Quantifying the Effect of Ribosomal Density on mRNA Stability

<div><p>Gene expression is a fundamental cellular process by which proteins are eventually synthesized based on the information coded in the genes. This process includes four major steps: transcription of the DNA segment corresponding to a gene to mRNA molecules, the degradation of the mRNA molecules, the translation of mRNA molecules to proteins by the ribosome and the degradation of the proteins. We present an innovative quantitative study of the interaction between the gene translation stage and the mRNA degradation stage using large scale genomic data of <i>S. cerevisiae</i>, which include measurements of mRNA levels, mRNA half-lives, ribosomal densities and protein abundances, for thousands of genes. The reported results support the conjecture that transcripts with higher ribosomal density, which is related to the translation stage, tend to have elevated half-lives, and we suggest a novel quantitative estimation of the strength of this relation. Specifically, we show that on average, an increase of <i>78%</i> in ribosomal density yields an increase of <i>25%</i> in mRNA half-life, and that this relation between ribosomal density and mRNA half-life is not function specific. In addition, our analyses demonstrate that ribosomal density along the entire ORF, and not in specific locations, has a significant effect on the transcript half-life. Finally, we show that the reported relation cannot be explained by different expression levels among genes. A plausible explanation for the reported results is that ribosomes tend to protect the mRNA molecules from the exosome complexes degrading them; however, additional non-mutually exclusive possible explanations for the reported relation and experiments for their verifications are discussed in the paper.</p></div

Biological process GO: RD profile at single nucleotide resolution, of the first <i>600</i> nts when all genes are aligned to the ORF's 5′ end

<p>: <b>A</b>. The RD median of the ORF's 5′ end RD profiles: the red/green bars represent the RD median of the <i>20%</i> of the genes with top/bottom half-life. <b>B</b>. Wilcoxon rank sum test between the RD profiles of the genes from the top and bottom <i>20%</i> half-life for different functional genes groups. The x-axis represents the log (Wilcoxon test P-value) and the y-axis represents the functional genes group (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102308#pone.0102308.s016" target="_blank">Table S5</a>). Red bars indicate that <i>P-value ≤0.05</i> whereas green bars indicate that <i>P-value >0.05</i>.</p

Biological process GO: RD profile at single nucleotide resolution, of the last <i>600</i> nts when all genes are aligned to the ORFs 3′ end

<p>: <b>A</b>. The RD median of the ORF's 3′ end RD profiles: the red/green bars represent the RD median of the <i>20%</i> of the genes with top/bottom half-life. B. Wilcoxon rank sum test between the ribosomal densities profiles of the genes from the top and bottom <i>20%</i> half-life for different functional genes groups. The x-axes represent the log (Wilcoxon test P-value) and the y-axes represent the functional genes group (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102308#pone.0102308.s016" target="_blank">Table S5</a>). Red bars indicate the <i>P-value ≤0.05</i> whereas green bars indicate the <i>P-value >0.05</i>.</p

Description of the ribosomal profiling approach

<p>: <b>A</b>. Cells are treated with cycloheximide (for example) to arrest translating ribosomes; <b>B</b>. RNA fragments that are protected from RNases by the ribosomes are isolated and <b>C</b>. processed for Illumina high-throughput sequencing. <b>D</b>. The next steps are computational – reads are mapped to the ORFs of the analyzed organism. Ribosomal footprint reads of a certain codon are generated when the codon is covered by ribosomes. Thus, highly translated genes tend to create a higher number of reads.</p

mRNA half-life distributions.

<p>Half-life distributions of the genes from the bottom <i>20%</i> RD (green curve), top <i>20%</i> RD (red curve), and of all genes (blue curve). The inset in each plot includes the median of each curve, which is represented by the intersection with the x-axis of a vertical line with the appropriate color: green, red and blue lines that indicate the half-life medians of the genes from the bottom and top <i>20%</i> RD and of all genes respectively; for a better visualization, the graphs are based on the log (mRNA HL) values. The number above the arrow in each inset is the P-value corresponding to the Wilcoxon rank sum test between the mRNA HL of genes with the top and bottom <i>20%</i> RD. <b>A</b>. Half-life distributions at the ORF's 5′ end according to the genes RD average of the first <i>50</i> nucleotides downstream the ORF's 5′ end. <b>B</b>. Half-life distributions at the ORF's 3′ end according to the genes RD average of the last <i>50</i> nucleotides upstream the ORF's 3′ end. <b>C</b>. Half-life distributions according to the genes total average of the ORF's RD.</p

Ribosomal density versus mRNA half-life given the protein abundance (binned data; details in Materials and Methods).

<p>The RD were calculated as: 1) the number of ribosomes on the mRNA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102308#pone.0102308-Arava1" target="_blank">[30]</a> divided by its length, 2) averaging the ribosomal profiling data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102308#pone.0102308-Ingolia1" target="_blank">[29]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102308#pone.0102308-Brar1" target="_blank">[32]</a> over the entire ORF, 3) the ORF's 5′ end –the first 50 nt and 4) the ORF's 3′ end – the last 50 nt.</p

Wilcoxon rank sum test between mRNA half-lives of genes with top and bottom <i>20%</i> RD for each <i>40</i> nts sliding window.

<p><b>A</b>. The ORF's first <i>600</i> nts; genes are aligned to the ORF's 5′ end. <b>B</b>. The ORF's last <i>600</i> nts; genes are aligned to the ORF's 3′ end. The x-axes represent the location of the sliding window downstream to the aligned ORF's 5′ end (positive numbers at <b>A</b>.) and upstream to the aligned ORF's 3′ end (negative number at <b>B</b>.) respectively; the y-axes represent the log (Wilcoxon test P-value); the black line indicates that <i>P-value  = 0.05</i>; the blue line indicates that <i>P-value  = 0.001</i>. The green cross indicates that there is no significant difference between the half-life medians of the two groups; the red cross indicates that the half-life median of the genes group with higher RD is significantly higher than the one of the genes group with lower RD (<i>P-value ≤0.05)</i>. There were no positions with significant signal in the opposite direction (i.e. genes with higher RD that have significantly lower HL).</p

Ribosomal density profile for highly translated genes at single nucleotide resolution.

<p><b>A</b>. The first <i>600</i> nts; all genes are aligned to the ORF's 5′ end. <b>B</b>. The last <i>600</i> nts; all genes are aligned to the ORF's 3′ end. The y-axes represent the mean RD in logarithmic scale at specific locations along the ORF; the x-axes represent the location of a nucleotide measured as the distance from the ORF's 5′ end (positive numbers at <b>A</b>.) or distance from the ORF's 3′ end (negative number at <b>B</b>.). The red line describes the <i>20%</i> of genes with the longest mRNA half-life; the green line describes the <i>20%</i> of genes with the shortest mRNA half-life, and the blue line describes all the genes. The inset in each plot includes the RD median for each group of genes, from left to right: the <i>20%</i> of the genes with the shortest half-life, all genes, and the <i>20%</i> of the genes with the longest half-life. The number above the arrow is the P-value corresponding to the Wilcoxon rank sum test between the local averaged RD of genes with the <i>20%</i> longest and shortest half-life.</p

Retinoic Acid is Required for Normal Morphogenetic Movements During Gastrulation

Retinoic acid (RA) is a central regulatory signal that controls numerous developmental processes in vertebrate embryos. Although activation of expression is considered one of the earliest functions of RA signaling in the embryo, there is evidence that embryos are poised to initiate RA signaling just before gastrulation begins, and manipulations of the RA pathway have been reported to show gastrulation defects. However, which aspects of gastrulation are affected have not been explored in detail. We previously showed that partial inhibition of RA biosynthesis causes a delay in the rostral migration of some of the earliest involuting cells, the leading edge mesendoderm (LEM) and the prechordal mesoderm (PCM). Here we identify several detrimental gastrulation defects resulting from inhibiting RA biosynthesis by three different treatments. RA reduction causes a delay in the progression through gastrulation as well as the rostral migration of the -positive PCM cells. RA inhibition also hampered the elongation of explanted dorsal marginal zones, the compaction of the blastocoel, and the length of Brachet\u27s cleft, all of which indicate an effect on LEM/PCM migration. The cellular mechanisms underlying this deficit were shown to include a reduced deposition of fibronectin along Brachet\u27s cleft, the substrate for their migration, as well as impaired separation of the blastocoel roof and involuting mesoderm, which is important for the formation of Brachet\u27s cleft and successful LEM/PCM migration. We further show reduced non-canonical Wnt signaling activity and altered expression of genes in the Ephrin and PDGF signaling pathways, both of which are required for the rostral migration of the LEM/PCM, following RA reduction. Together, these experiments demonstrate that RA signaling performs a very early function critical for the progression of gastrulation morphogenetic movements

Natural size variation among embryos leads to the corresponding scaling in gene expression

© 2020 The Authors Xenopus laevis frogs from laboratory stocks normally lay eggs exhibiting extensive size variability. We find that these initial size differences subsequently affect the size of the embryos prior to the onset of growth, and the size of tadpoles during the growth period. Even though these tadpoles differ in size, their tissues, organs, and structures always seem to be properly proportioned, i.e. they display static allometry. Initial axial patterning events in Xenopus occur in a spherical embryo, allowing easy documentation of their size-dependent features. We examined the size distribution of early Xenopus laevis embryos and measured diameters that differed by about 38% with a median of about 1.43 ​mm. This range of embryo sizes corresponds to about a 1.9-fold difference in surface area and a 2.6-fold difference in volume. We examined the relationship between embryo size and gene expression and observed a significant correlation between diameter and RNA content during gastrula stages. In addition, we investigated the expression levels of genes that pattern the mesoderm, induce the nervous system and mediate the progression of ectodermal cells to neural precursors in large and small embryos. We found that most of these factors were expressed at levels that scaled with the different embryo sizes and total embryo RNA content. In agreement with the changes in transcript levels, the expression domains in larger embryos increased proportionally with the increase in surface area, maintaining their relative expression domain size in relation to the total size of the embryo. Thus, our study identified a mechanism for adapting gene expression domains to embryo size by adjusting the transcript levels of the genes regulating mesoderm induction and patterning. In the neural plate, besides the scaling of the expression domains, we observed similar cell sizes and cell densities in small and large embryos suggesting that additional cell divisions took place in large embryos to compensate for the increased size. Our results show in detail the size variability among Xenopus laevis embryos and the transcriptional adaptation to scale gene expression with size. The observations further support the involvement of BMP/ADMP signaling in the scaling process