1. RNA-Binding Protein HuD Controls Insulin Translation
Eun Kyung Lee, Wook Kim, Kumiko Tominaga, Jennifer L. Martindale, Xiaoling Yang, Sarah S. Subaran, Olga D. Carlson, Evi M. Mercken, Rohit N. Kulkarni, Wado Akamatsu, Hideyuki Okano, Nora I. Perrone-Bizzozero, Rafael de Cabo, Josephine M. Egan, Myriam Gorospe 
Molecular Cell 
Volume 45, Issue 6, Pages (March 2012)
DOI: /j.molcel
Copyright © 2012 Elsevier Inc. Terms and Conditions

2. Figure 1 The IR Pathway Controls HuD Expression
(A) Immunostaining of sections of human and mouse pancreata to detect insulin (green, β cells), HuD, (red), and glucagon (blue, α cells); scale bar, 50 μm.
(B) Western blot analysis of HuD levels in mouse tissues.
(C–E) Western blot analyses of the levels of HuD, IR β subunit (IRβ), and loading control β-actin in βIRWT and βIRKO cells cultured for 16 hr in 2 mM glucose, and further cultured for 6 hr with the indicated glucose concentrations (C), in βIRKO cells transfected with either an control plasmid (vector) or with a plasmid that expressed IR (D), and in βIRWT cells cultured in 2 mM glucose + 0.1% FBS for 24 hr before insulin treatment for the indicated times (E).
(F–H) Expression of the indicated proteins in βTC6 cells was studied by western blot analysis 48 hr after transfecting siRNAs directed to Irs2 (F) or Akt (G) or after treatment for 24 hr with inhibitors of PI3K (LY294002) or Akt (H).
(I) Chromatin IP analysis of the interaction of FoxO1 with the HuD promoter in βTC6 cells cultured in 2 or 25 mM glucose for 30 min.
(J and K) Western blot analysis of the levels of HuD, FoxO1 and β-actin in βIRWT cells (J) and βTC6 cells (K) 48 hr after siRNA transfection.
(L) Western blot analysis of the expression of the indicated proteins in βTC6 cells 30 hr after transfecting a control plasmid (Vector) or plasmids to express Flag-tagged FoxO1, either WT or the transcription-deficient mutant (H215R-537). Bottom, diagram of constructs for Flag-tagged WT and H215R-537.
(M) Schematic of the proposed regulation of HuD expression by the IR signaling pathway in β cells. In (C)–(H) and (J)–(L), signals on western blots (WB) were quantified by densitometry and the errors (SEM) calculated. WBs are representative of at least three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

3. Figure 2 HuD Binds to the Ins2 5′ UTR and Represses Translation
(A) HuD interaction with Ins2 mRNA was studied by RIP analysis using anti-HuD or control IgG antibodies. RNA was isolated, and Ins2 mRNA levels measured by reverse transcription-quantitative PCR analysis (RT-qPCR) and normalized to Gapdh mRNA levels.
(B) Schematic of mouse insulin (Ins2) mRNA (top) and biotinylated segments (5′ UTR, CR, 3′ UTR) and subfragments of the 5′ UTR (5′A–5′D) tested (bottom).
(C) Biotin pull-down analysis of the interaction of HuD and biotinylated RNAs in (B). Biotinylated GAPDH RNA was included as negative control. IN, input (2 μg of βTC6 whole-cell lysate).
(D) Sequence alignment of the last 16 nt of fragment 5′D in different species.
(E) Biotinylated RNAs (1 μg each, top), including 5′D and mutants mut1-mut4 (white, mutated nucleotides) were incubated with βTC6 cell lysates (HuD endogenous) and with 1 μg of purified, recombinant GST-HuD (66 kDa) or control GST (26 kDa). Complexes were detected using anti-HuD or anti-GST antibodies. MWM, molecular weight marker; IN, input lysate or purified protein.
(F) Left: Reporter plasmids: parent vector (pEGFP), plasmid expressing full-length Ins2 5′ UTR [p(5′)EGFP], plasmid expressing one copy of fragment 5′D [p(5′D)EGFP], and plasmid expressing three copies of 5′D [p(3 × 5′D)EGFP]. Right: 24 hr after siRNA transfection, each plasmid was transfected and EGFP expression levels assessed 24 hr after that. Western blot analysis of the levels of EGFP, HuD, and loading control proteins. After quantification by densitometry, EGFP signals were expressed relative to EGFP levels in the Ctrl siRNA sample in each pair (top row) and relative to EGFP levels in the Ctrl siRNA, pEGFP transfection group (bottom row). Quantified WB signals (±SEM) are shown.
(G) Cells were transfected as explained in (F) and the levels of reporter EGFP mRNAs were quantified by RT-qPCR and plotted relative to those in the Ctrl siRNA of each group (main graph) and relative to those in the Ctrl siRNA of the pEGFP transfection group (inset graph).
(H and I) Ins2 mRNA levels in βTC6 cells transfected with either control or HuD-directed siRNA were quantified by RT-qPCR analysis (H) and the levels of protein expressed from Ins2 mRNA (proinsulin), as well as HuD and β-actin were assessed by western blot analysis and quantified (I).
(J) Polysome analysis of Ins2 mRNA. Lysates prepared from βTC6 cells (2 mM glucose) as explained in (H) were fractionated through sucrose gradients to generate polysome profiles (left). Arrow: direction of sedimentation; –, no ribosomal components; LMWP (fractions 6–8) and HMWP (fractions 9–12). The relative distribution of Ins2 mRNA and Actin mRNA on polysome gradients was studied by RT-qPCR analysis of the RNA present in each of 12 gradient fractions, and represented as percentage of total mRNA (right). Data are representative of three independent experiments.
(K) Nascent translation of GAPDH and proinsulin was assessed by incubation of βTC6 cells in the presence of 35S-Met/Cys. After IP using IgG (control), anti-GAPDH, and anti-insulin antibodies, the newly translated proteins were visualized by SDS-PAGE, transfer, and PhosphorImager analysis of the blots (top); bottom, quantification of 35S-proinsulin in three different experiments.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Glucose Challenge Dissociates Ins2 mRNA from HuD and Mobilizes Cytoplasmic HuD
(A) RIP analysis of HuD-Ins2 mRNA complexes in βTC6 cells. Glucose-starved (2 mM glucose, 16 hr) βTC6 cells were incubated for with 25 mM glucose (15 or 30 min), whereupon RIP analysis was performed to assess the enrichment of Ins2 mRNA in HuD IP compared with IgG IP.
(B and C) The effect of glucose stimulation (15 mM, 20 min) on insulin expression by western blot analysis (B, left) or by RT-qPCR analysis of Ins2 mRNA (B, right) was studied in βTC6 cells 48 hr after transfection with control plasmid (vector) or plasmid pMyc-HuD to express Myc-tagged HuD. (C) De novo proinsulin translation was assessed by incorporation of 35S-amino acids as explained in Figure 2K.
(D–F) Forty-eight hours after transfection with Ctrl siRNA or HuD siRNA, proinsulin levels were assessed by western blot analysis (D left), Ins2 mRNA levels by RT-qPCR (D right), intracellular and secreted mature insulin by ELISA (E), and C-peptide by ELISA (F). Graphs depict the means of three experiments and p values. Data were normalized to protein concentration.
(G) Immunofluorescent detection of HuD, PB marker Dcp1a, and SG marker TIAR in βTC6 cells that were stimulated with glucose as explained in (A). Bar, 10 μm. Graphs depict the quantification of signal intensities in the segment indicated (red arrows). In the graphs of (B) and (D)–(F), errors represent SEM.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Higher Insulin in HuD−/− Mice; Lower Insulin and Diminished Glucose Tolerance in HuD-Overexpressing Mice
(A) Left, immunofluorescent detection of insulin and HuD in pancreatic islets from HuD−/−, HuD+/−, and HuD+/+ mice; nuclei were visualized by TO-PRO-3 staining. Right, insulin fluorescence was assessed from more than ten sections per animal; n = 3 animals per genotype, >118 islets per genotype.
(B) Western blot analysis of HuD levels in whole pancreas (200 μg) and brain (40 μg) of HuD+/+ and HuD−/− mice.
(C–E) Proinsulin levels were assessed by western blot analysis (C), mature insulin levels by ELISA (D), and Ins2 mRNA levels by RT-qPCR (E) in total pancreatic lysates from HuD+/+, HuD+/−, and HuD−/− mice.
(F and G) Immunofluorescent detection of HuD-myc and insulin in pancreatic islets from HuD Tg and WT mice. (F) HuD-myc was visualized with anti-myc (left) and anti-HuD (right) antibodies. (G) Insulin fluorescence was quantified and plotted (187 islets from HuD WT mice and 169 islets from HuD Tg mice); n = 5 animals per genotype.
(H) Western blot analysis of whole pancreas lysates prepared from HuD WT and HuD Tg mice (200 μg, two mice per group).
(I) Intraperitoneal glucose tolerance test (IPGTT) in 3-month-old female HuD WT and HuD Tg mice, injected with 1g/kg (i.p.). Plasma glucose (top) and insulin (bottom) levels were measured at the times shown. ∗p < 0.05, ∗∗p < 0.01.
(J) Schematic of HuD expression and influence—positive (green) and negative (red)—on insulin biosynthesis. Bars in (A), (F), and (G) = 50 μm. In the graphs of (A), (D), (E), (G), and (I), errors represent SEM.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions