12 research outputs found
Interaction of HuDA and PABP at 5'UTR of mouse insulin2 regulates insulin biosynthesis
<div><p>Understanding the regulation of insulin biosynthesis is important as it plays a central role in glucose metabolism. The mouse insulin gene2 (Ins2) has two splice variants; long (Ins2L) and short (Ins2S), that differ only in their 5’UTR sequence and Ins2S is the major transcript which translate more efficiently as compared to Ins2L. Here, we show that cellular factors bind preferentially to the Ins2L 5’UTR, and that PABP and HuD can bind to Ins2 splice variants and regulate its translation. <i>In vitro</i> binding assay with insulin 5’UTR and different HuD isoforms indicate that the ‘N’ terminal region of HuD is important for RNA binding and insulin translation repression. Using reporter assay we showed that specifically full-length HuD A isoform represses translation of reporter containing insulin 5’UTR. We further show that PABP and HuD interact with each other in RNA-dependent manner and this interaction is affected by glucose and PDI (5’UTR associated translation activator). These results suggest that PABP interacts with HuD in basal glucose conditions making translation inhibitory complex, however upon glucose stimulation this association is affected and PABP is acted upon by PDI resulting in stimulation of insulin translation. Together, our findings snapshot the mechanism of post-transcriptional regulation of insulin biosynthesis.</p></div
HuD and PABP interaction is regulated by PDI.
<p><b>(A)</b> His-PABP was incubated with GST or GST-HuD B and GST-pull down was performed and analyzed by western blotting for presence of PABP, HuD-B or GST as indicated.<b>(B)</b> Immunoprecipitation of endogenous PABP was performed from the MIN6 cell transfected with GFP or GFP-PDI. The immunoprecipitate was analyzed for the presence of HuD, PABP, GFP or GFP-PDI by western blotting. <b>(C)</b> GST pull-down was performed from a protein mixture containing recombinant His-PABP, GST-HuD B, and His-PDI or His-PDIΔC (C-terminal deletion). The eluted proteins were analyzed for the presence of PABP, GST-HuD B and GST. <b>(D)</b> Recombinant purified HuD A, PABP, GFP and PDI Δ C were incubated with Ins2L-5’UTR (left panel) or without (right panel). The reaction was then immunoprecipitated with specific antibodies (indicated above the lanes) and the eluted fractions were analyzed by western blotting for the presence of HuD-A, PABP and GFP.</p
HuD and PABP interact in RNA-dependent manner under basal condition.
<p><b>(A)</b> Immunoprecipitation of endogenous PABP from MIN6 cell lysates treated with low (LG) or high glucose (HG) condition and immunoblotted for HuD. <b>(B)</b> Reciprocal immunoprecipitation of endogenous HuD from low or high glucose treated cells and immunoblotted for PABP. <b>(C)</b> MIN6 cells treated with low glucose condition were lysed and the lysate was used to immunoprecipitate PABP or HuD in the presence (+) or absence of (-) RNase and probed for HuD or <b>(D)</b> PABP respectively. Rabbit IgG was used as a non specific antibody control for immunoprecipitation.</p
HuD-A inhibits insulin translation.
<p><i>In vitro</i> translation assay of chimeric Ins2L-5’UTR-Luc-Ins2-3‘UTR <b>(A)</b> in presence of Long/Short (L/S) unlabeled RNA along with His-PABP <b>(B)</b> in presence of His-GFP or His-HuD A/B and D. Lower panel indicates the amount of luciferase and renilla RNA at the end of translation reaction detected by RT-PCR. Renilla was used as internal control for translation efficiency and His-GFP was used as nonspecific protein control and <b>(C)</b> in presence of recombinant PABP and/or HuD along with nonspecific protein (NSP). In all three experiments, the Luc/Renilla ratio was normalized and relative luciferase activity is expressed as a percentage of control and represented as bar graph. <b>(D)</b> MIN6 cells were transfected with HA-tagged HuD plasmid along with Long-5’UTR-Luc-Ins 3’UTR and Renilla luciferase plasmid. After 16 hours post transfection, the cells were treated with low glucose (2 mM) or high glucose (20 mM) for one hour and luciferase activity was measured. HuD expression was assessed by western blot analysis of the lysate (lower panel) <b>(E)</b> Secreted insulin levels in the low glucose condition media was assayed by ELISA in MIN6 cells overexpressing different HuD isoforms. The insulin readings were normalized to the control cells transfected with only empty vector (EV). <b>(F)</b> Intracellular proinsulin levels in low glucose condition were assayed in MIN6 cells by western blot analysis after overexpressing HA-tagged HuD isoforms along with empty vector control. HuD expression was assessed by Western blot analysis (lower panel). The graph represented in A, B, C and D are average of three experiments and the error bar represents the ± SEM and * indicates p<0.05 when compared with control.</p
PABP binds differentially to the middle region (L2, position 18–39) of insulin 5’UTR.
<p>Competitive RNA-EMSA was performed with recombinant PABP and <b>(A)</b> radiolabeled Ins2L-5’UTR RNA as probe in presence of increasing fold excess of unlabeled L or S RNA and <b>(B</b>) radiolabeled L2 (position 18–39) RNA as probe in presence of molar excess of unlabeled RNA (L, S, L1, L2, L3, L4). The shifted band was quantified and analyzed densitometrically and represented as the bar graph (lower panel). The values are an average of three independent experiments with error bars representing the mean ± SEM, and the significance indicated by p values. (<b>C</b>) m-fold predicted secondary structure of Ins2 splice variants (L/S). The boxed area shows the different folding of L2 region in both the splice variants.</p
Mechanism of differential translation regulation of Ins2 splice variants.
<p>Under basal condition, PABP and HuD bind to long variant preferentially causing translation inhibition by making stable inhibitory complex. Upon glucose stimulation activated/phosphorylated PDI brings disulfide bond rearrangement in PABP thereby interfering with the interaction between HuD and PABP. This results in displacement of HuD from insulin transcript into P-bodies. The bound activator complex upregulates insulin translation and insulin is produced.</p
‘N’ terminal of HuD isoform is important for RNA binding.
<p><b>(A)</b> RNA-EMSA of two different preparations of HuD isoforms. <b>(B)</b> Competitive RNA-EMSA with His-HuD A and radiolabeled Ins2L 5’UTR RNA in presence of increasing molar excess of unlabeled L and S RNA and <b>(C</b>) in presence of molar excess of fragments of Ins2L 5’UTR; L1, L2, L3, L4 or non-specific RNA (NS). The shifted band was quantified and analyzed densitometrically and represented as the bar graph (lower panel). The values are an average of three independent experiments with error bars representing the mean ± SEM and the significance indicated by <i>p</i> values.</p
Cellular PABP and HuD associates with mouse insulin transcripts.
<p><b>(A)</b> Schematic showing the regions of amplification of Ins2L and Ins2S with the indicated primers. RNA associated with PABP/ HuD specifically immunoprecipitated from UV cross-linked samples were analyzed by RT-PCR using insulin gene specific primers. IgG is used as control. Insulin 5’UTR RNA associated with <b>(B)</b> PABP or <b>(C)</b> HuD is analyzed by RT-PCR using Ins2L or Ins2S specific primers. Insulin transcript pulldown efficiency with respective proteins (PABP and HuD) is normalized with respect to input used for the experiment. <b>(D)</b> Analysis of mouse insulin 5’UTR associated proteins in βTC6 cell lysates using biotinylated Ins2L 5’UTR RNA. The biotin pull-down is carried out in the presence of 10-fold molar excess of non-biotinylated long (L) or short (S) 5’UTR RNA (as competitors) and PABP/HuD associated with insulin RNA was analyzed by western blotting.</p
Additional file 2: of Analysis of hepatic transcriptome demonstrates altered lipid metabolism following Lactobacillus johnsonii BS15 prevention in chickens with subclinical necrotic enteritis
Table S2. Details for genes and pathways relevant to lipid metabolism mediating the preventive effects of BS15 treatment bases on SNE chickens. (XLSX 13 kb
Additional file 3: of Analysis of hepatic transcriptome demonstrates altered lipid metabolism following Lactobacillus johnsonii BS15 prevention in chickens with subclinical necrotic enteritis
Table S3. Details for regulatory genes and pathways related to lipid metabolism in comparison of control group and BS15 group. (XLSX 42 kb