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Upstream open reading frames repress the translation from the iab-8 RNA - PubMed

  • ️Mon Jan 01 2024

Upstream open reading frames repress the translation from the iab-8 RNA

Yohan Frei et al. PLoS Genet. 2024.

Abstract

Although originally classified as a non-coding RNA, the male-specific abdominal (MSA) RNA from the Drosophila melanogaster bithorax complex has recently been shown to code for a micropeptide that plays a vital role in determining how mated females use stored sperm after mating. Interestingly, the MSA transcript is a male-specific version of another transcript produced in both sexes within the posterior central nervous system from an alternative promoter, called the iab-8 lncRNA. However, while the MSA transcript produces a small peptide, it seems that the iab-8 transcript does not. Here, we show that the absence of iab-8 translation is due to a repressive mechanism requiring the two unique 5' exons of the iab-8 lncRNA. Through cell culture and transgenic analysis, we show that this mechanism relies on the presence of upstream open reading frames present in these two exons that prevent the production of proteins from downstream open reading frames.

Copyright: © 2024 Frei et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. An element in the 3’ area of iab-8 exon 2 is able to repress downstream translation.

A. Summary of the results and constructs. The different constructs tested are schematized with their names indicated on the left. The exons of the iab-8 transcript are represented by rectangles and labeled accordingly. The coding sequence for MSAmiP is represented in purple with the GFP coding sequence fused to the MSAmiP sequence (without an ATG start codon) indicated by a solid green box for constructs that expressed GFP or by a grey hatched box for construct that did not (see panel C). The sizes in nucleotides are indicated by the scale bars above the first two constructs (iab-8 and msa cDNA). The “(“and “)” represent DNA fragment deletions. B. An example of the results from the S2-cell transfections visualized by confocal microscopy using the same settings for each fluorophore. The iab-8-MSAmiP-GFP transfection is shown on top and the msa-MSAmiP-GFP transfection is shown on the bottom. The left panels show the mCherry control signals, while the right panels show the GFP signals. C. Quantification of the fluorescence from the confocal images using the Fiji software and using Prism (bottom left graphic). The X axis lists the different plasmids tested while the y-axis indicates the relative fluorescence of individual cells (arbitrary units). Each dot represents one cell. The errors bars indicate the median with the interquartile range. To determine statistically different levels of expression, we used the multiple comparison Kruskal-Wallis test (non-parametric) in the PRISM software. The **** indicate p<0.0001 relative to the iab-8 construct.

Fig 2
Fig 2. An ORF in the 200bp fragment represses downstream translation.

A. Summary of the results and constructs. The different constructs tested are schematized with their names indicated on the left. The pink rectangles represent the 200 bp fragment being tested. The position of each start codon is indicated by a triangle above the construct. The coding sequence of GFP (without an ATG start codon) is indicated by a solid green rectangle for constructs that express GFP and by a grey hatched box for constructs that did not (see panel B). The 14 bp deletion centered on the ATG sequence in the 200 bp fragment is indicated by the “()” interrupting the pink rectangle. The added Kozak sequences and linkers are represented by labeled boxes. Note that the Kozak sequence here contains an ATG start codon (in yellow), but the linkers lack this element. The three last constructs represent the constructs generated for the translation frame test. They differ in that the 200 bp fragment is reduced by one or two bases to modify the reading frame relative to the GFP sequence. B. The graph indicates the relative fluorescence measured (arbitrary units, Y-axis) for each of the construct tested (X-axis). Each dot represents a measured S2 cell. The error bars indicate the median with the interquartile range. Statistical analysis was performed using the multiple comparison Kruskal-Wallis test (non-parametric) on the PRISM software. The **** indicate p<0.0001 relative to the construct indicated with the longer bar.

Fig 3
Fig 3. Multiple uORFs in iab-8 exon1&2 can repress downstream translation.

A. Summary of the results and constructs. The different constructs tested are schematized with their names indicated on the left. Above the constructs are schematic representations of the two firsts exons of the iab-8 ncRNA with all of the potential ORFs indicated by the directional boxes, color-coded based on their reading frame relative to the beginning of the transcript. The start codons of each ORF are indicated by colored triangles using the color code established for the ORFs. The exon-1&2 region was divided into sub-fragments delineated (Fragments A-F) and labeled in the top panel of A. The (and) represent DNA fragment deletions. The coding sequence of GFP (with the ATG start codon in yellow) is indicated by a solid green rectangle for constructs that express GFP and by a grey hatched box for constructs that did not based on the results displayed in panel B. Kozak sequences are indicated by light grey boxes. B. The graph shows the relative fluorescence measured (arbitrary units, Y-axis) for each of the constructs tested (X-axis). Each dot represents a measured S2 cell. The error bars indicate the median with the interquartile range. Statistical analysis was performed using the multiple comparison Kruskal-Wallis test (non-parametric) on the PRISM software. The **** indicate p<0.0001 relative to the construct indicated with the longer bar.

Fig 4
Fig 4. Context dependent translation from the multiple uORFs in the B and D fragments.

A. A schematic representation of the two firsts exons of the iab-8 transcript with all of the potential start codons indicated by triangles above the exons and stop codons indicated by hanging octagons below the exons. The start and stop codons are color coded as in Fig 3, based on the reading frames relative to the start of the transcript. The Bbig, Bsmall and D fragments are delineated and labeled. B. and C. Summary of the results and constructs. The different constructs tested are schematized with their names indicated on the left of each construct. The potential start and stop codons are indicated with a color code consistent with panel A. The coding sequence of GFP without a start codon was placed in each of the three frames to act as a readout for upstream translation initiation. Above each construct, potential open reading frames that are in frame with the GFP sequence are indicated. Constructs where GFP is expressed show the GFP coding sequence as a solid green rectangle, while constructs that do not show GFP expression are shown as grey hatched rectangles. In one case, an intermediate GFP expression was seen and that is shown as a green hatched rectangle (GFP expression levels are based on results shown in D). D. The graph shows the relative fluorescence measured (arbitrary units, Y-axis) for each of the constructs tested (X-axis). Each dot represents a measured S2 cell. The error bars indicate the median with the interquartile range. Statistical analysis was performed using the multiple comparison Kruskal-Wallis test (non-parametric) on the PRISM software. The **** indicate p<0.0001 relative to the construct indicated with the longer bar.

Fig 5
Fig 5. ORF1 translation represses ORF2 translation.

A. A schematic representation of the first exon of the iab-8 transcript with all of the potential start codons indicated by triangles above the exons and stop codons indicated by hanging octagons below the exons. The start and stop codons are color coded as in Fig 3 based on the reading frames relative to the start of the transcript. The Bsmall and Bsmall+big fragments are delineated and labeled. B. and C. Summary of the results and constructs. The different constructs tested are schematized with their names indicated to the left of each construct. The potential start and stop codons are indicated with a color code consistent with panel A. The coding sequence of GFP without a start codon was placed in frame each of the three frames to act as a readout for upstream translation initiation. Above each construct, open reading frame that are in frame with the GFP sequence are indicated. Constructs where GFP is expressed show the GFP coding sequence as a solid green rectangle, while constructs that do not show GFP expression are shown as grey hatched rectangles. Intermediate GFP expression is shown as a green hatched rectangle (GFP expression levels are based on results shown in Panels D and E).). D. and E. The graphs show the relative fluorescence measured (arbitrary units, Y-axis) for each of the constructs tested (listed along the X-axis). Each dot represents a measured S2 cell. The error bars indicate the median with the interquartile range. Statistical analysis was performed using the multiple comparison Kruskal-Wallis test (non-parametric) on the PRISM software. The **** indicate p<0.0001 relative to the construct indicated with the longer vertical bar on the graph. To compare the two constructs in frame 2 in C, we used the Mann-Whitney test.

Fig 6
Fig 6. uORF repression can be monitored in the context of a complete exon 1 and 2 sequence and in flies.

A. Summary of the results and constructs. The different constructs tested are schematized with their names indicated to the left of each construct. The potential start (triangles) and stop codons (hanging octagons) are indicated with a color code consistent with Fig 3. Mutated start codons (ATG to GCC) are represented by white triangles. The coding sequence of GFP with a start codon and Kozak sequence was placed downstream of the different forms of exons 1 and 2 of the iab-8 RNA. Constructs where GFP is expressed show the GFP coding sequence as a solid green rectangle, while constructs that do not show GFP expression are shown as grey hatched rectangles. B. The graph shows the relative fluorescence measured (arbitrary units, Y-axis) for each of the constructs tested (listed along the X-axis). Each dot represents a measured S2 cell. The error bars indicate the median with the interquartile range. Statistical analysis was performed using the multiple comparison Kruskal-Wallis test (non-parametric) on the PRISM software. The **** indicate p<0.0001 relative to the construct indicated with the longer bar. C. GFP immunohistochemistry (IHC) of the constructs (from panel A), in Drosophila tissues as visualized through confocal microscopy. GFP expression of the three constructs (listed to the left of the images was driven using the D1-Gal4 driver for the secondary cells of the male accessory gland (left) and the en-Gal4 driver for the posterior wing imaginal discs (right). The first column for each set of images shows DAPI staining to delimit the tissue. The second column shows GFP staining. And the third column shows the merged image. Slight differences in tissue shapes is due to experimental artifacts and could not be attributed to a genotype specific effect.

Fig 7
Fig 7. Loss of ORF 1 and 2 may result in a slight derepression of ABD-A in the CNS of Drosophila embryos.

The developing nerve chords were dissected from stage 15 embryos after staining against ABD-A using the goat anti-ABD-A, DH-17 antibody (red) and engrailed as a parasegment marker (green). The location of parasegment 13 is marked on the right of B. A. Mutation removing ORF 1 and 2 (ΔORF1&2). B. Replacement of the wild-type sequence (wt-rescue). A’ and B’ are enlargements of parasegment 13 of A. and B. respectively. Scale bar = 25μm.

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This work was supported by the Canton of Geneva (R.K.M and F.K.), the Swiss National Fund for Research (http://www.snf.ch/Seiten/VariationRoot.aspx) grant 31003A_149634 (to F.K.) and grant 310030_192621 (to R.K.M). and the Georges and Antoine Claraz Foundation Foundation (to F.K. and R.K.M.). Salaries for R.K.M. and F.K. were paid by the Canton of Geneva. The salaries of Y.F. and C.I. were paid by the Swiss National Fund for Research (http://www.snf.ch/Seiten/VariationRoot.aspx) grant 31003A_149634 (to F.K. and R.K.M.). The salary of M.R. was paid by the Swiss National Fund for Research (http://www.snf.ch/Seiten/VariationRoot.aspx) grant 310030_192621 (to R.K.M). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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