SUMO protease FUG1, histone reader AL3 and chromodomain protein LHP1 are integral to repeat expansion-induced gene silencing in Arabidopsis thaliana.

Epigenetic gene silencing induced by expanded repeats can cause diverse phenotypes ranging from severe growth defects in plants to genetic diseases such as Friedreich's ataxia in humans. The molecular mechanisms underlying repeat expansion-induced epigenetic silencing remain largely unknown. Using a plant model with a temperature-sensitive phenotype, we have previously shown that expanded repeats can induce small RNAs, which in turn can lead to epigenetic silencing through the RNA-dependent DNA methylation pathway. Here, using a genetic suppressor screen and yeast two-hybrid assays, we identified novel components required for epigenetic silencing caused by expanded repeats. We show that FOURTH ULP GENE CLASS 1 (FUG1)-an uncharacterized SUMO protease with no known role in gene silencing-is required for epigenetic silencing caused by expanded repeats. In addition, we demonstrate that FUG1 physically interacts with ALFIN-LIKE 3 (AL3)-a histone reader that is known to bind to active histone mark H3K4me2/3. Loss of function of AL3 abolishes epigenetic silencing caused by expanded repeats. AL3 physically interacts with the chromodomain protein LIKE HETEROCHROMATIN 1 (LHP1)-known to be associated with the spread of the repressive histone mark H3K27me3 to cause repeat expansion-induced epigenetic silencing. Loss of any of these components suppresses repeat expansion-associated phenotypes coupled with an increase in IIL1 expression with the reversal of gene silencing and associated change in epigenetic marks. Our findings suggest that the FUG1-AL3-LHP1 module is essential to confer repeat expansion-associated epigenetic silencing and highlight the importance of post-translational modifiers and histone readers in epigenetic silencing.

Quantifying splice-site usage: a simple yet powerful approach to analyze splicing.

RNA splicing, and variations in this process referred to as alternative splicing, are critical aspects of gene regulation in eukaryotes. From environmental responses in plants to being a primary link between genetic variation and disease in humans, splicing differences confer extensive phenotypic changes across diverse organisms (1-3). Regulation of splicing occurs through differential selection of splice sites in a splicing reaction, which results in variation in the abundance of isoforms and/or splicing events. However, genomic determinants that influence splice-site selection remain largely unknown. While traditional approaches for analyzing splicing rely on quantifying variant transcripts (i.e. isoforms) or splicing events (i.e. intron retention, exon skipping etc.) (4), recent approaches focus on analyzing complex/mutually exclusive splicing patterns (5-8). However, none of these approaches explicitly measure individual splice-site usage, which can provide valuable information about splice-site choice and its regulation. Here, we present a simple approach to quantify the empirical usage of individual splice sites reflecting their strength, which determines their selection in a splicing reaction. Splice-site strength/usage, as a quantitative phenotype, allows us to directly link genetic variation with usage of individual splice-sites. We demonstrate the power of this approach in defining the genomic determinants of splice-site choice through GWAS. Our pilot analysis with more than a thousand splice sites hints that sequence divergence in cis rather than trans is associated with variations in splicing among accessions of Arabidopsis thaliana. This approach allows deciphering principles of splicing and has broad implications from agriculture to medicine.

Translation of insulin granule proteins are regulated by PDI and PABP.

Glucose mediated insulin biosynthesis is tightly regulated and shared between insulin granule proteins such as its processing enzymes, prohormone convertases, PC1/3 and PC2. However, the molecular players involved in the co-ordinated translation remain elusive. The trans-acting factors like PABP (Poly A Binding Protein) and PDI (Protein Disulphide Isomerize) binds to a conserved sequence in the 5'UTR of insulin mRNA and regulates its translation. Here, we demonstrate that 5'UTR of PC1/3 and PC2 also associate with PDI and PABP. We show that a' and RRM 3-4 domains of PDI and PABP respectively, are necessary for RNA binding activity to the 5'UTRs of insulin and its processing enzymes.

MicroRNA, Proteins, and Metabolites as Novel Biomarkers for Prediabetes, Diabetes, and Related Complications.

Type 2 diabetes mellitus (T2DM) is no more a lifestyle disease of developed countries. It has emerged as a major health problem worldwide including developing countries. However, how diabetes could be detected at an early stage (prediabetes) to prevent the progression of disease is still unclear. Currently used biomarkers like glycated hemoglobin and assessment of blood glucose level have their own limitations. These classical markers can be detected when the disease is already established. Prognosis of disease at early stages and prediction of population at a higher risk require identification of specific markers that are sensitive enough to be detected at early stages of disease. Biomarkers which could predict the risk of disease in people will be useful for developing preventive/proactive therapies to those individuals who are at a higher risk of developing the disease. Recent studies suggested that the expression of biomolecules including microRNAs, proteins, and metabolites specifically change during the progression of T2DM and related complications, suggestive of disease pathology. Owing to their omnipresence in body fluids and their association with onset, progression, and pathogenesis of T2DM, these biomolecules can be potential biomarker for prognosis, diagnosis, and management of disease. In this article, we summarize biomolecules that could be potential biomarkers and their signature changes associated with T2DM and related complications during disease pathogenesis.

Interaction of HuDA and PABP at 5'UTR of mouse insulin2 regulates insulin biosynthesis.

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. In vitro 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.

Prolonged exposure to insulin with insufficient glucose leads to impaired Glut4 translocation.

Insulin maintains glucose homeostasis by stimulating glucose uptake from extracellular environment to adipose and muscle tissue through glucose transporter (GLUT4). Insulin resistance plays a significant role in pathologies associated with type2 diabetes. It has been previously shown that hyperinsulinemia can lead to insulin resistance. In these studies very high levels of insulin was used to achieve insulin resistance. We hypothesized that one of the causes of type 2 diabetes could be insulin synthesis in the absence of glucose stimulation. We used CHO cell line, stably expressing Myc-GLUT4-GFP along with human insulin receptor to study the effect of hyperinsulinemia in the presence of low glucose (6.5 mM) or high glucose (20 mM). The insulin responsiveness of these cells was assessed by FRAP, FACS and subcellular fractionation. The results suggest that exposure of cells to insulin in low glucose conditions made these cells insulin resistant within 10 passages, while the same level of insulin in the presence of high glucose did not result in insulin resistance. These results clearly suggest that hyperinsulinemia combined with hypoglycaemia may lead to insulin resistance and may be one of the causes for the typ2 diabetes.