In pancreatic β-cells, miRNAs regulate glucose-stimulated insulin secretion and β-cell survival as well as insulin biosynthesis (Kim and Lee 2012). Recently, the Jeyaseelan group reported two miRNAs (miR-25 and miR-92a) as direct regulators of insulin biosynthesis at the mRNA level (Setyowati Karolina et al. 2013). The 3′ UTR of INS mRNA contains binding sites for miR-25 and 92a that are conserved in the rat and mouse and are partially conserved in humans. Overexpression of miR-25 or miR-92a not only reduced INS mRNA but also its biosynthesis; conversely, introduction of anti-miR-25 or miR-92a increases insulin biosynthesis. Interestingly, significant up-regulation of miR-25 and miR92a is observed in the pancreas of diabetic rats, in which INS mRNA levels are decreased.

Although miRNAs typically target the 3′ UTR of the mRNA for functional inhibition(Pillai et al. 2007; Yekta et al. 2004), they can also target the 5′ UTR and coding regions of mRNA as well as increase translation of target mRNAs through the 3′ and 5′ UTR (Zhou et al. 2009; Orom et al. 2008; Vasudevan et al. 2007). Most recently, the Seshadri group has reported that miR-196b can directly increase INS mRNA translation by targeting the 5′ UTR (Panda et al. 2014). Mouse miR-196b specifically targets the 5′ UTR of INS mRNA and the binding site of miR-196b overlaps with that of HuD, a 5′ UTR-associated RBP that represses INS mRNA translation. Through this binding, miR-196b increases INS mRNA translation and this is likely Ago2-dependent process, because miR-196b-mediated activation of insulin expression is abolished when Ago2 levels are depleted (Panda et al. 2014). Interestingly, suppression of miR-196b expression causes increased association of HuD with the 5′ UTR of INS mRNA and, conversely, this association is modestly decreased by overexpression of miR-196b, probably due to altered stem-loop structure of the 5′ UTR. HuD and miR-196b might compete for binding to the same site in the 5′ UTR of INS mRNA, providing an example of coordinated regulation of translation by miRNA and RBP (Fig. 2.2).

The Hu/ELAV (human antigen/embryonic lethal abnormal vision-like) family includes the ubiquitously expressed HuR (HuA) and the primarily neuronal HuB, HuC, and HuD. HuD/ELAVL4 is reported to specifically and directly affect post-transcriptional regulation of INS mRNA (Kim and Lee 2012). Like HuB and HuC, HuD expression was believed to be restricted primarily to neurons (Hinman and Lou 2008). However, recently it has been shown to be expressed in insulin -producing pancreatic β-cells (Lee et al. 2012; Poy et al. 2009), in which its levels are controlled by the insulin signaling pathway, sequentially implicating Irs2, PI3K, Akt, and FoxO1 (Fig. 2.2). Like other Hu/ELAV family proteins, HuD controls stability and translation of target mRNAs by binding to 5′ or 3′ UTR bearing AU- and U-rich sequences through three highly conserved RNA recognition motifs (RRMs) (Hinman and Lou 2008; Pascale et al. 2008).

In pancreatic β-cells, HuD, but not HuR and other RBPs, including HuR, TIAR [T-cell-restricted intracellular antigen-1 (TIA-1)-related protein], heterogeneous nuclear ribonucleoprotein (hnRNP) K, and nuclear factor (NF) 90, as well as hnRNP C, Ago, and the fragile X syndrome protein (FMRP ), associates with a 22-nucleotide segment in the 5′ UTR , but not with the coding region (CR) or 3′ UTR of mouse INS2 mRNA (Lee et al. 2012). Binding of HuD to the INS2 5′ UTR represses insulin production by reducing the INS2 mRNA translation, and not by altering INS2 mRNA stability . This binding is repressed by glucose treatment, which induces a rapid and robust release of INS2 mRNA from the HuD complex within 30 min of glucose stimulation thereby enables translation of INS2 mRNA (Lee et al. 2012) (Fig. 2.2). Consistently, insulin levels are increased in pancreatic β-cells of HuD-null mice, and conversely, decreased in HuD-transgenic mice. This modulation of insulin by HuD in vivo likely contributes the homeostatic regulation of plasma glucose and insulin concentrations. In support of this is the finding that HuD-transgenic mice display impaired glucose clearance from the blood due to lower plasma insulin levels, thus defining HuD as a pivotal post-transcriptional regulator of insulin.

The polypyrimidine tract-binding protein (PTB ), also known as hnRNP I, binds to pyrimidine-rich sequences of single stranded target mRNAs through four RRMs and plays an important role in several cellular processes such as alternative splicing (Garcia-Blanco et al. 1989; Valcarcel and Gebauer 1997; Wagner and Garcia-Blanco 2001), polyadenylation of pre-mRNAs (Lou et al. 1999; Moreira et al. 1998), cytoplasmic RNA localization (Cote et al. 1999), mRNA stability (Tillmar et al. 2002; Knoch et al. 2004) and translation initiation (Hellen et al. 1993). Glucose-induced stabilization of INS mRNA is a key event in the control of insulin biosynthesis and PTB plays an essential role in this process. In pancreatic β-cells, glucose induces the binding of PTB to the polypyrimidine-rich sequence located in the 3′ UTR of INS mRNA to stabilize INS mRNA, resulting in a glucose-dependent increase in INS mRNA (Tillmar et al. 2002; Tillmar and Welsh 2002). Glucose stimulation of β-cells enhances the cytoplasmic function of PTB by promoting its accumulation in the cytoplasm, upon which PTB binds and stabilizes INS mRNAs (Knoch et al. 2006). The cytoplasmic accumulation of PTB is regulated by cAMP and protein kinase A (PKA)-dependent phosphorylation (Xie et al. 2003; Knoch et al. 2006). Activation of PKA by elevation of cAMP levels causes the direct phosphorylation and nucleo-cytoplasmic transport of PTB (Fig. 2.3). Consistently, glucagon-like peptide 1 (GLP-1), which activates PKA and potentiates glucose-stimulated insulin gene expression and secretion by increasing cAMP levels in β-cells, promotes the phosphorylation of PTB, and conceivably, its cytoplasmic accumulation, which in turn enhances the levels of mRNAs containing PTB binding sites in their 3′ UTR (Knoch et al. 2006). However, GLP-1does not stabilize INS mRNA significantly in either rat insulinoma INS-1 cells or freshly isolated islets (Knoch et al. 2006).

Poly(A)-binding protein (PABP ) is a multifunctional RNA binding protein with an N-terminal RNA binding domain composed of four RRMs, and a C-terminal helical domain (Adam et al. 1986), which is important for its interaction with other proteins and for cooperative binding to poly(A) tails (Melo et al. 2003). Through poly(A) tail binding, PABP modulates the stability and translation of target mRNAs (Sonenberg and Dever 2003). Moreover, PABP can also bind to the A-rich sequence in the 5′ UTR to regulate key steps in mRNA translation (de Melo Neto et al. 1995; Oberer et al. 2005). In pancreatic β-cells, PABP binds specifically to the 5′ UTR of INS mRNA and increases the rate of ribosome recycling through its interaction with other translation factors; this leads to higher insulin translation during glucose stimulation (Kulkarni et al. 2011). The binding of PABP to the 5′ UTR of INS mRNA during glucose stimulation is regulated by protein disulfide isomerase (PDI), which was the first identified ER-resident catalyst of native disulfide bond formation (Anfinsen 1973). Glucose stimulation of β-cells induces PDI activation by promoting its phosphorylation. Activated PDI interacts with PABP and catalyzes the reduction of the PABP disulfide bond resulting in specific binding of PABP to the 5′ UTR of INS mRNA and increased insulin translation (Kulkarni et al. 2011).

Although various splice variants of IGF-I and IGF-II have been characterized (Goldspink and Yang 2004; Velloso and Harridge 2010; Weller et al. 1993; Yang et al. 1995), the factors that govern the spicing factors are largely unknown. The serine-arginine protein splicing factor-1/alternative splicing factor (SF2/ASF) binds to a purine-rich sequence in exon 5 of IGF-I mRNA to promote its inclusion in the mature transcript (Smith et al. 2002). Exon 5 inclusion is responsible for myogenic differentiation, muscle hypertrophy, and myopathy (Barton et al. 2002; Matheny and Nindl 2011). The 5′ UTR composition of IGF-II mRNA varies throughout development due to alternative splicing (Ohlsson et al. 1994; Ekstrom et al. 1995; Monk et al. 2006), although the mechanisms and regulators required for this currently remain uncharacterized.