An investigation of exon repetition

Exon repetition consists in the presence of tandemly repeated exons in mRNA. In all the cases reported so far, the trivial explanation that the phenomenon arises from a duplication of specific exons in the gene was ruled out by southern blot analysis. It is therefore assumed that exon repetition is the result of a post-transcriptional process involving two pre-mRNA molecules transcribed from the same gene. Exon repetition of SA is of particular interest, since it is both tissue and strain-specific. This at first suggested that repetition in SA might be caused by a tissue and strain-specific trans-acting factor. This work has shown that the property of exon repetition is allele-specific in the two best characterised examples, represented by the SA and COT (carnitine octanoyltransferase) genes in rat. Allele-specificity of exon repetition is therefore the underlying cause of strain-specificity of exon repetition in SA and also in COT.;The biological significance of exon repetition remains unknown. One possible function is the generation of new proteins. Quite often though, repetition of the exon that contains the AUG translational start codon creates a short upstream open reading frame (uORF) that precedes the ORF encoding the full length protein. The effect of this has been investigated and the results show that translation of the downstream open reading frame is significantly reduced by the presence of uORFs.;The mechanism that generates exon repetition is not well understood. It was initially proposed for the COT gene that the presence of a putative exonic splicing enhancer (ESE) in exon 2 conferred exon repetition. This work demonstrates that the presence of the ESE is not sufficient to cause exon repetition in-vivo.

GLP-1-stimulated ERK activation in MIN6 cells is mediated via local Ca²⁺ signalling.

<p>MIN6 cells incubated in KRB plus 1 mM glucose were loaded with 100 µM EGTA-AM or BAPTA-AM at room temperature prior to treatment with 10 nM GLP-1 plus 16.7 mM glucose for the times indicated. a) Proteins were resolved by SDS-PAGE and Western blotted using anti-phospho-ERK1/2 (pERK) and anti-ERK2 (ERK2) antibodies. A representative blot is shown with densitometric analysis of the results below showing mean +S.E.M. (n = 3). Data were analysed by two-way ANOVA with Bonferroni's multiple comparison test compared to GLP-1 plus glucose at each time point; *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001. b) MIN6 cells were treated as in a) but in addition, loaded with 2 µM fura-2-AM and [Ca²⁺]_i levels measured by epifluorescence microscopy. i) The mean increase in [Ca²⁺]_i represented as area under the curve (A.U.C.) during ii) 10 min or iii) 30 min stimulation, mean +S.E.M. (n>30). Statistical comparisons were by one-way ANOVA with Dunnett's range test compared to GLP-1 plus glucose in the absence of chelator. ***, <i>P</i><0.001.</p

A sustained global increase in [Ca²⁺]_i is insufficient for the prolonged activation of ERK.

<p>(a and b) MIN6 cells were preincubated for 1 h in KRB supplemented with 1 mM glucose. Cells were incubated in 50 mM K⁺ (K50) in the presence or absence of 10 µM nifedipine for the times indicated (All statistical comparisons were by one-way ANOVA with Bonferroni's multiple comparison test compared to K50 at each time point; *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001). a) Proteins were resolved by SDS-PAGE and Western blotted using anti-phospho-ERK1/2 and anti-ERK2 antibodies. A representative blot is shown above densitometric analysis of the results showing mean +S.E.M. (n = 3). b) In cells loaded with 2 µM fluo-4-AM, fluorescence (as an index of [Ca²⁺]_i) was measured using a NOVOstar platereader. (c and d) MIN6 cells were preincubated for 1 h in KRB supplemented with 1 mM glucose. Cells were then treated with 10 µM ionomycin or 50 mM K⁺ (K50) for the times indicated. c) In cells loaded with 2 µM fluo-4-AM, fluorescence (as an index of [Ca²⁺]_i) was measured using a NOVOstar platereader. ***, <i>P</i><0.001 for K50 versus ionomycin at equivalent time points. d) After treatments, proteins were resolved by SDS-PAGE and Western blotted using anti-phospho-ERK1/2 (pERK) or anti-ERK2 (ERK2) antibodies. A representative blot is shown with densitometric analysis of the results below showing mean +S.E.M. (n = 3).</p

The sustained activation of L-type VGCCs is required for the sustained activation of ERK.

<p>MIN6 cells were preincubated for 1 h in KRB supplemented with 1 mM glucose. The cells were then incubated in KRB containing 10 nM GLP-1 and 16.7 mM glucose in the absence or presence of 10 µM nifedipine applied at either 0, 10 or 20 min post-GLP-1/glucose addition. Proteins were resolved by SDS-PAGE and Western blotted using anti-phospho-ERK1/2 (pERK) and anti-ERK2 (ERK2) antibodies. A representative blot is shown with densitometric analysis of the results below showing mean +S.E.M. (n = 3). Results were analysed using two-way ANOVA with Bonferroni's multiple comparison test compared to GLP-1 plus glucose; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

The role of intracellular Ca²⁺ stores in GLP-1-stimulated ERK activation.

<p>a) MIN6 cells were either pre-incubated in KRB supplemented with 1 mM glucose in the absence (control) or presence of 100 µM ryanodine or 1 µM thapsigargin for 30 min prior to treatment with 10 nM GLP-1 plus 16.7 mM glucose for the times indicated. Where indicated, cells were also treated with 16.7 mM glucose alone. Proteins were separated by SDS-PAGE and Western blotted using anti-phospho-ERK1/2 (pERK) and anti-ERK1/2 (ERK1/2) antibodies. A representative blot is shown with densitometric analysis of the results below showing mean +S.E.M. (n = 3). Data were analysed by two-way ANOVA with Bonferroni's multiple comparison test compared to GLP-1 plus glucose at each time point. No significant differences were observed. b) MIN6 cells incubated in the absence of extracellular Ca²⁺ calcium were preincubated without or with 100 µM ryanodine for 30 min prior to the addition 10 mM caffeine. Changes in fluorescence as an index of [Ca²⁺]_i were determined in fluo-4-loaded cells using a NOVOstar platereader. c) MIN6 cells incubated in the absence of extracellular Ca²⁺ were pretreated without or with 1 µM thapsigargin for 30 min prior to the addition 100 µM carbachol (Carb.) . Changes in [Ca²⁺]_i were determined in fluo-4-loaded cells using a NOVOstar platereader ***, <i>P</i><0.001 by Student's t test.</p

L-type VGCC activation is sufficient to mediate sustained ERK activation in MIN6 cells via local Ca²⁺ signalling.

<p>MIN6 cells incubated in KRB plus 2 mM glucose were loaded with 100 µM of EGTA-AM or BAPTA-AM prior to treatment with 10 µM Bay-K 8644 at room temperature for the times indicated. a) Proteins were resolved by SDS-PAGE and Western blotted using anti-phospho-ERK1/2 (pERK) or anti-ERK2 (ERK2) antibodies. A representative blot is shown with densitometric analysis of the results below showing mean +S.E.M. (n = 4). Results were analysed by two-way ANOVA with Bonferroni's multiple comparison test compared to Bay-K 8644 alone at each time point; ***, <i>P</i><0.001. b) MIN6 cells were treated as in (a) but in addition were loaded with 2 µM fura-2-AM and [Ca²⁺]_i levels measured by epifluorescence microscopy (n>30). A mean trace is shown. In addition, a representative trace obtained from MIN6 cells stimulated with 10 nM GLP-1 plus 16.7 mM glucose was included for comparison.</p

GLP-1-stimulated ERK activation requires L-type VGCC activation.

<p>a) MIN6 cells were preincubated for 1 h in KRB supplemented with 1 mM glucose. Cells were then incubated in 16.7 mM glucose in the absence or presence of 10 nM GLP-1. Proteins were resolved by SDS-PAGE and Western blotted using anti-phospho-ERK1/2 (pERK) or anti-ERK2 (ERK2) antibodies. A representative blot is shown above densitometric analysis of the results showing mean +S.E.M. (n = 5). All statistical comparisons were by one-way ANOVA with Bonferroni's multiple comparison test compared to glucose alone at each time point; *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001. b) MIN6 cells were preincubated for 1 h in KRB supplemented with 1 mM glucose. Cells were stimulated with 10 nM GLP-1 plus 16.7 mM glucose in the presence or absence of 10 µM nifedipine for the times indicated. Proteins were resolved by SDS-PAGE and Western blotted using anti-phospho-ERK1/2 and anti-ERK2 antibodies. A representative blot is shown above densitometric analysis of the results showing mean +S.E.M. (n = 3). All statistical comparisons were by one-way ANOVA with Bonferroni's multiple comparison test compared to GLP-1 plus glucose at each time point; *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001. c) MIN6 cells were preincubated for 1 h in KRB supplemented with 1 mM glucose. Cells were stimulated with 10 nM GLP-1 plus 16.7 mM glucose in the presence or absence of 50 µM dilitiazem for the times indicated. Cell lysates were analysed by SDS-PAGE and Western blotting using anti-phospho-ERK1/2 or anti-ERK2 antibodies. A representative blot is shown above densitometric analysis of the results ± S.E.M. All statistical comparisons were by one-way ANOVA with Bonferroni's multiple comparison test compared to glucose plus GLP1; *, <i>P</i><0.05; **, <i>P</i><0.01; (n = 3). d) MIN-6 cells were loaded with fura-2-AM and [Ca²⁺]_i levels measured using epifluorescence microscopy. i) Representative traces from single cells incubated with 1 mM glucose (control), 16.7 mM glucose (glucose), 10 nM GLP-1 plus 16.7 mM glucose (GLP-1/glucose) or 10 nM GLP-1 plus 16.7 mM glucose in the presence of 10 µM nifedipine (GLP-1/glucose + nifedipine). ii) Area under the curve (A.U.C.) across the 30 min stimulation showing mean +S.E.M. (n>30). Statistical comparisons were by one-way ANOVA with Dunnett's range test compared to GLP-1 plus glucose; ***, <i>P</i><0.001. e) Membrane potential recordings from MIN6 cells recorded in the perforated-patch, current-clamp mode. i) The effect of addition of 10 nM GLP-1 plus 16.7 mM glucose on membrane potential. ii) A membrane potential recording after 5.5 min in the continuous presence of 10 nM GLP-1 and 16.7 mM glucose. iii) After 10 min in the presence of 10 nM GLP-1 and 16.7 mM glucose, the effect on excitability of bath application of nifedipine (10 µM) was recorded.</p