role of these UTRs (especially the 5' UTR) in translational regulation is not assessed here.

Reviewer #3 (Public Review):

Summary:

In their manuscript titled "Multiplexed Assays of Human Disease‐relevant Mutations Reveal UTR Dinucleotide Composition as a Major Determinant of RNA Stability" the authors aim to investigate the effect of sequence variations in 3'UTR and 5'UTRs on the stability of mRNAs in two different human cell lines.

To do so, the authors use a massively parallel reporter assay (MPRA). They transfect cells with a set of mRNA reporters that contain sequence variants in their 3' or 5' UTRs, which were previously reported in human diseases. They follow their clearance from cells over time relative to the matching non-variant sequence. To analyze their results, they define a set of factors (RBP and miRNA binding sites, sequence features, secondary structure etc.) and test their association with differences in mRNA stability. For features with a significant association, they use clustering to select a subset of factors for LASSO regression and identify factors that affect mRNA stability.
They conclude that the TA dinucleotide content of UTRs is the strongest destabilizing sequence feature. Within that context, elevated GC content and protein binding can protect susceptible mRNAs from degradation. They also show that TA dinucleotide content of UTRs affects native mRNA stability, and that it is associated with specific functional groups. Finally, they link disease associated sequence variants with differences in mRNA stability of reporters.

Strengths:

(1) This work introduces a different MPRA approach to analyze the effect of genetic variants. While previous works in tissue culture use DNA transfections that require normalization for transcription efficiency, here the mRNA is directly introduced into cells at fixed amounts, allowing a more direct view of the mRNA regulation.

(2) The authors also introduce a unique analysis approach, which takes into account multiple factors that might affect mRNA stability. This approach allows them to identify general sequence features that affect mRNA stability beyond specific genetic variants, and reach important insights on mRNA stability regulation. Indeed, while the conclusions to genetic variants identified in this work are interesting, the main strength of the work involve general effect of sequence features rather than specific variants.

(3) The authors provide adequate supports for their claims, and validate their analysis using both their reporter data and native genes. For the main feature identified, TA di-nucleotides, they perform follow-up experiments with modified reporters that further strengthen their claims, and also validate the effect on native cellular transcripts (beyond reporters), demonstrating its validity also within native scenarios.

(4) The work provides a broad analysis of mRNA stability, across two mRNA regulatory segments (3'UTR and 5'UTR) and is performed in two separate cell-types. Comparison between two different cell-types is adequate, and the results demonstrate, as expected, the dependence of mRNA stability on the cellular context. Analysis of 3'UTR and 5'UTR regulatory effects also shows interesting differences and similarities between these two regulatory regions.

Weaknesses:

(1) The authors fail to acknowledge several possible confounding factors of their MPRA approach in the discussion.
First, while transfection of mRNA directly into cells allows to avoid the need to normalize for differences in transcription, the introduction of naked mRNA molecules is different than native cellular mRNAs and could introduce biases due to differences in mRNA modifications, protein associations etc. that may occur co-transcriptionally.
Second, along those lines, the authors also use in-vitro polyadenylation. The length of the polyA tail of the transfected transcripts could potentially be very different than that of native mRNAs and also affect stability.

(2) The analysis approach used in this work for identifying regulatory features in UTRs was not previously used. As such, lack of in-depth details of the methodology, and possibly also more general validation of the approach, is a drawback in convincing the reader in the validity of this approach and its results.
In particular, a main point that is not addressed is how the authors decide on the set of "factors" used in their analysis? As choosing different sets of factors might affect the results of the analysis. For example, the choice to use 7-mer sequences within the factors set is not explained, particularly when almost all motifs that are eventually identified (Figure 3B-E) are shorter.
In addition, the authors do not perform validations to demonstrate the validity of their approach on simulated data or well-established control datasets. Such analysis would be helpful to further convince the reader in the usefulness and robustness of the analysis.

(3) The analysis and regression models built in this work are not thoroughly investigated relative to native genes within cells. The effect of sequence "factors" on native cellular transcripts' stability is not investigated beyond TA di-nucleotides, and it is unclear to what degree do other predicted factors also affect native transcripts.

Author response:

Public Reviews:

Reviewer #1 (Public Review):

In the manuscript by Su et al., the authors present a massively parallel reporter assay (MPRA) measuring the stability of in vitro transcribed mRNAs carrying wild-type or mutant 5' or 3' UTRs transfected into two different human cell lines. The goal presented at the beginning of the manuscript was to screen for effects of disease-associated point mutations on the stability of the reporter RNAs carrying partial human 5' or 3' UTRs. However, the majority of the manuscript is dedicated to identifying sequence components underlying the differential stability of reporter constructs. This shows that TA dinucleotides are the most predictive feature of RNA stability in both cell lines and both UTRs.

The effect of AU rich elements (AREs) on RNA stability is well established in multiple systems, and the present study confirms this general trend but points out variability in the consequence of seemingly similar motifs on RNA stability. For example, the authors report that a long stretch of Us has extreme opposite effects on RNA stability depending on whether it is preceded by an A (strongly destabilizing) or followed by an A (strongly stabilizing). While the authors interpretation of a context- dependence of the effect is certainly well-founded, it seems counterintuitive that the preceding or following A would be the (only) determining factor. This points to a generally reductionist approach taken by the authors in the analysis of the data and in their attempt to dissect the contribution of "AU rich sequences" to RNA stability, with a general tendency to reduce the size and complexity of the features (e.g. to dinucleotides). While this certainly increases the statistical power of the analysis due to the number of occurrences of these motifs, it limits the interpretability of the results. How do TA dinucleotides per se contribute to destabilizing the RNA, both in 5' and 3' UTRs, but (according to limited data presented) not in coding sequences? What is the mechanism? RBPs binding to TA dinucleotide containing sequences are suggested to "mask" the destabilizing effect, thereby leading to a more stable RNA. Gain of TA dinucleotides is reported to have a destabilizing effect, but again no hypothesis is provided as to the underlying molecular mechanism. In addition to reducing the motif length to dinucleotides, the notion of "context dependence" is used in a very narrow sense; especially when focusing on simple and short motifs, a more extensive analysis of the interdependence of these features (beyond the existing analysis of the relationship between TA- diNTs and GC content) could potentially reveal more of the context dependence underlying the seemingly opposite behavior of very similar motifs.

The contribution of coding region sequence to RNA stability has been extensively discussed (For example: doi.org/10.1016/j.molcel.2022.03.032; doi.org/10.1186/s13059-020-02251-5; doi.org/10.15252/embr.201948220; doi.org/10.1371/journal.pone.0228730; doi.org/10.7554/eLife.45396). While TA content at the third codon position (wobble position) has been implicated as a pro-degradation signal, codon optimality has emerged as the most prominent determinant for RNA stability. This indicates that the role of coding regions in RNA stability differs from that of UTRs due to the involvement of translation elongation. We did not intend to suggest that TA-dinucleotides in UTRs and coding regions have the same effect.

We hypothesize that TA-dinucleotide may recruit endonucleases RNase A family, whose catalytic pockets exhibit a strong bias for TA dinucleotide (doi.org/10.1016/j.febslet.2010.04.018). Structures or protein bindings that blocks this recognition might stabilize RNAs. To gain further insight into the motif interactions, we plan to investigate the interactions between TA and other 15 dinucleotides through more detailed analyses.

The present MPRAs measures the effect of UTR sequences in one specific reporter context and using one experimental approach (following the decay of in vitro transcribed and transfected RNAs). While this approach certainly has its merits compared to other approaches, it also comes with some caveats: RNA is delivered naked, without bound RBPs and no nuclear history, e.g. of splicing (no EJCs), editing and modifications. One way to assess the generalizability of the results as well as the context dependence of the effects is to perform the same analysis on existing datasets of RNA stability measurements obtained through other methods (e.g. transcription inhibition). Are TA dinucleotides universally the most predictive feature of RNA half-lives?

Our system studies the stability control of RNA synthesized in vitro and delivered into human cells. While we did not intend to generalize our conclusions to endogenous RNAs, our approach contributes to the understanding of in vitro synthesized RNA used for cellular expression, such as in vaccines. It is known that endogenous RNAs undergo very different regulation. The most prominent factors controlling endogenous RNA stability are the density of splice junctions and the length of UTRs (doi.org/10.1186/s13059-022-02811-x; doi.org/10.1186/s12915-021-00949-x). To decipher the sequence regulation, these factors are controlled in our experiments. Therefore we do not expect the dinucleotide features found by our approach to be generalized as the most predictive feature of RNA half-life in vivo.

The authors conclude their study with a meta-analysis of genes with increased TA dinucleotides in 5' and 3'UTRs, showing that specific functional groups are overrepresented among these genes. In addition, they provide evidence for an effect of disease-associated UTR mutations on endogenous RNA stability. While these elements link back to the original motivation of the study (screening for effects of point mutations in 5' and 3' UTRs), they provide only a limited amount of additional insights.

We utilized the Taiwan Biobank to investigate whether mutations significantly affecting RNA stability also impact human biochemical measurements. Our findings indicate that these mutations indeed have a significant effect on various biochemical indices. This highlights the importance of our study, as it bridges basic science with potential applications in precision medicine. By linking specific UTR mutations with measurable changes in biochemical indices, our research underscores the potential for these findings to inform targeted medical interventions in the future.

In summary, this manuscript presents an interesting addition to the long-standing attempts at dissecting the sequence basis of RNA stability in human cells. The analysis is in general very comprehensive and sound; however, at times the goal of the authors to find novelty and specificity in the data overshadows some analyses. One example is the case where the authors try to show that TA-dinucleotides and GC content are decoupled and not merely two sides of the same coin. They claim that the effect of TA dinucleotides is different between high- and low-GC content contexts but do not control for the fact that low GC-content regions naturally will contain more TA dinucleotides and therefore the effect sizes and the resulting correlation between TA-diNT rate and stability will be stronger (Fig. 5A). A more thorough analysis and greater caution in some of the claims could further improve the credibility of the conclusions.

Low GC content implies a higher TA content but does not directly equate to a high TA-diNT rate. For instance, the sequence ATTGAACCTT has a lower GC content (0.3) compared to TATAGGCCGC (0.6), yet it also has a lower TA-diNT rate (0 vs. 0.22). To address this concern more rigorously, we performed a stratified analysis based on TA-diNT rate. As shown in our Fig. S7C, even after stratifying by TA-diNT rate (upper panel high TA-diNT rate / lower panel low TA-diNT rate), we still observe that the destabilizing effect of TA is stronger in the low GC content group.

Reviewer #2 (Public Review):

Summary of goals:

Untranslated regions are key cis-regulatory elements that control mRNA stability, translation, and translocation. Through interactions with small RNAs and RNA binding proteins, UTRs form complex transcriptional circuitry that allows cells to fine-tune gene expression. Functional annotation of UTR variants has been very limited, and improvements could offer insights into disease relevant regulatory mechanisms. The goals were to advance our understanding of the determinants of UTR regulatory elements and characterize the effects of a set of "disease-relevant" UTR variants.

Strengths:

The use of a massively parallel reporter assay allowed for analysis of a substantial set (6,555 pairs) of 5' and 3' UTR fragments compiled from known disease associated variants. Two cell types were used.

The findings confirm previous work about the importance of AREs, which helps show validity and adds some detailed comparisons of specific AU-rich motif effects in these two cell types.

Using a Lasso regression, TA-dinucleotide content is identified as a strong regulator of RNA stability in a context dependent manner based on GC content and presence of RNA binding protein binding motifs. The findings have potential importance, drawing attention to a UTR feature that is not well characterized.

The use of complementary datasets, including from half-life analyses of RNAs and from random sequence library MRPA's, is a useful addition and supports several important findings. The finding the TA dinucleotides have explanatory power separate from (and in some cases interacting with) GC content is valuable.

The functional enrichment analysis suggests some new ideas about how UTRs may contribute to regulation of certain classes of genes.

Weaknesses:

It is difficult to understand how the calculations for half-life were performed. The sequencing approach measures the relative frequency of each sequence at each time point (less stable sequences become relatively less frequent after time 0, whereas more stable sequences become relatively more frequent after time 0). Since there is no discussion of whether the abundance of the transfected RNA population is referenced to some external standard (e.g., housekeeping RNAs), it is not clear how absolute (rather than relative) half-lives were determined.

We estimated decay constant λ and half-life () by the following equations:

where Ci(t) and Ci(t=0) are read count values of the ith replicate at time points and (see also Methods). The absolute abundance was not required for the half-life calculation.

Fig. S1A and B are used to assess reproducibility. They show that read counts at a given time point correlate well across replicate experiments. However, this is not a good way to assess reproducibility or accuracy of the measurements of t1/2 are. (The major source of variability in read counts in these plots - especially at early time points - is likely the starting abundance of each RNA sequence, not stability.) This creates concerns about how well the method is measuring t1/2. Also creating concern is the observation that many RNAs are associated with half-lives that are much longer than the time points analyzed in the study. For example, based upon Figure S1 and Table S1 correctly, the median t1/2 for the 5' UTR library in HEK cells appears to be >700 minutes. Given that RNA was collected at 30, 75, and 120 minutes, accurate measurements of RNAs with such long half lives would seem to be very difficult.

We estimated the half-life based on the following equations:

Where Ci(t) and Ci(t=0) are read count values of the ith replicate at time points and (see also Methods). The calculation of the half-life involves first determining the decay constant 𝜆, which represents a constant rate of decay. Since 𝜆 is a constant, it is possible to accurately calculate it without needing data over the entire decay range. Our experimental design considers this by selecting appropriate time points to ensure a reliable estimation of 𝜆, and thus, the half-life. To determine the most suitable time points, we conducted preliminary experiments using RT-PCR. These experiments indicated that 30, 75, and 120 minutes provided an effective range for capturing the decay dynamics of the transcripts.

There is no direct comparison of t1/2 between the two cell types studied for the full set of sequences studied. This would be helpful in understanding whether the regulatory effects of UTRs are generally similar across cell lines (as has been shown in some previous studies) or whether there are fundamental differences. The distribution of t1/2's is clearly quite different in the two cell lines, but it is important to know if this reflects generally slow RNA turnover in HEK cells or whether there are a large number of sequence-specific effects on stability between cell lines. A related issue is that it is not clear whether the relatively small number of significant variant effects detected in HEK cells versus SH-SY5Y cells is attributable to real biological differences between cell types or to technical issues (many fewer read counts and much longer half lives in HEK cells).

For both cell lines, we selected oligonucleotides with R2 > 0.5 and mean squared error (MSE) < 1 for analysis when estimating half-life (λ) by linear regression. This selection criterion was implemented to minimize the effect of experimental noise. Additionally, we will further analyze the MSE distribution to determine if the two cell lines exhibit significantly different levels of experimental noise. We will also provide a direct comparison of half-lives between the two cell lines to assess the similarity in stability regulation.

The general assertion is made in many places that TA dinucleotides are the most prominent destabilizing element in UTRs (e.g., in the title, the abstract, Fig. 4 legend, and on p. 12). This appears to be true for only one of the two cell lines tested based on Fig. 3.

TA-dinucleotides and other TA-rich sequences exhibit similar effects on RNA stability, as illustrated in Fig. S5A-C. In two cell lines, TA-dinucleotide and WWWWWW sequences were representatives of the same stability-affecting cluster. While the impact of TA-dinucleotides can be generalized, we will rephrase some statements for clarification to avoid any potential misunderstanding.

Appraisal and impact:

The work adds to existing studies that previously identified sequence features, including AREs and other RNA binding protein motifs, that regulate stability and puts a new emphasis on the role of "TA" (better "UA") dinucleotides. It is not clear how potential problems with the RNA stability measurements discussed above might influence the overall conclusions, which may limit the impact unless these can be addressed.

It is difficult to understand whether the importance of TA dinucleotides is best explained by their occurrence in a related set of longer RBP binding motifs (see Fig 5J, these motifs may be encompassed by the "WWWWWW cluster") or whether some other explanation applies. Further discussion of this would be helpful. Does the LASSO method tend to collapse a more diverse set of longer motifs that are each relatively rare compared to the dinucleotide? It remains unclear whether TA dinucleotides are associated with less stability independent of the presence of the known larger WWWWWWW motif. As noted above, the importance of TA dinucleotides in the HEK experiments appears to be less than is implied in the text.

To ensure the representativeness of the features entered into the LASSO model, we pre-selected those with an occurrence greater than 10% among all UTRs. There is no evidence to support a preference for dinucleotides by LASSO. To address whether the destabilizing effect of TA dinucleotides is part of the broader WWWWWW motif, we will divide TA dinucleotides into two groups: those within the WWWWWW motif and those outside of it. We will then examine whether TA dinucleotides in these two groups exhibit the same destabilizing effect.

The inclusion of more than a single cell type is an acknowledgement of the importance of evaluating cell type-specific effects. The work suggests a number of cell type-specific differences, but due to technical issues (especially with the HEK data, as outlined above) and the use of only two cell lines, it is difficult to understand cell type effects from the work.

The inclusion of both 3' and 5' UTR sequences distinguishes this work from most prior studies in the field. Contrasting the effects of these regions on stability is of interest, although the role of these UTRs (especially the 5' UTR) in translational regulation is not assessed here.

We examined the role of UTR and UTR variants in translation regulation using polysome profiling. By both univariate analysis and an elastic regression model, we identified motifs of short repeated sequences, including SRSF2 binding sites, as mutation hotspots that lead to aberrant translation. Furthermore, these polysome-shifting mutations had a considerable impact on RNA secondary structures, particularly in upstream AUG-containing 5’ UTRs. Integrating these features, our model achieved high accuracy (AUROC > 0.8) in predicting polysome-shifting mutations in the test dataset. Additionally, metagene analysis indicated that pathogenic variants were enriched at the upstream open reading frame (uORF) translation start site, suggesting changes in uORF usage underlie the translation deficiencies caused by these mutations. Illustrating this, we demonstrated that a pathogenic mutation in the IRF6 5’ UTR suppresses translation of the primary open reading frame by creating a uORF. Remarkably, site-directed ADAR editing of the mutant mRNA rescued this translation deficiency. Because the regulation of translation and stability does not converge, we illustrate these two mechanisms in two separate manuscripts (this one and doi.org/10.1101/2024.04.11.589132).

Reviewer #3 (Public Review):

Summary:

In their manuscript titled "Multiplexed Assays of Human Disease‐relevant Mutations Reveal UTR Dinucleotide Composition as a Major Determinant of RNA Stability" the authors aim to investigate

the effect of sequence variations in 3'UTR and 5'UTRs on the stability of mRNAs in two different human cell lines.

To do so, the authors use a massively parallel reporter assay (MPRA). They transfect cells with a set of mRNA reporters that contain sequence variants in their 3' or 5' UTRs, which were previously reported in human diseases. They follow their clearance from cells over time relative to the matching non-variant sequence. To analyze their results, they define a set of factors (RBP and miRNA binding sites, sequence features, secondary structure etc.) and test their association with differences in mRNA stability. For features with a significant association, they use clustering to select a subset of factors for LASSO regression and identify factors that affect mRNA stability.

They conclude that the TA dinucleotide content of UTRs is the strongest destabilizing sequence feature. Within that context, elevated GC content and protein binding can protect susceptible mRNAs from degradation. They also show that TA dinucleotide content of UTRs affects native mRNA stability, and that it is associated with specific functional groups. Finally, they link disease associated sequence variants with differences in mRNA stability of reporters.

Strengths:

(1) This work introduces a different MPRA approach to analyze the effect of genetic variants. While previous works in tissue culture use DNA transfections that require normalization for transcription efficiency, here the mRNA is directly introduced into cells at fixed amounts, allowing a more direct view of the mRNA regulation.

(2) The authors also introduce a unique analysis approach, which takes into account multiple factors that might affect mRNA stability. This approach allows them to identify general sequence features that affect mRNA stability beyond specific genetic variants, and reach important insights on mRNA stability regulation. Indeed, while the conclusions to genetic variants identified in this work are interesting, the main strength of the work involve general effect of sequence features rather than specific variants.

(3) The authors provide adequate supports for their claims, and validate their analysis using both their reporter data and native genes. For the main feature identified, TA di-nucleotides, they perform follow-up experiments with modified reporters that further strengthen their claims, and also validate the effect on native cellular transcripts (beyond reporters), demonstrating its validity also within native scenarios.

(4) The work provides a broad analysis of mRNA stability, across two mRNA regulatory segments (3'UTR and 5'UTR) and is performed in two separate cell-types. Comparison between two different cell-types is adequate, and the results demonstrate, as expected, the dependence of mRNA stability on the cellular context. Analysis of 3'UTR and 5'UTR regulatory effects also shows interesting differences and similarities between these two regulatory regions.

Weaknesses:

(1) The authors fail to acknowledge several possible confounding factors of their MPRA approach in the discussion.

First, while transfection of mRNA directly into cells allows to avoid the need to normalize for differences in transcription, the introduction of naked mRNA molecules is different than native cellular mRNAs and could introduce biases due to differences in mRNA modifications, protein associations etc. that may occur co-transcriptionally.

Second, along those lines, the authors also use in-vitro polyadenylation. The length of the polyA tail of the transfected transcripts could potentially be very different than that of native mRNAs and also affect stability.

The transcripts used in our study were polyadenylated in vitro with approximately 100 nucleotides (Fig. S1C), similar to the polyA tail lengths typically observed in vivo (dx.doi.org/10.1016/j.molcel.2014.02.007). Additionally, these transcripts were capped to emulate essential mRNA characteristics and to minimize immune responses in recipient cells. This design allows us to study RNA decay for in vitro-synthesized RNA delivered into human cells, akin to RNA vaccines, but it does not necessarily extend to endogenous RNAs. As mentioned, endogenous RNAs undergo nuclear processing and are decorated by numerous trans factors, resulting in distinct regulatory mechanisms. We will provide a more in-depth discussion on these differences and their implications in the revised manuscript.

(2) The analysis approach used in this work for identifying regulatory features in UTRs was not previously used. As such, lack of in-depth details of the methodology, and possibly also more general validation of the approach, is a drawback in convincing the reader in the validity of this approach and its results.

In particular, a main point that is not addressed is how the authors decide on the set of "factors" used in their analysis? As choosing different sets of factors might affect the results of the analysis.

In our study, we employed the calculation of the Variance Inflation Factor (VIF) as a basis for selecting variables. This well-established method is widely used to detect variables with high collinearity, thus ensuring the robustness and reliability of our analysis. By identifying and excluding highly collinear variables, we aimed to minimize multicollinearity and improve the accuracy of our regression models. For more detailed information on the use of VIF in regression analysis, please refer to Akinwande, M., Dikko, H., and Samson, A. (2015). Variance Inflation Factor: As a Condition for the Inclusion of Suppressor Variable(s) in Regression Analysis. Open Journal of Statistics, 5, 754-767. doi: 10.4236/ojs.2015.57075. We will include the method details in the revised manuscript.

For example, the choice to use 7-mer sequences within the factors set is not explained, particularly when almost all motifs that are eventually identified (Figure 3B-E) are shorter.

The known RBP motifs are primarily 6-mer. To explore the possibility of discovering novel motifs that could significantly impact our model, we started with 7-mer sequences. However, our analysis revealed that including these additional variables did not improve the explanatory power of the model; instead, it reduced it. Consequently, our final model focuses on motifs shorter than 7-mer. We will explain the motif selections in the revised manuscript.

In addition, the authors do not perform validations to demonstrate the validity of their approach on simulated data or well-established control datasets. Such analysis would be helpful to further convince the reader in the usefulness and robustness of the analysis.

We acknowledge the importance of validating our approach on simulated data or well-established control datasets to demonstrate its robustness and reliability. However, to the best of our knowledge, there are currently no well-established control datasets available that perfectly correspond to our specific study context. Despite this, we will continue to search for any relevant datasets that could be utilized for this purpose in future work. This effort will help to further reinforce the confidence in our methodology and its findings.

(3) The analysis and regression models built in this work are not thoroughly investigated relative to native genes within cells. The effect of sequence "factors" on native cellular transcripts' stability is not investigated beyond TA di-nucleotides, and it is unclear to what degree do other predicted factors also affect native transcripts.

Our system studies the stability control of RNA synthesized in vitro and delivered into human cells. While we validated the UTR TA-dinucleotide effect in vivo, we did not intend to conclude that this is the most influential regulation for endogenous RNAs. It is known that endogenous RNAs undergo very different regulation. The most prominent factors controlling endogenous RNA stability are the density of splice junctions and the length of UTRs (doi.org/10.1186/s13059-022-02811-x; doi.org/10.1186/s12915-021-00949-x). To decipher the sequence regulation, we controlled for these factors in our experiments. Therefore, we acknowledge that several endogenous features, which were excluded by our approach, may serve as predictive features of RNA half-life in vivo.