Community effects in regulation of translation

Certain forms of translational regulation, and translation itself, rely on long-range interactions between proteins bound to the different ends of mRNAs. A widespread assumption is that such interactions occur only in cis, between the two ends of a single transcript. However, certain translational regulatory defects of the Drosophila oskar (osk) mRNA can be rescued in trans. We proposed that inter-transcript interactions, promoted by assembly of the mRNAs in particles, allow regulatory elements to act in trans. Here we confirm predictions of that model and show that disruption of PTB-dependent particle assembly inhibits rescue in trans. Communication between transcripts is not limited to different osk mRNAs, as regulation imposed by cis-acting elements embedded in the osk mRNA spreads to gurken mRNA. We conclude that community effects exist in translational regulation

Multiomic Profiling Identifies cis-Regulatory Networks Underlying Human Pancreatic β Cell Identity and Function.

EndoC-βH1 is emerging as a critical human β cell model to study the genetic and environmental etiologies of β cell (dys)function and diabetes. Comprehensive knowledge of its molecular landscape is lacking, yet required, for effective use of this model. Here, we report chromosomal (spectral karyotyping), genetic (genotyping), epigenomic (ChIP-seq and ATAC-seq), chromatin interaction (Hi-C and Pol2 ChIA-PET), and transcriptomic (RNA-seq and miRNA-seq) maps of EndoC-βH1. Analyses of these maps define known (e.g., PDX1 and ISL1) and putative (e.g., PCSK1 and mir-375) β cell-specific transcriptional cis-regulatory networks and identify allelic effects on cis-regulatory element use. Importantly, comparison with maps generated in primary human islets and/or β cells indicates preservation of chromatin looping but also highlights chromosomal aberrations and fetal genomic signatures in EndoC-βH1. Together, these maps, and a web application we created for their exploration, provide important tools for the design of experiments to probe and manipulate the genetic programs governing β cell identity and (dys)function in diabetes

Translational activation of oskar mRNA: reevaluation of the role and importance of a 5' regulatory element [corrected].

Local translation of oskar (osk) mRNA at the posterior pole of the Drosophila oocyte is essential for axial patterning of the embryo, and is achieved by a program of translational repression, mRNA localization, and translational activation. Multiple forms of repression are used to prevent Oskar protein from accumulating at sites other than the oocyte posterior. Activation is mediated by several types of cis-acting elements, which presumably control different forms of activation. We characterize a 5' element, positioned in the coding region for the Long Osk isoform and in the extended 5' UTR for translation of the Short Osk isoform. This element was previously thought to be essential for osk mRNA translation, with a role in posterior-specific release from repression. From our work, which includes assays which separate the effects of mutations on RNA regulatory elements and protein coding capacity, we find that the element is not essential, and conclude that there is no evidence supporting a role for the element only at the posterior of the oocyte. The 5' element has a redundant role, and is only required when Long Osk is not translated from the same mRNA. Mutations in the element do disrupt the anchoring function of Long Osk protein through their effects on the amino acid sequence, a confounding influence on interpretation of previous experiments

Community effects in regulation of translation

Abstract Certain forms of translational regulation, and translation itself, rely on long-range interactions between proteins bound to the different ends of mRNAs. A widespread assumption is that such interactions occur only in cis, between the two ends of a single transcript. However, certain translational regulatory defects of the Drosophila oskar (osk) mRNA can be rescued in trans. We proposed that inter-transcript interactions, promoted by assembly of the mRNAs in particles, allow regulatory elements to act in trans. Here we confirm predictions of that model and show that disruption of PTB-dependent particle assembly inhibits rescue in trans. Communication between transcripts is not limited to different osk mRNAs, as regulation imposed by cis-acting elements embedded in the osk mRNA spreads to gurken mRNA. We conclude that community effects exist in translational regulation

Sequence conservation in the 5' region of the <i>osk</i> gene.

<p>The diagram at top shows the 5' region of <i>osk</i>, with the extended 5' UTR as a black line and the <i>osk</i> coding region as a rectangle. The AUG start codons for Long and Short Osk are shown, and the region containing the 5' activation element is shaded. The two analyses of conservation are shown below, with the phastCons output above and the consecCons output below, as indicated. For the latter, each vertical line indicates the presence of 2 consecutive positions that are perfectly conserved among the species analyzed (Methods and Materials). At bottom are segments of the <i>osk</i> sequence showing the short regions most highly conserved in the extended 5' UTR. Within the coding region, codons are indicated by spacing, and perfectly conserved positions are identified with asterisks. Endpoints of the indicated deletion mutations are marked.</p

The 5' element is required for the early phase of Osk protein accumulation.

<p>A-B,A'-B'. Detection of transgenic OskHA protein expressed from single copies of the indicated transgenes in the <i>osk</i>^<i>A87</i><i>/Df(3R)osk</i> background (RNA null). Panels A'-B' are magnified views of the posterior of the oocyte to better show the proteins. Green is OskHA and red is DNA detected with ToPro-3. C-J,C'-J'. Detection of transgenic Short OskHA protein expressed from single copies of the indicated transgenes in the presence of endogenous Long Osk for anchoring. Panels C'-J' are magnified views of the posterior of the oocyte to better show the proteins. I. Quantification of protein levels from the imaging experiments of C-J. OskHA signal intensities (Methods and Materials) are shown normalized to that from the <i>oskHA</i> transgene. The number of oocytes scored is indicated at the bottom of each bar. Error bars indicate standard error. J. Western blot analysis of transgenes expressed as single copies in the presence of a wild type copy of <i>osk</i>. Only the transgenic Osk protein is detected using anti-HA antibodies. Tubulin is detected as a loading control.</p

Effects of mutating the 5' element on translation and Long Osk function.

<p>A. Diagram of the 5' region of the transgene mRNAs, using the conventions from Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125849#pone.0125849.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125849#pone.0125849.g002" target="_blank">2</a> but with the deletions marked by brackets and gaps. B. Western blot analysis of transgenes expressed as single copies in the <i>osk</i>^<i>A87</i><i>/Df(3R)osk</i> background. C. Patterning activity of <i>osk</i> transgenes, tested as single copies in the <i>osk</i>^<i>A87</i><i>/Df(3R)osk</i> background (RNA null). The number of abdominal segments corresponds to the level of <i>osk</i> activity, with wild type embryos having eight. n values were <i>oskHA</i>, 511; <i>oskHA ∆121–150</i>, 260; <i>oskM1RHA ∆121–150</i>, 324. D. Levels of <i>osk</i> mRNA produced from a single copy of the indicated transgenes. All values are normalized against the level of mRNA from a single copy of the <i>oskHA</i> transgene. Levels of <i>rp49</i> were monitored to normalize for amount of RNA used in each assay. Error bars indicate standard error. E. Detection of transgenic OskHA expressed from single copies of the indicated transgenes. For the panels at left, the transgenes were tested in the <i>osk</i>^<i>A87</i><i>/Df(3R)osk</i> background, revealing the anchoring defect of the OskHA∆121–150 mutant, which lacks aa 36–45. This defect is rescued when coexpressed with wild type Long Osk, as shown in the panels at right. F-G. The amino terminal domain of Osk confers anchoring on GFP. F shows the distribution of GFP, and G shows the distribution of the Osk::GFP fusion protein from transgene <i>UAS-osk1-534</i>::<i>GFP</i>, which includes the first 534 bp of the <i>osk</i> mRNA and encodes a fusion protein with the first 173 amino acids of Osk, including the entire Long Osk amino terminal domain. The fusion protein is highly enriched at the oocyte cortex and at nurse cell boundaries. White boxes outline the types of regions shown in panels H,J,L (solid lines) and I,K,M (dashed lines), although these are not the same egg chambers as in those panels. Green is GFP (or Osk::GFP), red is DNA (nuclei) stained with ToPro-3. H-M. Detection of Osk::GFP fusion proteins in stage 10 egg chambers. All panels are at higher magnification than in F and G to highlight anchoring at the oocyte cortex (H,J,L) or at nurse cell boundaries (I,K,M)(the scale bar is 5 μm). For panels I, K, M and M' the images show a portions of several nurse cells and the boundaries between them. Signal intensities can only be compared between panels J-M, which were imaged under identical conditions. Panels L' and M' are identical to L and M except that the green signal was enhanced to better show the absence of any anchoring. The level of protein from the <i>UAS-GFP</i> transgene is much higher than from the <i>UAS-osk1-534</i>::<i>GFP</i> transgenes, and lower intensity laser settings were used to obtain images in F, H and I with signal intensity comparable to G, J and K. Anchoring of the Osk::GFP protein is manifested in the enrichment at the cortex, along nurse cell boundaries, and the punctate appearance in the cytoplasm. Neither GFP alone nor the Osk INV121-182::GFP protein shows any similar anchoring. Green is GFP (or Osk::GFP), red is DNA stained with ToPro-3.</p

The 5' element is required for translational activation.

<p>A and B. Western blot analysis of transgenes expressed as single copies in the <i>osk</i>^<i>A87</i><i>/Df(3R)osk</i> background. Tubulin is detected as a loading control. C. Diagram of the <i>oskM1R</i> 5' region, showing the positions of the two iORFs and how the ∆311–360 deletion fuses iORF2 to the Osk reading frame, and thus can produce the novel protein band detected in A. The partial sequence shown has the reading frame for Osk protein indicated by spaces, and the reading frame for iORF2 indicated by vertical hash marks. D-G. In situ hybridization to detect transgene mRNAs in the <i>osk</i>^<i>A87</i><i>/Df(3R)osk</i> background (panels D'-G' are magnified views of the posterior region to better show the mRNA distributions). For the <i>oskHA</i> transgene, which makes both Long and Short Osk, the mRNA is tightly restricted to a posterior crescent (D,D'). The <i>oskM1R</i> transgene lacks Long Osk and its anchoring function, and the mRNA has a more punctate distribution (E,E'). Similarly, both of the mutants tested, one with normal <i>osk</i> activity (F,F'; the ∆61–90 deletion) and one largely lacking <i>osk</i> activity (G,G'; the ∆118–135 deletion), have the same punctate distribution of mRNA.</p