miR-196b-Mediated Translation Regulation of Mouse Insulin2 via the 5′UTR

The 5′ and the 3′ untranslated regions (UTR) of the insulin genes are very well conserved across species. Although microRNAs (miRNAs) are known to regulate insulin secretion process, direct regulation of insulin biosynthesis by miRNA has not been reported. Here, we show that mouse microRNA miR-196b can specifically target the 5′UTR of the long insulin2 splice isoform. Using reporter assays we show that miR-196b specifically increases the translation of the reporter protein luciferase. We further show that this translation activation is abolished when Argonaute 2 levels are knocked down after transfection with an Argonaute 2-directed siRNA. Binding of miR-196b to the target sequence in insulin 5′UTR causes the removal of HuD (a 5′UTR-associated translation inhibitor), suggesting that both miR-196b and HuD bind to the same RNA element. We present data suggesting that the RNA-binding protein HuD, which represses insulin translation, is displaced by miR-196b. Together, our findings identify a mechanism of post-transcriptional regulation of insulin biosynthesis.


Introduction
Insulin is a small peptide hormone secreted by pancreatic b cells and is important for glucose homeostasis in mammals. Insulin expression begins at embryonic E9.5 day in the gut endoderm [1]. Insulin expression in b cells is regulated by many nutrients, but mainly by glucose. Interestingly, glucose stimulation results in insulin secretion within minutes [2] and is immediately followed by specific increase in insulin translation [3]. The 59 and 39untranslated regions (UTR's) of insulin mRNA have been shown to have a role in this translation regulation [4;5]. In mouse, two nonallelic genes encode for insulin and specific splice variants from these genes have also been reported [6;7]. Some of the splice variants have altered 59UTR and have differential translation efficiency and hence have been implicated in diabetes [6].
MicroRNAs (miRNAs) are short (,22-nt) regulatory RNAs that influence a number of pancreatic events, including the development of pancreatic islets and b cells [8;9], insulin secretion [10][11][12], insulin resistance and diabetes [13][14][15]. Normally, miRNAs target the 39UTRs, causing degradation and/or translational repression of the target mRNA [16]; occasionally, miRNAs have also been found to activate translation through the 39UTR [17]. In addition, bioinformatics studies have suggested the presence of a large number of potential target sites in the 59UTRs and coding regions of mRNAs [18]. 59UTR-targeted miRNA-mediated translation increase has been shown for miR-10a and the target ribosomal protein mRNA [19]. miRNA can increase translation by targeting the 59UTR and 39UTRs with or without involvement of Argonaute 2 (Ago2), a key player of the RISC (RNA-induced Silencing Complex) [19;20]. However, the mechanism of miRNAmediated increase in translation is not fully understood.
In our previous study, we showed that mouse insulin2 mRNA undergoes alternative splicing, resulting in a shorter 59UTR splice variant insulin2-S lacking 12 nucleotides in the 59UTR. The short 59UTR splice variant constitutes 75% of the insulin2 mRNA pool, and has an increased translation efficiency [7]. In the present study, we show that miR-196b can specifically target the 59UTR of insulin2 mRNA (the longer 59UTR containing a splice variant) and regulates its translation in an Ago2-dependent manner. Interestingly, miR-196b increases target gene expression without affecting insulin2 mRNA levels but by enhancing the size of insulin2 mRNA polysomes, indicating that miR-196b upregulates insulin2 translation.

Results
Identification of miRNA that target 59UTR of mouse insulin mRNA Insulin mRNA expressed in mouse pancreas consists of a pool of transcripts containing different 59UTRs. The variations in the 59UTR are due to two non-allelic genes as well as alternative splicing, resulting in at least three different 59UTRs in insulin mRNA. In light of recent reports that microRNAs can function through the 59UTRs of target mRNAs, we explored the possibility that the insulin mRNA 59UTR isoforms could contribute to their differential regulation via miRNA actions. miRNAs that can potentially target insulin 59UTR were identified by MicroInspector web tool. Four miRNAs (miR-196b, miR-323-5p, miR-338-5p and miR-370) with high complementarity to seed sequences (at least 5 base pairs between nucleotide position 2-8 of the miRNA) and a free energy of less than 222 kCal/Mole were selected (Table 1) for further analysis.
Mouse miR-196b activates the expression of insulin2-59UTR reporter construct The translation regulation ability of these miRNAs was tested using a luciferase reporter system. Insulin 59UTR corresponding to mouse insulin1, insulin2 and insulin2-S were cloned at the 59UTR of a luciferase reporter gene. The luciferase reporter plasmids, the control renilla plasmids, and the microRNAs were transfected into human embryonic kidney (HEK) 293T cells; 48 hours later, luciferase activity was measured. Transfection of miR-323-5p (data not shown), miR-338-5p, and miR-370 plasmids did not affect the expression of luciferase reporter from constructs containing the respective target sequence in the 59UTR ( Fig  S1A-B). Additionally, transfection of miR-370 and miR-338-5p duplexes did not show any significant change in reporter activity when transfected with insulin1 and insulin2-S reporters, respectively (Fig. S1C,D). Similarly, transfection of miR-370 and miR-338-5p duplexes along with insulin2 reporter also did not show any change in reporter activity when compared to control miRNA (Fig. S1E). However, transfection of miR-196b plasmid resulted in an increase in luciferase activity from constructs containing the target insulin2 59UTR by about 50% (Fig. 1A). Luciferase mRNA levels, assessed by quantitative PCR, showed that miR-196b transfection did not result in increased luciferase mRNA levels, but actually caused a modest decrease in luciferase mRNA levels. Thus, the increase in relative luciferase activity upon miR-196b transfection was likely due to an increase in translation and not due to increased mRNA abundance. The expression of miR-196b had no effect on the other 59UTR luciferase constructs that did not contain the target sequence in the 59UTR.
Expression of miR-196b did not change significantly the luciferase activity derived from insulin2-luciferase in bTC6 cells, an insulin-producing cell line (Fig. S2A), possibly because of low expression levels of the miRNA and/or because of the large amount of endogenous insulin2 mRNA already present in the cell. In order to overcome this limitation, we synthesized the duplex miR-196b RNA and co-transfected it with the insulin2-59UTRluciferse construct in HEK293T and bTC6 cells. A ,140% increase in luciferase expression was observed in HEK293T cells when compared to control miRNA without any significant change in the mRNA level (Fig. 1B). Interestingly, in bTC6 cells miR-196b expression increased the relative luciferase activity of the insulin2 59UTR-containing reporter (Fig. 1C). Surprisingly, The miR-196b target site is just at the exon1-exon2 junction of the insulin2 mRNA which makes it very specific to this insulin2 isoform, not the insulin2-S splice variant, which lacks the miR-196b seed sequence. Therefore, insulin2-S 59UTR containing reporter was used as a control reporter in all further experiments ( Fig. 2A). The control insulin2-S luciferase reporter did not show any significant change in the expression with miR-196b transfection in bTC6 cells (Fig. S2B). These data suggest that the underlying mechanism of the miR-196b-mediated translation regulation of the insulin2 59UTR containing mRNA can occur in both insulin-producing and non-producing cells.
We assessed the specificity of the miR-196b-mediated translation regulation by using a miR-196 antagonist, antisense miR-196b. The Luc reporter plasmid was co-transfected with miR-196b or control miRNA and 29O-methylated antisense inhibitor of miR-196b, in HEK293T cells. miR-196b inhibitor blocks the miR-196 mediated activation of insulin2-59UTR-Luciferase translation (Fig. 2B). The control reporter without the miR-196b target site showed no significant change in expression with the inhibitor (Fig. 2C). Similar results were obtained with bTC6 cells (Fig. 2D). The mutant insulin2-reporter expression was not affected by miR-196b in bTC6 cells (Fig. 2E). These results indicate that mouse miR-196b regulates the translation of the mouse insulin2 mRNA by targeting its 59UTR.

Argonaute 2 is required for miR-196b-mediated translation upregulation
Since miR-196b targets the 59UTR and activates translation, we tested whether Ago2, an important component of the RISC, is required for translation activation mediated by this microRNA. We knocked down Ago2 using a specific siRNAs in HEK293T cells and transfected the reporter containing insulin2 59UTR along with miR-196b. Ago2 siRNA reduced Ago2 expression levels by almost 70% and the miR-196b-mediated translation activation was almost completely inhibited, while the control siRNA did not show any significant alterations in the expression levels (Fig. 3A).

miR-196b inhibits the formation of Insulin2-59UTRprotein complexes
We have previously shown that specific factors bind to the insulin 59UTR with differential efficiency [7]. A specific RNAprotein complex is formed with insulin2 59UTR and cytoplasmic protein factors, but in case of insulin2-S, the complex formation is reduced. In vitro and in vivo translation experiments showed a correlation between the complex formation and reduced translation efficiency, suggesting that the trans-acting factor that associates with insulin2-59UTR is likely to be a translation inhibitor. The translation efficiency of insulin2 mRNA is the lowest among mouse insulin mRNAs (Fig. S3). We hypothesized that the miRNA could activate translation by interfering with the RNA-protein interactions at the 59UTR. Therefore, we analysed the RNA-protein complex formation in the presence and absence of miRNA-196b.   The miR-196b sense strand or antisense strand were incubated with the insulin2 59UTR before the addition of lysate, and the RNA-protein complex formation was assessed by RNA electrophoretic mobility shift assay (REMSA). The sense strand miR-196b inhibits the complex formation while the antisense strand had no effect on the complex formation in bTC6 cells (Fig. 3B). We observed similar results using extracts from HEK293T cells (Fig. 3C). These data suggest that the binding of miR-196b disrupts the RNP (ribonucleoprotein) complex formation.

miR-196b causes reduced association of mouse Insulin2 mRNA with HuD
To study the effect of miR-196b on insulin translation, we analyzed the association of Insulin2 reporter with the cell9s polysomes in Ctrl siRNA-or miR-196b transfected bTC6 cells. miR-196b did not affect the global translation profile of bTC6 cells (Fig.4A). RT-qPCR analysis of polysomal fractions show an increased association of the insulin 2 reporter with the polysome upon miR-196b treatment, suggesting that miR-196b promotes the translation of Insulin2 in bTC6 cells (Fig.4B). However, we did not see specific change in polysome association of endogenous mouse insulin2 mRNA following miR-196b transfection (Fig.S4), perhaps because the vast abundance of endogenous insulin mRNA.
Recently, Lee et al. have shown that the RNA-binding protein HuD bind to insulin 59UTR and repressed the translation of insulin mRNA [21]. As miR-196b also targets the 59UTR of insulin2 mRNA, we sought to determine if there is any interplay between HuD and miR-196 in controlling Insulin2 translation. Following transfection of control RNA, miR-196b miRNA or miR-196b-as inhibitor into bTC6 cells along with the Insulin2 reporter, the interaction of HuD with the reporter mRNA was assessed by ribonucleoprotein immunoprecipitation (RIP) analysis followed by detection of the reporter RNA in the IP material. Interestingly, we found increased association of HuD with the insulin 59UTR reporter after antagonizing miR-196b function (miR-196b-as group), while expression of miR-196b modestly decreased the association of the insulin 59UTR reporter mRNA with HuD. These results suggest that HuD and miR-196b might compete for binding to the 59UTR of Ins2 mRNA (Fig. 4C). We also assessed the effect of the miR-196b expression on the association of HuD with endogenous insulin mRNA and find similar trend, although to a lesser degree (Fig. S5).

miR-196b competes with HuD for binding to 59UTR of Insulin2 mRNA
To analyse the functional interplay between miR-196b and HuD upon insulin 2 translation we followed the gain of function and loss of function experiments. The over expression of HuD in HEK293T cells along with the insulin2 reporter and miR-196b abolished the miR-196b mediated translation up regulation (Fig. 4D). This finding suggests that HuD/miR-196b compete for binding to 59UTR of insulin2 mRNA. Further, we silenced HuD in bTC6 cells and studied the effect of HuD knock down on translation upregulation by miR-196b (Fig. 4E). Upon HuD silencing, we observed even higher insulin2 translation as compared with miR-196b overexpression alone (Fig. 4F).

miR-196b is expressed in mouse bTC6 cells and embryonic pancreas
The expression levels of various miRNAs were analysed in mouse bTC6 cells. miR-196b, miR-338-5p, miR-370 and miR-375 were previously reported to be expressed in adult mouse pancreas [22]. We detected miR-196, miR-30d and miR-375 in bTC6 cells using QuantiMir RT-PCR kit (Fig. 5A-B). We then calculated the copy number of the above miRNAs using known amounts of miRNA using the QuantiMiR assay system by RT-qPCR (Fig. 5C). This result shows the relatively lower expression of miR-196b in bTC6 cells as compared to other two wellcharacterized miRNAs. We also detected miR-196b expression in mouse embryonic pancreas by using the stem-loop primer RT-PCR method (Experimental Methods). In mouse embryonic pancreas at day e14.5, specific PCR products were detected corresponding to miR-196b and miR-375 (Fig. 5D), and isoforms of insulin2 mRNA such as insulin2 and insulin2-S (Fig. 5E). These data indicate that during embryonic development miR-196b and insulin2 were expressed together in the same tissue.

Glucose increases the miR-196b expression in bTC6 cells
Recent reports suggested that miRNAs are regulated by glucose in MIN6 cells. In order to analyse the effect of glucose on the expression on miR-196b in bTC6 cells, we incubated bTC6 cells in the absence of glucose or in the presence of 25 mM glucose for 16 h, and prepared total RNA. Analysis of the miRNA cDNA (using QuantiMiR kit) indicated that miR-196b along with miR-30D was upregulated in the presence of high glucose, whereas miR-375 level did not change significantly (Fig. 5F). The insulin2 reporter expression also increased under these conditions, suggesting a potential positive influence of the heightened miR-196b levels (Fig. 5G). These results suggest that increased miR-196b expression in response to glucose may be an additional mechanism for glucose-stimulated insulin synthesis.
Discussion miRNAs regulate the expression of an estimated one-third of genes in mammals [23;24]. miRNAs typically target the 39UTR of the mRNA and suppress its expression by inhibiting translation and/or by degrading the target mRNA [16;25]. Recently, several miRNAs have been shown to target other regions in the mRNA and some of these miRNAs have also been shown to increase the expression of target genes. In pancreatic b cells, several miRNAs and RNA binding proteins have been reported to regulate the glucose-induced insulin expression [26;27]. Here, we report the role of mouse miR-196b in the regulation of insulin2 mRNA translation. miR-196b exists in the HOX gene cluster and regulates HOXB8 gene by targeting the 39UTR expressed in mouse embryonic stages [25] as well as in adult pancreas [22]. We show that miR-196b is expressed at day e14.5 in the embryonic pancreas and propose that it could be involved in the regulation of translation of insulin genes specifically during embryonic development. This increased translation of insulin could be important since embryonic pancreas needs to maximize the production of insulin for organogenesis and growth. We believe that miR-196bmediated translation upregulation of embryonic insulin is an adaptation to this specific need for increased insulin. miR-196b targets the 59UTR of mouse insulin2 mRNA and we hypothesize that the miRNA mediated up-regulation is due to  reduced association of HuD to insulin 59UTR (Fig. 6). The decreased binding of HuD is probably due to altered stem-loop structure of the 59UTR or due to competition with miRNA for the binding to mRNA. These results suggest a coordinated regulation of translation by miRNA and UTR-binding proteins, similar to those observed in case of miR-466I [28]. Ago2 is an important player in miRNA-mediated regulation of gene expression; our results indicate that the miR-196b-mediated activation of insulin expression also requires Ago2, suggesting that the miRNA-196bmediated displacement of HuD might involve Ago2, although the exact mechanism of the translation regulation is still unknown. This regulation is similar to the regulation observed for mRNA of some of the ribosomal genes that contain the TOP element in the 59UTR. These mRNAs are positively regulated by miR-10b and negatively regulated by binding of specific factors to the 59UTR of the mRNA [29].
The physiological relevance of this regulation is still unclear, as insulin2 isoform contributes only about 13% of total Insulin mRNA and is poorly translated when compared to the mouse Insulin1 or the insulin2-S isoform [7]. Thus, under normal physiological conditions, the protein product from this mRNA isoform is less than 10% of the total insulin produced (Fig. S3). But this regulation could be important in embryonic development where it can significantly increase the insulin production from a reduced pool of insulin mRNA transcripts.
The competition between HuD and miR-196 for binding to insulin2 mRNA may explain the mechanism of translation upregulation by miR-196b. The requirement of Ago2 for such an upregulation suggests the involvement of RISC complex in this activity and not simply an antisense RNA activity. Further, regulation of miR-196b expression by glucose suggests an additional level of regulation of insulin biosynthesis by a novel mechanism that could play an important role in the case of diabetes.
In summary, the present study demonstrates for the first time that a microRNA can target a specific splice variant of the insulin mRNA and can promote its translation. We propose that miR-196b interaction with insulin mRNA disrupts the interaction of HuD to the mRNA thereby resulting in translation activation.

miRNA search
Potential miRNAs that can target mouse insulin mRNAs were identified by using the MicroInspector web tool (http://bioinfo. uni-plovdiv.bg/microinspector/). This search engine predicts the miRNAs based on the seed sequence complementarity and binding energy (DG) of miRNA-mRNA duplex.

Pancreas isolation and cell culture
These procedures were carried out in strict accordance with the recommendations of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Government of India. The protocol was approved by the Institutional Animal Ethics Committee (IAEC) of National Centre for Cell Science (protocol Number B-192), and all efforts were made to minimize suffering of the experimental animals. BALB/c mice (6-8-weeks old) were used for pancreas isolation. The embryos from BALB/c mice were dissected from pregnant mothers at day e14.5, and the embryonic pancreas was dissected and cut off from the surrounding tissue. Adult pancreatic islets were prepared by ficoll gradient method, as described [30]. RNA was prepared using Trizol (Invitrogen, Carlsbad, CA).
Transfection, reporter assay and RNA quantitation HEK293T or bTC6 cells were cultured in 24-well plates and transfected with various plasmids/RNA using Lipofectamine-2000 (Invitrogen). Ten ng of pRL-Tk (Promega) was included in all the transfection reaction to serve as transfection control. Fifty ng of luciferase reporter containing various insulin 59UTRs, 500 ng of miR-196b-pSUPER or empty vector were transfected; 48 hr later, the cells were harvested and luciferase activity was measured with the DLR kit (Promega). The luciferase activity was normalized with the renilla expression levels.
Biotin-miR-196b RNA duplex was prepared by annealing both strands (synthesized by IDT, USA) in annealing buffer (30 mM HEPES-KOH pH 7.4, 2 mM MgCl 2 , 100 mM KCl, 50 mM NH 4 CH 3 COOH) at a final concentration of 20 mM. miR-196 or control duplexes were transfected at 40 nM concentration for 4 hr in the presence of Opti-MEM without serum and then substituted with complete medium with serum for further 2 hr before transfecting with the reporter plasmids. Unlabeled mature miR-196b duplex RNA was obtained from QIAGEN. For the miRNA inhibitor experiment, 100 pmoles of miR-196b inhibitor (59CC-CAACAACAGGAAACUACCUA39-29O-methyl anti-miR-196b, IDT) was transfected along with the duplex miRNA 6 hr before prior to reporter miRNA transfection. For siRNA studies, the control siRNA and the Ago2 siRNA (target sequence: GCAG-GACAAAGATGTATTA, Dharmacon, USA) were transfected and recovered for 24 hr before transfection with the luciferase reporter and the miRNA constructs.
Myc-HuD plasmid (500 ng) was transfected to HEK29T cells before the transfection of insulin2 reporter and miR-196. HuD was silenced in bTC6 cells by sequential transfection of HuD siRNA (Santa Cruz), 24 hr apart, followed by the reporter and miR-196b transfection and 48 hr later the reporter expression was measured.
RNA from the transfected cells was prepared using TRIzol and the luciferase mRNA levels were assessed by RT-PCR and normalised to human beta actin or mouse GAPDH mRNA levels by quantitative PCR Assay (ABI). TaqMan qPCR for luciferase was performed using oligos 21 and 22 as primers, and 23 as probe (Table S1).

RT-PCR analysis of miRNA and insulin mRNA
First-strand synthesis was performed from 200 ng of total RNA from embryonic pancreas using the miR-196b-RT stem-loop expressing normal or reduced HuD levels (F). The fold change in relative luciferase activity was measured with the activity of the luciferase construct with control miRNA set to 1. The graphs in (C,D,F) represent the means 6 SD of 3-9 independent experiments; P values (Student's t-test) are indicated. doi:10.1371/journal.pone.0101084.g004 miR-196b Promotes Insulin Biosynthesis PLOS ONE | www.plosone.org Comparison of miR-196b, miR-30d, and miR-375 expression from RT-qPCR data of bTC6 total. Copy numbers per 1 pg of total RNA were calculated using standard curve based on known amount of miRNA. (D) cDNA for miR-196b was prepared by miR-196b-RT stem-loop primer and amplified with specific primers (primers 18 and 20, Table S1). The cDNA for miR-375 was prepared by miR-375-RT stem-loop primer and amplified with specific primers (primers 19 and 20, Table S1) from e14.5 day pancreas and the PCR product was resolved on a 3.5% agarose gel. (E) Insulin2 RT-PCR detection miR-196b Promotes Insulin Biosynthesis PLOS ONE | www.plosone.org primer and the ImProm II reverse transcriptase kit (Promega) as described previously [31]. The RT-PCR was performed with a common reverse primer 20, and forward primers 18 and 19 for miR-196b and miR-375, respectively (Table S1) (95uC for 5 min, followed by 5 cycles of 95uC for 15 s, 36uC for 30 s and 72uC for 5 s, followed by 40 cycles of 95uC for 15 s, 50uC for 30 s and 72uC for 5 s).
For detecting miRNA in bTC6 cells, 1 mg of TRizol prepared total RNA was used for cDNA synthesis using the QuantiMir cDNA Kit (System Biosciences, Mountain View, CA) following the manufacturer's protocol. miRBase database (http://www.mirbase. org) was used to make the forward primers for RT-qPCR of the miRNAs. A universal reverse primer was supplied by the kit. The real-time PCR was performed with primers specific to the miRNA and the universal reverse primer using the manufacture's protocol.
Immunoblotting and RNA-EMSA HEK293T or bTC6 cells were lysed in a ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM DTT 1 mM PMSF and protease inhibitor) by 10 strokes of Dounce homogenizer and the lysate was cleared by centrifugation at 20,000g for 20 minute at 4uC. Labelled insulin2 59UTR was in vitro transcribed from the annealed double stranded oligomer using T7 RNA polymerase (Ambion) in presence of 50 mCi [a-32 P] UTP. For EMSA reactions, 20000 cpm probe was heated at 65uC for 3 min with 10 pmoles of miR-196b sense or antisense RNA and quickchilled on ice for 10 minute with gel shift buffer (5 mM Tris (pH 7.5), 15 mM KCl, 5 mM MgCl 2 , 0.25 mM DTT, 40 U of RNasin, and 10% glycerol) followed by the addition of 5 mg of the extract and further incubated on ice for 30 minutes. The RNP complex was analyzed by 6% PAGE, as described previously [7]. The Ago2 siRNA/Cont siRNA lysates were resolved on a 10% SDS-PAGE and proteins were transferred on to PVDF membrane. The membrane was probed with anti-Ago2 antibody (Cell Signalling) and b-Actin (Abcam) and detected by ECL TM Advance (Amersham). HuD overexpression or HuD siRNA samples were resolved in 4-20% TGX gels (BioRad) and transferred to nitrocellulose membrane. The membranes were probed with HuD, GAPDH or HSP90 antibodies from Santa Cruz.

Polysome Analysis
bTC6 cells were transfected with insulin2-reporter along with Ctrl miRNA or miR-196b for 48hr before it was taken for Polysome fractions. The cells were preincubated with cycloheximide (Sigma; 100 mg/ml for 15 min), and cytoplasmic lysates were fractionated into 12 fractions through 15%-60% linear sucrose gradients using ultracentrifuge. RNA was prepared from the collected fractions using TRIzol (Invitrogen) and followed by RT-qPCR analysis of reporter mRNA and GAPDH mRNA [21].

Endogenous HuD-mRNA complex immunoprecipitation
To analyze the association of HuD to endogenous mRNAs in bTC6 cells immunoprecipitation (IP) of RNP complexes were performed as described previously [32]. Briefly the bTC6 cells were lysed in 20 mM Tris-HCl at pH 7.5, 100 mM KCl, 5 mM MgCl 2 , and 0.5% NP-40 for 10 min on ice and cleared by centrifugation at 15,000 6 g for 10 min at 4uC. The lysate was incubated with protein-A Dynabeads beads coated with antibodies recognizing HuD or control IgG (Santa Cruz Biotechnology) for 2 hr at 4uC. The beads were washed thrice with ice cold NT2 using gene-specific primer (primers 1 and 2, Table S1) using RNA isolated from e14.5 day mouse pancreas. (F) The change in expression of various miRNAs in high glucose treated bTC6 cells using QuantiMiR RT-qPCR kit. (G) Expression of insulin2 reporter in high glucose treated bTC6 cells normalized to renilla expression. In (C,F,G) the graphs represent means 6 SD of 3-8 independent experiments; P values (Student's t-test) are indicated. doi:10.1371/journal.pone.0101084.g005 Figure 6. Mechanism of miR-196b action. The miR-196b target site is at the 59UTR stem loop structure of the insulin2 mRNA. Targeting of miR-196b to the stem-loop region of the insulin2 mRNA disrupts the secondary structure and prevents binding of the translational inhibitor, resulting in the activation of insulin translation. doi:10.1371/journal.pone.0101084.g006 buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM MgCl 2 , and 0.05% NP-40) followed by DNase treatment with 20 units of DNase I for 15 min at 37uC to remove the DNA. The samples are then incubated with 0.5 mg/ml Proteinase K supplemented with 0.1% SDS/ for 15 min at 55uC to digest the proteins. For analysis of individual mRNAs the RNA from the IP samples were prepared by phenol-chloroform and used for qRT-PCR.