Regulatory roles of Escherichia coli 5' UTR and ORF-internal RNAs detected by 3' end mapping

  1. Philip P Adams
  2. Gabriele Baniulyte
  3. Caroline Esnault
  4. Kavya Chegireddy
  5. Navjot Singh
  6. Molly Monge
  7. Ryan K Dale
  8. Gisela Storz
  9. Joseph T Wade

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Abstract

Many bacterial genes are regulated by RNA elements in their 5´ untranslated regions (UTRs). However, the full complement of these elements is not known even in the model bacterium Escherichia coli . Using complementary RNA-sequencing approaches, we detected large numbers of 3´ ends in 5´ UTRs and open reading frames (ORFs), suggesting extensive regulation by premature transcription termination. We documented regulation for multiple transcripts, including spermidine induction involving Rho and translation of an upstream ORF for an mRNA encoding a spermidine efflux pump. In addition to discovering novel sites of regulation, we detected short, stable RNA fragments derived from 5´ UTRs and sequences internal to ORFs. Characterization of three of these transcripts, including an RNA internal to an essential cell division gene, revealed that they have independent functions as sRNA sponges. Thus, these data uncover an abundance of cis - and trans -acting RNA regulators in bacterial 5´ UTRs and internal to ORFs.

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  1. Version published to 10.7554/elife.62438 on eLife
  2. Version published to 10.1101/2020.07.18.207399v2 on bioRxiv
  3. eLife

    ###Reviewer #4:

    PREreview of "Regulatory roles of 5′ UTR and ORF-internal RNAs detected by 3′ end mapping"

    Authored by Philip P. Adams et al. and posted on bioRxiv DOI: 10.1101/2020.07.18.207399

    Review authors in alphabetical order: Monica Granados, Runhua Han, Katrina Murphy, Nik Tsotakos

    This review is the result of a virtual, live-streamed journal club organized and hosted by PREreview and eLife. The discussion was joined by 20 people in total, including researchers from several regions of the world, four of the preprint authors, and the event organizing team.

    Overview and take-home message:

    Adams et al. have demonstrated with both their genome-wide and targeted analyses of RNA elements in E.coli how two labs can collaboratively come together to make significant advances in their respective fields while also producing a model paper to open the door for more research. Their research not only looked at non-coding sRNA regulation but identified so called gene "mistakes" that also have functions. In addition, they bridged the gaps in our knowledge about how sRNAs derived from internal open reading frames can act as sponges and how termination spots of 5' untranslated regions affects sRNA regulation on their target mRNAs. Although this work is of interest in microbial gene expression, below are a few concerns that could be addressed in the next version of this paper.

    Positive feedback:

    • All this work took only a year?! Congratulations, this is really great work.
    • The journal club definitely recommends this preprint for others in the field and for peer review. This work could be an important contribution.
    • The conclusions are supported by data. Many of the newly discovered sRNA and their regulatory mechanism were experimentally confirmed and investigated. All the data analysis support the hypothesis and conclusion of each section.
    • Really appreciate the manuscript. The introduction had enough background for why the researchers took this approach. Really enjoyed reading this paper - even with a neuroscience background.
    • This preprint provides a lot of information of RNA termination sites by 3' end mapping in the model bacteria (E. coli), which also enlightens the studies relevant to sRNA discovery and RNA regulation mechanisms in other bacteria.
    • One of the most exciting and novel findings is that the 3'-end termination of the 5'-UTR of some known sRNA targets can reinforce the sRNA regulation.
    • A potentially exciting opportunity for studying differential regulation via sRNAs during the exponential growth and plateau phases.
    • The paper employs very high-throughput sequencing technologies on a bacterial model that normally doesn't get so much attention, especially on the non-coding RNAs and post-transcriptional regulation.
    • The current knowledge of sRNA regulation mechanisms are expanded.
    • Primes more questions by trying out new techniques to find new regulatory areas.
    • Just the beginning of the deeper dive model into gene regulation and Rho-dependent termination - opens the door for more research and makes this paper extra referenceable moving forward. For future research or consideration, what can be extrapolated from this research for other organisms?
    • I thought the methods were very thorough, they also have a data availability statement and uploaded the sequencing data.
    • Data is available, and UCSC browser tracks made available!
    • Gold star for having code used for calling the 3' ends open and available on github (always a pro in my eyes!) +2 for GITHUB!
    • Genome-wide data can give other researchers a chance to find new mechanisms relevant to their genes and circuits of interest.
    • So much detail was available! I am not familiar with the standard techniques in the field, but from what I read the detail seemed to be reproducible.
    • I always appreciate when the Results subsections are bolded which helps gather my thoughts.

    Major concerns:

    1. The authors may consider adding another figure panel or some additional text summarising how the 3' ends they mapped are distributed over the genome - e.g. are they enriched in any specific region or well-distributed?

    2. The authors mention that they identified 412 genomic loci putatively associated with a Rho termination event, based on a Rho score of 2.0, indicated in Table S2. However, in Fig. 1C the total number of Rho-dependent termination events mentioned is 433. The discrepancy between these two numbers can be slightly confusing. Could the authors describe the methodological differences that led to the two different numbers?

    3. The authors identify the 280nt mdtJI transcript that is the result of premature termination, and show very nicely how this transcript is susceptible to read through in the presence of spermidine under elevated pH conditions (see Figure 3). In Figure 2F, however, the Northern blot indicates the presence of a longer transcript as well in the presence of the mutant Rho. Do the authors have any indications what this longer transcript (~400bp) is?

    4. With regards to the results presented in Figure 4, the authors consider the possibilities of MicA-directed cleavage of the ompA mRNA or protection from degradation due to base pairing with the sRNA. If the first possibility were true, could the probe used in the Northern blot detect smaller fragments, or was it designed to only detect the full length transcript?

  4. eLife

    ###Reviewer #3:

    The paper of Adams et al. attempts to provide a resource of Rho-dependent and independent transcript 3' ends in the model bacterium Escherichia coli, focusing especially on 3' ends identified in 5' UTRs and within coding sequences. Studying several of these termini in detail, the authors present interesting novel types of regulatory loops involving products of pre-mature transcription termination or of mRNA transcript processing. These include, for example, small RNAs derived from 5' UTRs of targets of canonical sRNAs, which sponge the canonical sRNAs and, in turn, affect the target they are derived from. The paper will be of interest to the microbiology and RNA communities, and may inspire in-depth investigation of regulatory loops and novel sRNAs discovered here, as well as the discovery of additional novel regulatory RNAs and new structures of regulatory loops inferred from the resource that the authors provide.

    Major comments:

    Additional analyses of the data are needed, as detailed below.

    1. Comparison between the large-scale data sets of 3' ends provided by the current and previous studies. It is very important that the comparisons between the current data set of 3' ends and previous ones will be done properly, especially the comparison with a data set generated by the same protocol (Term-seq) by the developers of the protocol, Dar and Sorek (2018). There are several issues that should be considered in regard to the comparisons to previous data and evaluation of the statistical significance:

    a) Computation of the statistical significance of overlapping results by the hypergeometric test. It is not clear how the reported p-values were computed, and it is not possible to re-compute them as the value of N was not provided. For this test, the p-value of a result at least as good as the one obtained should be computed ("cumulative p-value"). Looking at the results in the Venn diagrams presented in Supplementary Figure S1, it is hard to see how p-values of <10-100 were obtained. The authors should check their computation. They should provide the details of the computation for all hypergeometric tests included in the manuscript, to enable their assessment.

    b) Data processing to reveal 3' ends. The computational method used to process the Term-seq data is different from the one presented in the paper of Dar and Sorek. The authors should explain why they turned to a different computational scheme and what is it’s advantages. It would be more appropriate to compare the current data set and Dar and Sorek's data set when analyzed by the same computational methodology. The authors should apply their new computational method to Dar and Sorek's data, or analyze their results by Dar and Sorek's computational method, and re-assess the overlap in the determined 3' ends.

    c) Rho-dependent termination. It is not clear why the authors followed Dar and Sorek for determining Rho-dependent termination. Dar and Sorek used available data of BCM treated cells from Peters et al. (2012), and therefore could only evaluate the readthrough in the vicinity of determined 3' ends. Since the authors made the effort to treat the cells with BCM and generate sequencing libraries, it is not clear why they did not simply carry out Term-seq following BCM treatment and compared the identified 3' ends to those determined without BCM. Secondly, in evaluation of the readthrough the authors, again, modified the computational method of Dar and Sorek. This needs justification and the parameters used need explanation (window size of 500 nt and threshold of the Rho score of at least 2). For the comparison of the results, the Dar and Sorek data set and the current data set should be analyzed by the same method and the results compared. In connection to that, since the BCM experiment was conducted in the current study only once, it would be important to analyze the Peters et al. data by the new computational method and compare the results. The analyses described in comments (1b) and (1c) might improve the overlap between the results of the different studies and reduce the inconsistencies.

    d) If the present large discrepancies between the current data set and previous one stay despite the new analyses, the authors need to carefully examine the similarities and inconsistencies, try to understand the reasons for that, and assess the reliability of their data.

    e) The authors can compare their own data sets in the different growth phases and conditions. It would be interesting to examine if the same or different 3' ends were deciphered in the three experiments. I believe it is expected that many of the termini will be re-discovered but some will be different between the different growth phases and conditions. This analysis will provide an assessment of the consistency of the results and might provide new biological insights.

    1. Experimental results

    a) Several 3' termination sites were tested experimentally by molecular experiments. From the reported results it seems that all tested sites were re-confirmed by the molecular experiments. How were the studied sites selected? Were there sites from the large-scale data that were tested by the molecular experiments and were not confirmed as 3' ends? A report of true positives and false positives would provide another important assessment of the reliability of the data.

    b) It would be informative to assess the correspondence between the Rho score and the ratio of beta galactosidase activity between rho mutant and WT cells (Figure 2 and Supplementary Figure S2). It seems that genes with Rho scores below 2, such as sugE, may show high ratios. How should users of the provided resource consider the Rho score values?

  5. eLife

    ###Reviewer #2:

    Adams et al. have comprehensively identified the 3' ends of transcripts in E. coli and demonstrate that many transcripts are prematurely terminated either by Rho-dependent or intrinsic manner. Strikingly, in addition to small RNAs prevalently discovered in 3'UTR, the authors reveal that several premature transcripts generated from 5'UTR or internal CDS also function as sponges of Hfq-dependent small RNAs, i.e. pairs of ChiZ-ChiX, IspZ-OxyS and FtsO-RybB. It remains unclear which RNA chaperones and RNases are involved in the regulation. This study introduces new members to an emerging class of bona fide regulatory RNAs derived from mRNAs.

    1. Pages 10 - 12; The results of LacZ reporter assay and northern blot seem contradictory at a glance. Expectedly the reporter experiments which are carried out with the cells of OD0.4~0.6 showed a significant increase of LacZ activity in the rhoR66S mutant, which is defective in Rho-dependent termination (Figs. 2DE and S2B). On the other hand, in many cases, the northern blot analysis of total RNA extracted from the cells of OD0.4 revealed the increase of premature terminated 5'UTR fragments in the rhoR66S strain (Figs. 2F and S2C). Moreover, some 5'UTRs exhibited different patterns at OD2.0. This cannot be accounted for simply by the difference in growth phase (the last sentence of Page 10). The authors' suggestion that higher levels of longer transcripts in the absence of Rho are processed to give the shorter products (Page 12, Lines 7-8) is confusing since the increased LacZ reporter should be expressed from the longer transcripts. This point can be clarified by rehybridizing the northern blots with probes for corresponding genes downstream of the premature termination regions.

    2. In the same direction as the comment above, the northern blot analysis for mdtJI shows that the premature termination product of mdtU (~280 nt) is increased in the rhoR66S strain during growth in a normal LB medium (Fig. 2F). In stark contrast, the increase of mdtU transcript seems not significant in the LB pH9.0 without spermidine (Fig. 3E; lanes 1 and 3). However, in the presence of spermidine, the level of mdtJI long transcript was rather decreased in the rhoR66S strain (Fig. 3E; lanes 2 and 4). This result is contradictory to the result of LacZ reporter assay (Figs. 2DE). The influence of spermidine to the mdtU-lacZ reporter expression should also be tested.

    3. Pages 20-21; The effect of RybB on FtsO has not been clarified in the manuscript. When RybB is abundant, the level of FtsO was lower than the other situations (Fig. 7B, lane 6). This is indicative of coupled degradation upon base-pairing between FtsO and RybB. However, when RybB was induced by ethanol, the level of FtsO was rather increased (Fig. 7E), probably attributable to transcriptional activation of ftsI. To clarify the reciprocal regulation between RybB and FtsO and its consequence, this reviewer suggests quantifying the half-life of each sRNA in the presence or absence of its counterpart sRNA.

  6. eLife

    ###Reviewer #1:

    In this study, Adams et al. apply various RNA-seq-based approaches to map transcript 3'ends in E. coli in a genome-wide manner and distinguish between 3' ends derived from processing, Rho-dependent, or intrinsic termination. Strikingly, classification of 3'ends revealed that less than one quarter located within a 50 bp window downstream of annotated coding sequences (CDSs), whereas a substantial fraction fell within 5'UTRs and CDSs. The authors show that several transcription termination sites (TTSs) in 5'UTRs locate downstream of known cis-regulatory elements (riboswitches, uORFs) and may arise from premature transcription termination, leading to the hypothesis that other cis-regulatory elements may be discovered by characterizing 3'ends within 5'UTRs. Indeed, further supporting this, the authors present mechanistic data for a uORF (termed mdtU) affecting Rho-dependent transcription termination of the downstream operon in response to the polyamine spermidine.

    Other 3'ends were adjacent to known sRNA target sites within mRNA 5'UTRs or ORFs. Since several of these RNA fragments accumulate to high levels under physiological conditions, the authors go on demonstrating function for three such representatives (namely two 5'-derived RNAs, termed ChiZ, IspZ, and one ORF-internal candidate, FtsO). Interestingly, all three of them were found to be "sponges" of bona fide intergenic sRNAs, affecting either the activity of the latter (ChiZ on ChiX) or their steady-state levels (IspZ on OxyS; FtsO on RybB).

    Together, this important study expands our definition of bacterial sRNAs, demonstrates functionality of several "nonconventional" sRNAs, blurs the discrimination between regulator and target, and is expected to boost future studies looking into bacterial sRNAs derived from 5'UTRs or ORFs. The study is timely - as several recent studies proposed the existence of noncanonical sRNAs - and highly relevant as it provides data to support functionality of some of these RNAs (e.g. FtsO is the first ORF-internal sRNA with a reported function).

  7. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.

    ###Summary:

    In the present study, you have comprehensively identified the 3' ends of transcripts in E. coli and demonstrated that many arise from premature transcription termination in either Rho-dependent or intrinsic manner. As a result, you discovered numerous stable RNAs derived from 5'UTRs or CDSs and functionally characterized several of these "unconventional" RNAs as sponges of well-studied Hfq-dependent small RNAs. The reviewers all agreed that this is impressive work, the findings are novel and relevant for researchers within the microbiology and RNA communities and may inspire future studies of non-canonical bacterial sRNAs. Overall, they deem the results convincingly supported by the experimental data, but would like to see a few more experimental and analytical amendments to your work.

  8. Version published to 10.1101/2020.07.18.207399v1 on bioRxiv