Journal: bioRxiv

Article Title: Regulation of protein abundance in neurons by selective translation of 3′UTR isoforms

doi: 10.64898/2026.07.08.737200

Figure Lengend Snippet: (A) Workflow of polysome profiling performed in adult Drosophila heads and mouse brains. A lysate prepared from freshly separated fly heads or mouse brains in the presence of cycloheximide was subjected to ultracentrifugation in a sucrose gradient, in which the number of ribosomes bound to RNAs increases with density. RNA was isolated from each fraction for total RNA sequencing to calculate the translation status of individual mRNA isoforms, based on the expression of each transcript relative to lysate. (B) Differential translation of short or long 3’UTR isoforms for each gene in fly heads and mouse brains. Shown are genes in which the indicated 3’UTR isoforms (short or long) are enriched in the translating fractions. (C) Proportion of genes in fly heads and mouse brains that display differential translation of 3’UTR isoforms. Values indicate the number of genes for which isoform expression ratios are biased (compared to lysate) towards the short (blue) or long (red) 3’UTR isoform. (D) Heat maps representing the differential usage of proximal or distal poly(A) sites in the indicated polysome profile fractions, in 1064 Drosophila genes clustered by Ward’s method. All genes with differential translation of 3’UTR isoforms are shown, with one line representing one gene across all fractions. Relative amounts of short and long 3’UTRs are represented by relative poly(A) site usage, with 50% signifying equal relative abundance of both isoforms. A 260 , UV absorbance of the gradient at 260nm (arbitrary unit). (E) Gene ontology analysis of genes for which short (blue) or long (red) 3’UTR isoforms are enriched in either translating or non-translating fractions from fly heads. The top four terms are shown for each category. Background set: all expressed genes.

Article Snippet: Cells were transfected with RNP complexes and StemMACSTM eGFP mRNA (Miltenyi Biotec, 130-101-114).

Techniques: Isolation, RNA Sequencing, Expressing

Journal: bioRxiv

Article Title: Regulation of protein abundance in neurons by selective translation of 3′UTR isoforms

doi: 10.64898/2026.07.08.737200

Figure Lengend Snippet: (A) elav gene model (drawn to scale) and total RNA-seq tracks in fly heads of control and elav ΔnUTR flies. elav ΔnUTR flies are homozygous for an elav allele lacking the neuronal 3’UTR. (B) RT-qPCR quantification of the expression of the long elav 3’UTR isoform relative to total (short) elav mRNA in fly head polysome profile fractions, normalized to levels in tissue lysate. Error bars represent mean ±SD of two biological replicates. (C) ELAV protein expression in control and elav ΔnUTR adult fly heads. ELAV band intensity was normalized to the respective Histone H3 band intensity, with ELAV levels in control flies set to 1. Mean ±SD is indicated for three biological replicates. Ten heads of 3–5-day-old flies were used per replicate. (D) elav alleles used to create the fly genotypes used in ( E ). In elav FLAG , a Flag tag was inserted into the endogenous elav locus to generate a visibly larger protein. (E) ELAV protein expression by Western blot (top) and relative band intensity quantification (bottom) in adult fly heads of the indicated genotypes. Each genotype (all females) consists of two of the alleles shown in (D) . The proportion of ELAV expressed from each allele (control and elav FLAG , red; or elav ΔnUTR , light pink) was normalized to total ELAV per lane, with ELAV levels in elav FLAG /elav FLAG set to 1. Error bars represent mean ±SD of three biological replicates. *p ≤ 0.05 (Welch’s t-test). (F) Model of 3’UTR-mediated regulation of elav translation, in which mRNA isoforms containing the longer 3’UTR (nUTR, red) are translationally repressed and short isoforms produce the bulk of the encoded protein. ELAV protein in turn promotes the formation of longer 3’UTRs, negatively feeding back on ELAV abundance. (G, H) elav RNA ( G , males and females) and ELAV protein ( H , males) expression in adult heads of flies carrying increasing numbers of elav transgenes, to obtain the indicated gene dosage. Transgenes consisted of either the wild-type elav gene region (control) or lacked the elav nUTR ( ΔnUTR ). Genotypes are as follows, with + denoting a wild-type chromosome and tsg a chromosome carrying a transgene. Males, 1x (+/Y ; ; +/+ ) , 2x ( +/Y ; ; tsg/ +), 3x ( +/Y ; ; tsg/tsg ). Females, 0.5x ( Δelav/+ ; ; +/+) , 1x (+/+ ; ; +/+), 2x ( +/+; ; tsg/tsg ). For RT-qPCR, elav mRNA levels were normalized to RpL32 mRNA and levels in flies “control transgene, 1x dosage” were set to 1. For Western Blot, ELAV band intensity was normalized to the respective GAPDH band intensity, with ELAV levels in flies “1x dosage” set to 1. Error bars represent mean ±SD of three biological replicates for each genotype. Ten heads of adult flies were used per replicate.

Article Snippet: Cells were transfected with RNP complexes and StemMACSTM eGFP mRNA (Miltenyi Biotec, 130-101-114).

Techniques: RNA Sequencing, Control, Quantitative RT-PCR, Expressing, FLAG-tag, Western Blot

Journal: bioRxiv

Article Title: Regulation of protein abundance in neurons by selective translation of 3′UTR isoforms

doi: 10.64898/2026.07.08.737200

Figure Lengend Snippet: (A) Viability of elav ΔnUTR flies, measured as the rate of eclosion of live adult flies, compared to expected Mendelian ratios. Eclosion rates were normalized to those of control flies. Error bars indicate mean ±SD of four biological replicates. 375 (control) and 414 ( elav ΔnUTR ) adult flies were scored. **p ≤ 0.01 (Welch’s t-test). (B, C) Fraction of elav ΔnUTR and control flies that hatched from embryos into first instar larvae (B) , and that eclosed from pupae into live adults (C) , at the indicated time points after egg laying. Mean ±SEM of 10 biological replicates (10 embryos/pupae per replicate) is represented for each genotype. χ²=12.93, p=3.23 e-0.4 (B) , χ²=133.9, p=<0.0001 (C) (Mantel-Cox log-rank test). (D) Differential expression of the short (500 bp preceding the pPAS) and long 3′UTR in 313 ELAV target genes (genes that undergo ELAV-mediated 3′UTR extension), in elav ΔnUTR compared to control fly heads. Expression was quantified from RNA-seq data. ** p≤0.01 (Wilcoxon test). (E) Differential protein expression in elav ΔnUTR compared to control fly heads. Peptide intensity was measured by mass spectrometry. Proteins encoded by ELAV target genes are marked in red, with ELAV in black. (F) Expression of proteins encoded by ELAV target genes (top 50 genes with long 3’UTR isoform upregulated), in elav ΔnUTR compared to control fly heads. (G) RT-qPCR quantification of the long and short elav 3’UTR isoforms in adult heads of flies kept in control (with food) conditions or under starvation stress for 24h (starved). RNA levels were normalized to RpL32 mRNA and levels in control conditions were set to 1. Error bars represent mean ±SD of three biological replicates. (H, I) ELAV protein expression in adult heads of flies that were subjected to starvation stress for 24h. In (H), the Western blot membrane was cut below the ELAV band at 50 kDa and exposed separately to visualize degradation products. Intensities of the ELAV band and the ELAV degradation smear were normalized to the respective Histone H3 band intensity, with levels in control conditions set to 1. Mean ±SD is indicated for four (H) and three (I) biological replicates. (J) Stress resilience of control and elav ΔnUTR flies, measured as the proportion of flies alive after being subjected to starvation stress for the indicated number of hours. Mean ±SEM of five biological replicates (10 males and 10 females per replicate) is represented for each genotype. χ²=23.12, p=1.5 X10 −6 (Mantel-Cox log-rank test). (K) Principal component analysis of gene expression in adult heads of control and elav ΔnUTR flies kept in control (with food) conditions or under starvation stress for 24h (starved). (L) Differential expression of genes of the indicated gene sets in adult heads of flies kept under starvation stress for 24h compared to control fly heads. Expression was quantified from RNA-seq data. * p≤0.05 (Wilcoxon test).

Article Snippet: Cells were transfected with RNP complexes and StemMACSTM eGFP mRNA (Miltenyi Biotec, 130-101-114).

Techniques: Control, Quantitative Proteomics, Expressing, RNA Sequencing, Mass Spectrometry, Quantitative RT-PCR, Western Blot, Membrane, Gene Expression

Journal: bioRxiv

Article Title: Regulation of protein abundance in neurons by selective translation of 3′UTR isoforms

doi: 10.64898/2026.07.08.737200

Figure Lengend Snippet: (A) Flag xRIP-seq signal tracks for the elav 3′UTR, in flies in which Pumilio was endogenously Flag-tagged ( pum Flag ), and in untagged control flies (w 1118 ) . Signal is shown normalized to respective input. (B) ELAV protein expression in brains of control and Δpum larvae. Flies are homozygous for the elav FLAG allele on the first chromosome. (C) Western blot (top) and relative band intensity quantification (bottom) of ELAV protein in brains of control and Δpum larvae. All larvae are females expressing both Flag-ELAV (from the elav FLAG allele, red arrowhead) and untagged ELAV (from the elav ΔnUTR allele, light pink arrowhead). Histone H3 serves as a loading control. The proportion of ELAV expressed from each allele was normalized to total ELAV per lane, with ELAV levels in Control set to 1. Error bars represent mean ±SD of three biological replicates. *** p ≤ 0.001 (Welch’s t-test). (D) Enrichment of neuronal 3′UTRs in Pum xRIP relative to input. The heatmap profile plot displays 1 kb upstream, and the neuronal 3′UTR downstream (scaled region, indicated in red), of the proximal poly(A) site. Pum xRIP was performed in adult fly heads. (E) Quantification of Pum binding to short and nUTR-containing mRNA isoforms of ELAV target genes, by Pum xRIP-seq signal compared to input. p=0.0438 (Wilcoxon test). (F) Differential protein expression in Δpum compared to control fly heads. Peptide intensity was measured by mass spectrometry. Proteins encoded by Pum target genes (mRNAs enriched in Pum xRIP-seq compared to input) and proteins encoded by ELAV target genes (genes that undergo ELAV-mediated 3′UTR extension) are marked in purple and red, respectively. Pum is black. (G) Proportion and numbers of proteins encoded by transcripts of the indicated groups that are upregulated (up) and downregulated (down) in Δpum mutant larval brains compared to control. All mRNAs are compared to mRNAs whose long 3’UTR isoform is preferentially bound by Pum compared to the short isoform of the same gene. (H) Model of nUTR-mediated translational regulation of neuronal genes. nUTR-containing mRNAs are translationally repressed. Low levels of ELAV protein promotes expression of short 3’UTR isoforms, which in turn increases global protein abundance. The feedback loop is supported by nUTR-specific binding and translational repression by Pumilio.

Article Snippet: Cells were transfected with RNP complexes and StemMACSTM eGFP mRNA (Miltenyi Biotec, 130-101-114).

Techniques: Control, Expressing, Western Blot, Binding Assay, Mass Spectrometry, Mutagenesis, Quantitative Proteomics

Journal: bioRxiv

Article Title: Regulation of protein abundance in neurons by selective translation of 3′UTR isoforms

doi: 10.64898/2026.07.08.737200

Figure Lengend Snippet: (A) Gene model and mRNA-seq tracks of the ELAVL1 3’UTR (drawn to scale) in human iPSCs, and in the course of differentiation into neural progenitor cells and neurons. (B) Profile from NSC polysome profiling experiment. The four fractions used for downstream RNA-seq analysis are indicated. A 260 , UV absorbance of the gradient at 260nm (arbitrary unit). (C) Proportion of genes in NSCs that display differential translation of 3’UTR isoforms. Values indicate the number of genes for which isoform expression ratios are biased (compared to lysate) towards the short (blue) or long (red) 3’UTR isoform. (D) RT-qPCR quantification of the expression of the long ELAVL1 3’UTR isoform relative to total (short) elav mRNA isoforms in polysome profile fractions, normalized to levels in tissue lysate. Error bars represent mean ±SD of three biological replicates. (E) Light microscopy images of cerebral organoids grown from control and ELAVL1 ΔnUTR human iPSCs on day 14 and day 34. Scale bar: 500µm. (F) Mean diameter of control and ELAVL1 ΔnUTR (two independent mutants) cerebral organoids at the indicated number of days post-seeding. Error bars represent mean ±SD for six organoids per genotype and time point. (G) Apotome imaging of control and ELAVL1 ΔnUTR cerebral organoids stained for SOX2, ELAVL1 and DAPI. Scale bar: 400µm. (H) Quantification of neural rosette size in control and ELAVL1 ΔnUTR cerebral organoids. 36 (control), 31 (mutant #1) and 40 (mutant #2) rosettes were scored. ****p < 0.0001 (Mann-Whitney U test). (I) Quantification of the mean signal intensity for SOX2 (background intensity-corrected) in control and ELAVL1 ΔnUTR organoids. Error bars represent mean ±SD for four replicates. ****p < 0.0001 (Mann-Whitney U test). (J) Western blot comparing ELAVL1 protein expression in control and ELAVL1 ΔnUTR day 5 organoids. ELAVL1 band intensities were normalized to the respective Histone H3 band intensity, with ELAVL1 levels in control organoids set to 1. Mean ±SD is indicated for three biological replicates. (K) Quantification of the ELAVL1/SOX2 signal intensity ratio in control and ELAVL1 ΔnUTR organoids. Error bars represent mean ±SD for four replicates. *p < 0.05 (Welch’s t-test).

Article Snippet: Cells were transfected with RNP complexes and StemMACSTM eGFP mRNA (Miltenyi Biotec, 130-101-114).

Techniques: RNA Sequencing, Expressing, Quantitative RT-PCR, Light Microscopy, Control, Imaging, Staining, Mutagenesis, MANN-WHITNEY, Western Blot

Journal: Development (Cambridge, England)

Article Title: CSF1R + macrophage and osteoclast depletion impairs neural crest proliferation and craniofacial morphogenesis

doi: 10.1242/dev.205423

Figure Lengend Snippet: Gestational exposure to PLX5622 significantly depletes CSF1R-expressing cells. (A) Schematic illustrating collection of Csf1r EGFP + cells for flow cytometry. (B) Quantification of EGFP + cells from Csf1r EGFP craniofacial tissues ( n =3-10 embryos per sex/treatment/time-point from two to four dams). (C-W) Immunofluorescence images and quantification of CSF1R + and Csf1r EGFP+ cells in and around the E15.5 nasal septum (C-E), Meckel's cartilage (F-H), ear (I-K), maxillary incisor (L-N), eye (O-Q), trigeminal (R-T) and tongue (U-W). Arrows mark CSF1R and Csf1r EGFP double-positive cells ( n =3 embryos per sex/treatment from two or three dams). c.d., cochlear duct; d.p., dental papilla; e.k., enamel knot; ey, eye; l.s.c., lateral semicircular canal; m.c., Meckel's cartilage; n.s., nasal septum; s.r., stellate reticulum; tg, tongue; ut, utricle. Blue dots represent male and pink dots represent female. Counts represent mean±s.e.m. and were analyzed by a two-way ANOVA with Tukey's post-hoc test.

Article Snippet: Csf1r EGFP mice (JAX: 005304; RRID: IMSR_JAX:005304; The Jackson Laboratory) were crossed to C57BL/6 mice to generate heterozygous embryos for flow cytometry and immunofluorescence experiments.

Techniques: Expressing, Flow Cytometry, Immunofluorescence

Journal: Development (Cambridge, England)

Article Title: CSF1R + macrophage and osteoclast depletion impairs neural crest proliferation and craniofacial morphogenesis

doi: 10.1242/dev.205423

Figure Lengend Snippet: Prenatal exposure to PLX5622 disrupts osteoclast development and function. (A-D′) Csf1r EGFP + osteoclasts (arrows) in the E15.5 premaxilla (A-B′) and maxilla (C-D′). (E-I) Immunofluorescence images (E-H) and quantification (I,I′) of multinucleated CTSK/ Csf1r EGFP double-positive osteoclasts (arrows) (I) and Csf1r EGFP+ single-positive osteoclasts (I′) in the E15.5 mandible. (J-S′) TRAP staining (arrows) in E15.5 premaxilla (J-K′), mandible (L-M′), maxilla (N-O′), frontal (P-Q′) and basioccipital (R-S′) bones. (T-V) Quantification of total TRAP + area in premaxilla (T), mandible (U) and maxilla (V). n =3 embryos per sex/treatment from two or three dams. bo, basioccipital; fr, frontal; m.c, Meckel's cartilage; md, mandible; mx, maxilla pmx, premaxilla. Counts represent mean±s.e.m. and were analyzed by an ART ANOVA (I,I′) or a two-way ANOVA (T-V) with Tukey's post-hoc test. A′-F′ show magnifications of the respective boxed areas in A-F.

Article Snippet: Csf1r EGFP mice (JAX: 005304; RRID: IMSR_JAX:005304; The Jackson Laboratory) were crossed to C57BL/6 mice to generate heterozygous embryos for flow cytometry and immunofluorescence experiments.

Techniques: Immunofluorescence, Staining

Journal: Bioactive Materials

Article Title: Splenic dendritic cell-targeting mRNA transfection of H-type ionizable lipid-based LNPs for enhancing tumor immunotherapy

doi: 10.1016/j.bioactmat.2026.02.018

Figure Lengend Snippet: Schematic illustration of in vivo tumor immunotherapy enhanced by mRNA/HNPs through intravenous injection. H18 lipid, DOPE, cholesterol, DMG-PEG 2000 and mRNA were mixed to form mRNA/H 18 NPs with the special multilamellar concentric nanostructures. Following intravenous administration, mRNA/H 18 NPs demonstrated preferential adsorption of complement C3 proteins to form a characteristic protein corona, resulting in specific mRNA transfection in the spleen, especially in splenic dendritic cells. When encapsulating tumor antigen-encoding mRNA, the mRNA/H 18 NPs achieved precise transfection of the antigen mRNA in splenic dendritic cells. This targeted delivery stimulated dendritic cell maturation and subsequent antigen presentation, initiating robust T cell priming. The activated antigen-specific cytotoxic T lymphocytes then infiltrated into tumor tissues, ultimately inducing tumor cell elimination.

Article Snippet: Luciferase mRNA, OVA mRNA, and EGFP mRNA were purchased from Novoprotein (Suzhou, China).

Techniques: In Vivo, Injection, Adsorption, Transfection, Immunopeptidomics

Journal: Bioactive Materials

Article Title: Splenic dendritic cell-targeting mRNA transfection of H-type ionizable lipid-based LNPs for enhancing tumor immunotherapy

doi: 10.1016/j.bioactmat.2026.02.018

Figure Lengend Snippet: Preparation and Characterization of Optimal mRNA/H 18 NPs. (A) Schematic illustration of mRNA/H 18 NPs preparation. (B) The size distribution and (C) zeta potential of optimized mRNA/H 18 NPs. (D) The apparent p K a of mRNA/H 18 NPs. (E) Cryo-EM image of optimized mRNA/H 18 NPs. Scale bar = 50 nm. (F) Representative image and percentage of bioluminescence in major organs of mice following intravenous injection of mLuc/H 18 NPs. (G)-(I) Stability test for mRNA/H 18 NPs. (G) Size and PDI of mRNA/H 18 NPs when stored at 4 °C for different days (0, 3, 5, 7). (H) Left: Total bioluminescence flux in the spleen of mice 6 h after intravenous injection of mLuc/H 18 NPs stored at 4 °C for different days (0, 3, 5, 7). Right: Representative bioluminescence images of major organs of mice 6 h after intravenous injection of different mLuc/H 18 NPs stored at 4 °C for different days (0, 3, 5, 7). (I) Size and PDI of mRNA/H 18 NPs when diluted with PBS by different times. Data were shown as mean ± SD (n = 3).

Article Snippet: Luciferase mRNA, OVA mRNA, and EGFP mRNA were purchased from Novoprotein (Suzhou, China).

Techniques: Zeta Potential Analyzer, Cryo-EM Sample Prep, Injection

Journal: Bioactive Materials

Article Title: Splenic dendritic cell-targeting mRNA transfection of H-type ionizable lipid-based LNPs for enhancing tumor immunotherapy

doi: 10.1016/j.bioactmat.2026.02.018

Figure Lengend Snippet: In vivo splenic DC-specific transfection of mRNA/H 18 NPs and in vitro protein corona analysis of mRNA/H 18 NPs. (A) EGFP protein expression in splenic cell subsets of C57BL/6J mice 24 h post intravenous injection of different formulations. (B) The top 5 most abundant plasma proteins adsorbed on mRNA/H 18 NPs (C3: Complement C3; Ighm: Immunoglobulin heavy constant mu; Hbat1: Alpha-globin; Itih4: Inter alpha-trypsin inhibitor, heavy chain 4; Cnn2: Calponin). (C) Heatmap plot of major proteins in the protein corona adsorbed on mRNA/MC3-LNPs and mRNA/H 18 NPs. PBS group was used as a negative control. (D) Quantification of major adsorbed protein categories of different formulations. (E) Complement C3 abundance in protein corona adsorbed on mRNA/MC3-LNPs and mRNA/H 18 NPs. (F) Bioluminescence images of major organs and (G) Quantification of total bioluminescence flux in the spleen from C57BL/6J mice 6 h after intravenous injection of mLuc/H 18 NPs (mLuc dose of 0.25 mg kg −1 ). Mice were pre-treated with cobra venom factor (CVF) or PBS. (H) Fluorescence quantification of Cy5 mRNA delivered by uncoated or complement C3-coated Cy5-mRNA/H 18 NPs in BMDCs. BMDCs were pre-incubated with anti-CD11b (CR3) or anti-IgG blocking antibody. (I) Bioluminescence intensity of luciferase protein translated from mRNA delivered by uncoated or complement C3-coated mLuc/H 18 NPs in BMDCs. BMDCs were pre-incubated with anti-CD11b (CR3) or anti-IgG blocking antibody. Data were shown as mean ± SD (n = 3).

Article Snippet: Luciferase mRNA, OVA mRNA, and EGFP mRNA were purchased from Novoprotein (Suzhou, China).

Techniques: In Vivo, Transfection, In Vitro, Expressing, Injection, Clinical Proteomics, Negative Control, Combined Bisulfite Restriction Analysis Assay, Fluorescence, Incubation, Blocking Assay, Luciferase

Journal: STAR Protocols

Article Title: Protocol for an in vivo CRISPR screen for germinal center B cells in mice using ecotropic retrovirus

doi: 10.1016/j.xpro.2026.104586

Figure Lengend Snippet: Immunization experiment scheme for in vivo screen Host mice (IOMA gl) were immunized with OVA in alum. At week 2 after immunization, B cells from B1-8 hi , LSL-Cas9, AID-Cre, Kappa KO animals were transduced with pooled sgRNA library retrovirus and adoptively transferred to the hosts. Subsequently, the host animals were boosted with NP-OVA in alum at day 0 and 2 and administered with anti-DEC205-OVA mAb at day 6.5. Spleens were harvested and processed for cell sorting at day 10.

Article Snippet: Mouse: 6-weeks-old, sex-matched LSL-Cas9: B6J.129(B6N)-Gt(ROSA)26Sortm1(CAG-cas9∗,-EGFP)Fezh/J , The Jackson Laboratory , cat# 026175.

Techniques: In Vivo, Transduction, FACS