Regulation of <i>Rpl22l1</i> mRNA expression is mediated by a hairpin structure.

<p>(<b>A</b>) Schematic representation of the biosensor quantification assay. (<b>B–D</b>) Stereoimages of zebrafish embryos illustrate that co-injection of zRpl22 repressed fluorescence derived from an EGFP-Rpl22l1 fusion protein upon injecting mRNA for both and assessing fluorescence at 6 hours post fertilization. Rpl22, Rpl22l1 or mutated Rpl22l1 (Rpl22l1mt) coding sequence was fused to <i>EGFP</i> mRNA and co-injected with <i>mCherry</i> mRNA (injection control) along with the corresponding inhibitor mRNAs (<i>zRpl22</i> or <i>zRpl22l1</i>) into 1-cell stage zebrafish embryos. (<b>F</b>) Schematic representation of the experimental procedure. A <i>zRpl22l1-150h-EGFP</i> heterologous reporter mRNA, containing the minimal sequence identified by mFOLD to form the hairpin structure, was co-injected with <i>mCherry</i> mRNA (injection control) and (<b>G</b>) <i>Rpl22</i> mRNA or (<b>I</b>) Rpl22-Morpholino (Rpl22-MO) into 1-cell stage zebrafish embryos. (<b>E, H, J</b>) At 10 hpf, the relative fluorescence intensity was calculated and normalized to control injections (n = 3, each group). Data are shown as mean ± standard deviation (s.d.).</p

Patterns of GFP expression in <i>GBT-B4</i> gene trap lines that include hematopoietic tissues.

<p>Lateral views of embryos at 48 hpf or 6 dpf are shown. The <i>Tg(GBT-B4)fcc</i> line number is indicated to the left of the panels. The embryo age is indicated. CHT = caudal hematopoietic tissue; AGM = aorta-gonad-mesonephros. Note that the lines are grouped by hematopoietic expression patterns, but the embryos can express GFP in additional tissues.</p

The <i>GBT-B4</i> genetic screen approach and results.

<p>(A) Design features of the <i>GBT-B4</i> gene trap and the nucleotide sequence of the 14XUAS used in the parental <i>GBT-B1</i> and the 4.5xUAS syUAS engineered of <i>GBT-B4</i>. The consensus Gal4 binding site based on the 14XUAS is in red bold letters, sequences based on the Saccharomyces UAS are in bold green. The core Gal4 binding site CGGN₁₁CCG is highlighted in yellow. The non-consensus nucleotide in the 4^th binding site of the syUAS is in lower case (t instead of G). (B) Diagram of the genetic screen; arrows indicate the workflow. (C) Summary of the screen results.</p

Decreased expression of lymphoid markers in gene-trap mutant embryos.

<p>(A-F) Whole mount RNA in situ hybridization (WISH) of <i>lck</i> in 5 dpf larvae from the lines <i>fcc143</i> (143) (A-B), <i>agtpbp1</i>^{<i>fcc301</i>} (301) (C-D) and <i>eps15L1</i>^{<i>fcc436-P1</i>} (436) (E-F). Representative embryos displaying no GFP (A, E) or normal expression of <i>lck</i> (C) are compared to siblings with strong (str) GFP (B, F) or low levels of <i>lck</i> expression (D). In line <i>agtpbp1</i>^{<i>fcc301</i>}, the GFP expression levels varied, making it difficult to distinguish heterozygous and homozygous carriers. The number of embryos that showed the representative phenotype is indicated in panels C and D.</p

Rpl22 directly binds <i>Rpl22l1</i> mRNA to regulate its expression levels.

<p>(<b>A</b>) In the absence of Rpl22, <i>Rpl22l1</i> mRNA levels are more stable in the presence of Actinomycin D. Rpl22+/+ or Rpl22−/− 3T9 cells were treated with Actinomycin D (1 µM final concentration) and total RNA was harvested at the time points shown. Levels of <i>Rpl22l1</i> mRNA were quantitated by qRT-PCR. Results are the average ± SEM of 3 independent experiments and the statistical significance indicated is (*, p<0.01, compared to <i>Rpl22+/+</i> untreated; ** p<0.001, compared to <i>Rpl22+/+</i> at each time point). (<b>B</b>) M-fold analysis <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003708#pgen.1003708-Zuker1" target="_blank">[54]</a> of <i>zRpl22l1</i> mRNA reveals the presence of a consensus Rpl22 RNA-binding motif. In green are the residues deleted to remove the hairpin (<i>zRpl22l1Δhp</i>). In blue are the residues known to be essential for Rpl22 binding. (<b>C</b>) Autoradiogram of ribonuclease protection assay reveals Rpl22 protein binds to <i>Rpl22l1</i> mRNA and this binding is abrogated upon removal of the hairpin. 32P labeled <i>EBER1</i> (positive control), <i>EBER 2</i> (negative control), <i>zRpl22l1</i> or <i>zRpl22l1Δhp</i> RNAs were incubated in the absence or presence of GST-Rpl22 (41.7 kDa), GST (27 kDa) or m88, a GST-Rpl22 RNA binding mutant (41.6 kDa), as indicated, then UV-cross-linked, digested with RNase A, and run on a SDS protein gel. GST-Rpl22 was detected, hence, bound to <i>EBER1</i> and <i>zRpl22l1</i> RNAs but not <i>Rpl22l1Δhp</i> RNA, indicating Rpl22 binds to <i>Rpl22l1</i> mRNA and this binding is abrogated upon removal of the hairpin. Numbers indicate molecular weight protein ladder in kDa.</p

Summary of lines with embryonic marking of hematopoietic tissue and identification of the disrupted genes.

<p>*<i>GBT-B4</i> = <i>Gt(LOXP-GAL4-VP16-FRT</i>, <i>syUAS</i>:<i>EGFP-FRT-LOXP)</i></p><p>Summary of lines with embryonic marking of hematopoietic tissue and identification of the disrupted genes.</p

Mouse Rpl22 has an expressed paralog, Rpl22l1.

<p>(<b>A</b>) Alignment of Rpl22 and Rpl22l1 protein sequences. Lung, liver, spleen, kidney and pancreas, harvested from <i>Rpl22^−/−</i> mice and their littermate controls, were analyzed for relative <i>Rpl22</i> and <i>Rpl22l1</i> mRNA levels by qRT-PCR (<b>B</b>) or protein expression by Western blot analysis (<b>C</b>). Results are representative of 3 independent experiments.</p

Both mouse Rpl22 and Rpl22l1 proteins can be incorporated into ribosomes.

<p>Liver tissue was isolated from <i>Rpl22^+/+</i> (<b>A, C</b>) and <i>Rpl22^−/−</i> (<b>B, D</b>) mice then, after sedimentation of the lysates on sucrose gradients, fractions were collected and loaded onto an SDS-page gel for western blot analysis (<b>C</b> and <b>D</b>, respectively). Images are representative of 3 independent experiments. Multiple Reaction Monitoring Mass spectrometry (MRM-MS) analysis was performed on free 60S subunits and 80S monosomes from actively translating polysomes. Liver lysates from <i>Rpl22^+/+</i> and <i>Rpl22^−/−</i> mice were subjected to a brief treatment with low amounts of RNase A to degrade mRNA between ribosomes in polysomes and release the ribosomes as 80S monomers. After inhibiting the RNase with KCl and heparin, the samples were fractionated on 10–30% sucrose gradients containing 800 mM KCl to disrupt any “nonproductive couples” of 40S and 60S subunits <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003708#pgen.1003708-Martin1" target="_blank">[79]</a>. (<b>E, F</b>) Representative gradient profiles for Rpl22+/+ and Rpl22−/−samples, respectively. Fraction 6 was used to isolate 60S subunits for MRM-MS, while fraction 7 was used to isolate 80S monosomes. Summation of the integrated MRM peak areas for all transitions from all observed peptides for Rpl22 (<b>G</b>) and Rpl22l1 (<b>H</b>) proteins yielded the total MRM peak areas plotted for each of the four tested samples (WT60S, WT80S, KO60S, KO80S), indicating the relative amounts of Rpl22 and Rpl22l1 in these samples. The height of each bar represents the average of the three technical replicates performed for the given sample, and each error bar represents +/−1 standard deviation. p-values>3E-3 in both cases by paired student's t-test. Rpl22 peptides: AGNLGGGVVTIER; ITVTSEVPFSK; YFQINQDEEEEEDED. Rpl22l1 peptides: TGNLGNVVHIER; ITVVSEK.</p

Acute knockdown of Rpl22 enhances Rpl22l1 expression.

<p>3T9 cells were transduced with doxycycline-inducible shRNA lentiviral constructs directed at <i>Rpl22</i> (shRNA 1 and shRNA 2) or a non-specific (ns) shRNA construct and treated with doxycycline for 3 days to induce shRNA expression. NS, shRNA 1, or shRNA 2 expressing cells were analyzed for relative <i>Rpl22</i> and <i>Rpl22l1</i> mRNA levels by qRT-PCR (<b>A</b>) or protein expression by Western blot analysis (<b>B</b>). Results are the average ± SEM of 4 independent experiments (<b>A</b>) or representative of 3 independent experiments (<b>B</b>). Statistical significance is indicated (*; p<0.001 compared to untreated control).</p

Morpholino knockdown of <i>agtpbp1</i> and <i>eps15L1</i> results in decreased lymphoid <i>rag1</i> expression.

<p>(A-B) WISH of <i>rag1</i> in 4 dpf control (A) and <i>agtpbp1</i> (B) morpholino-injected embryos. (C) RT-PCR of <i>agtpbp1</i> and <i>ß-actin</i> in pooled control or morphant embryo samples. Quantitation of the normal transcript band normalized to <i>ß-actin</i> is indicated. (D-E) WISH of <i>rag1</i> in 4 dpf control (A) and <i>eps15L1</i> (B) morpholino-injected embryos. (C) RT-PCR of <i>eps15L1</i> and <i>ß-actin</i> in pooled control or morphant embryo samples. Quantitation of the normal transcript band normalized to <i>ß-actin</i> is indicated. Quantitation is in arbitrary units (A.U.), and relative to wild-type level which is set at 1. Head region of the embryos is shown in lateral views, anterior to the left. P values were determined using Fisher’s exact test.</p