Translational repression and expression of high confidence targets.

<p>(A) Ratio of polysome to mRNP occupancy across genotypes as compared to wildtype demonstrating substantially higher polysome occupancy in compound heterozygotes when compared to single heterozygotes. Data represented as average across biological duplicates. (B) Expression of transcripts with altered mRNP and polysome occupancy compared across genotypes demonstrating minimal change in overall expression demonstrating altered polysome occupancy is not driven by differences in total expression. (C) Cell-type expression of high confidence targets demonstrating enriched expression in YBX2 and 3 positive cell types. Expression—log₂(average TPM). Black dot—median value. (D) Polysome to mRNP ratio of transcripts relevant to spermatogenesis showing increased polysome occupancy in compound heterozygotes relative to wildtype. Note the only transcript with a well-characterized YRS is <i>Spata18</i>. Data is represented as the average polysome to mRNP ratio ± SD. (E) Western blot detection of MAEL, NSUN2, and GAPDH in whole adult testis protein lysates from wildtype, single heterozygote, and compound heterozygous mutants demonstrating increase MAEL and NSUN2, but not GAPDH, abundance in the compound heterozygous mutant testis relative to wildtype.</p

Reproductive parameters of compound heterozygous mutant mice.

<p>(A) Body weight and (B) testis weight remained unchanged, while (C) epididymal sperm count is significantly decreased in compound heterozygous relative to single heterozygous (<i>Ybx2</i>^+/- or <i>Ybx3</i> ^+/-) or wildtype mice. Values are mean ± SD, N = 3, * indicates p < 0.05. Fertility assessed by (D) embryos produced from mating with C3H/HeJ fertile females and sperm function quantified by (E) motile sperm and (F) progressive sperm motility assessed by computer-assisted sperm analysis demonstrating defects in sperm motility in compound heterozygotes. The data are expressed as percentage of motile sperm ± SD. ** p < 0.01 relative to wildtype sperm. N = 3. (G) 2-cell embryos derived from <i>in vitro</i> fertilization (IVF) of oocytes from superovulated C3H/HeJ females demonstrating infertility in compound but not single heterozygous males. The data are expressed as number of embryos ± SD per dam or percentage ± SD of 2-cell embryos. N = 3, *** p < 0.001.</p

Testis transcript mRNP and polysome occupancy in single and compound heterozygotes.

<p>(A) Comparison of mutant versus wildtype mRNP or polysome abundance of expressed transcripts demonstrating global decreases in mRNP abundance and global increases in polysome abundance in compound heterozygotes but not single heterozygotes relative to wildtype. Data represented as mRNP or polysome abundance normalized by total RNA abundance and averaged across biological replicates. Red points—transcripts with significant differences between wildtype and mutant (t-test, N = 2, p-value < 0.05) within either the mRNP or polysome fractions. Yellow points—transcripts with significant differences between wildtype and mutant in both mRNP and polysome fractions. (B) Intersect of transcripts with differential mRNP or polysome occupancy in the three mutant models demonstrating limited overlap between the single and compound heterozygotes. (C) Intersect of transcripts with differential mRNP and polysome occupancy in compound heterozygotes. Transcripts within the overlap represent high confidence targets for abnormal translation repression.</p

Morphological analysis of spermatogenesis and sperm chromatin condensation in compound heterozygous mutant mice.

<p>(A) Periodic acid–Schiff–stained sections of adult testis shows tubule cross-sections containing abnormal elongating spermatids (asterisks). Step 9 spermatids demonstrate defective elongation and abnormal condensation within tubule cross-sections (arrowheads). (B) The presence of single stranded DNA indicative of DNA damage was evaluated by sperm acridine orange staining. In compound heterozygous mutant sperm acridine orange fluoresced in a manner consistent with single-stranded DNA. Quantification of acridine orange–stained sperm demonstrating increases in compound heterozygous relative to wildtype sperm. Values expressed as a percentage ± SD. N = 3. p = 0.052. (C). Western blot analysis confirming the presence of PRM2* in compound heterozygous mutant sperm, while mature PRM2 levels were decreased (Lane 4). A representative blot is shown (N = 3). PRM1 levels remained unchanged (Lower panel). (D) Quantitation of the PRM2*/PRM2 ratio by densitometric scan of the PRM2 western blot revealed a significant increase in PRM2*/PRM2 ratio in compound heterozygous mutant relative to wildtype or <i>Ybx2</i>^+/- or <i>Ybx3</i>^+/- mutants. p< 0.05, N = 3.</p

YRS-independent regulation in compound heterozygote testes.

<p>(A) RNA-sequencing derived polysome to mRNP ratio of known YRS-containing transcripts across wildtype and heterozygote testes demonstrating no impact on polysome occupancy in compound heterozygotes. Data is represented as the average polysome to mRNP ratio ± SD. (B) Stage specific expression of PRM1 and (C) PRM2 in elongating spermatids (arrowhead) in wildtype and compound heterozygous testis. <i>Prm1</i> and <i>Prm2</i> mRNAs are not prematurely translated in compound heterozygous mutant mice as shown by the absence of PRM1 staining in stage IX (step 9 elongating spermatids). Scale bar = 100 μm. egs: elongating spermatid. (D) Significance of degenerate YRS motifs identified in the 3’ UTR of transcripts with altered mRNP abundance across genotypes. Comparison with known YRS-containing UTRs demonstrates substantial under-representation of high confidence YRSs in mis-regulated transcripts.</p

Image2_A-Kinase Anchor Protein 1 deficiency causes mitochondrial dysfunction in mouse model of hyperoxia induced acute lung injury.TIFF

Background: Critically ill patients on supplemental oxygen therapy eventually develop acute lung injury (ALI). Reactive oxygen species (ROS) produced during ALI perturbs the mitochondrial dynamics resulting in cellular damage. Genetic deletion of the mitochondrial A-kinase anchoring protein 1 (Akap1) in mice resulted in mitochondrial damage, Endoplasmic reticulum (ER) stress, increased expression of mitophagy proteins and pro-inflammatory cytokines, exacerbating hyperoxia-induced Acute Lung Injury (HALI).Objective: Despite a strong causal link between mitochondrial dysfunction and HALI, the mechanisms governing the disease progression at the transcriptome level is unknown.Methods: In this study, RNA sequencing (RNA-seq) analysis was carried out using the lungs of Akap1 knockout (Akap1−/−) mice exposed to normoxia or 48 h of hyperoxia followed by quantitative real time PCR and Ingenuity pathway analysis (IPA). Western blot analysis assessed mitochondrial dysfunction, OXPHOS complex (I-V), apoptosis and antioxidant proteins. Mitochondrial enzymatic assays was used to measure the aconitase, fumarase, citrate synthase activities in isolated mitochondria from Akap1−/− vs. Wt mice exposed to hyperoxia.Results: Transcriptome analysis of Akap1−/− exposed to hyperoxia reveals increases in transcripts encoding electron transport chain (ETC) and tricarboxylic acid cycle (TCA) proteins. Ingenuity pathway analysis (IPA) shows enrichment of mitochondrial dysfunction and oxidative phosphorylation in Akap1−/− mice. Loss of AKAP1, coupled with oxidant injury, significantly decreases the activities of TCA enzymes. Mechanistically, a significant loss of dynamin-related protein 1 (Drp1) phosphorylation at the protein kinase A (PKA) site Serine 637 (Ser637), decreases in Akt phosphorylation at Serine 437 (Ser47) and increase in the expression of pro-apoptotic protein Bax indicate mitochondrial dysfunction. Heme oxygenase-1 (HO-1) levels significantly increased in CD68 positive alveolar macrophages in Akap1−/− lungs, suggesting a strong antioxidant response to hyperoxia.Conclusion: Overall these results suggest that AKAP1 overexpression and modulation of Drp1 phosphorylation at Ser637 is an important therapeutic strategy for acute lung injury.</p

Primers for qRT-PCR.

<p>Primers for qRT-PCR.</p

qRT-PCR analysis of RPE and retinal- specific genes in homozygous <i>Mfrp^rd6</i>, <i>Tulp1^tvrm124 and Rpe65^tvrm148</i> mutants.

<p>(A) In homozygous <i>Mfrp^rd6</i> mutant mice, the transcripts in the visual cycle (<i>Rpe65, Lrat</i> and <i>Rgr</i>), phototransduction pathway (<i>Rgs9</i>, <i>GuCa1b</i>, <i>Pde6a</i>) and structural components of rods and cones (<i>Fscn2</i> and <i>RpGrip1</i>) were significantly decreased relative to the wild-type control (B6/J), validating the microarray results. (B) qRT-PCR analysis in <i>Tulp1^tvrm124/Tulp1^tvrm124</i> mutants at P14 revealed no significant change in any of the transcripts tested. (C) In <i>Rpe65^tvrm148/Rpe65^tvrm148</i> mutants, there was only a significant increase in <i>RpGrip1</i> from transcripts tested, relative to wild-type (B6/J) controls. The data are expressed as relative fold change (RFC) after normalizing to the wild-type control. RFC was calculated using ΔΔC_T method after internal calibration to β-Actin control. Each value represents RFC ± S.E.M. * <i>P</i><0.05 and ** <i>P</i><0.001 relative to controls. N = 3–6 per group.</p

Volcano plots showing the relationship between fold change (represented as mean A – mean B) and the level of significance (represented by the <i>Fs</i> permutated <i>p</i>-value).

<p>Differentially expressed probe sets (q<0.05 shown in red across all fold change levels) at and fold change greater than 2 are depicted in volcano plots in three pairwise comparisons. (A) <i>rd6/rd6</i> (<i>Mfrp^rd6</i>/<i>Mfrp^rd6</i>) P0 vs B6 (C57BL/6J) P0, (B) <i>rd6/rd6</i> P14 vs B6 P14 and (C) <i>rd6/rd6</i> P14 vs <i>rd6/rd6</i> P0.</p

Cellular localization of <i>Prss56 and Glul</i> in B6 (C57BL/6J) and <i>Mfrp^rd6/Mfrp^rd6</i> mice.

<p>(A) By <i>in situ</i> hybridization, in B6 (C57BL/6J) controls at P14, we observed <i>Prss56</i> transcript in only very few cells of the inner nuclear layer (INL) of the retina (top panel), whereas in <i>Mfrp^rd6/Mfrp^rd6</i> mutants, an intense staining of <i>Prss56</i> transcript was observed in INL of the retina (bottom panel). (B) By 2-plex <i>in situ</i> hybridization, in B6 controls, we observed co-localization of <i>Prss56</i> (red) and <i>Glul</i> (pseudo colored green) transcripts in only a few cell body of the (INL) of the retina (top panel), whereas in <i>Mfrp^rd6/Mfrp^rd6</i>, strong co-localization of <i>Prss56</i> and <i>Glul</i> transcripts in the cell body of the INL of the retina was observed (bottom panel). (C) Glutamine synthetase (GS) staining of Müller cells. In both C57BL/6J (B6) and <i>Mfrp^rd6/Mfrp^rd6</i> mice, Müller cells marked with glutamine synthetase showed a similar localization pattern (inset, top and bottom panels) as observed for <i>Prss56 in situ</i> hybridization staining, suggesting that Müller cells in the INL of retina express <i>Prss56</i> transcripts at P14.</p