SHARAKU: an algorithm for aligning and clustering read mapping profiles of deep sequencing in non-coding RNA processing.

MOTIVATION: Deep sequencing of the transcripts of regulatory non-coding RNA generates footprints of post-transcriptional processes. After obtaining sequence reads, the short reads are mapped to a reference genome, and specific mapping patterns can be detected called read mapping profiles, which are distinct from random non-functional degradation patterns. These patterns reflect the maturation processes that lead to the production of shorter RNA sequences. Recent next-generation sequencing studies have revealed not only the typical maturation process of miRNAs but also the various processing mechanisms of small RNAs derived from tRNAs and snoRNAs. RESULTS: We developed an algorithm termed SHARAKU to align two read mapping profiles of next-generation sequencing outputs for non-coding RNAs. In contrast with previous work, SHARAKU incorporates the primary and secondary sequence structures into an alignment of read mapping profiles to allow for the detection of common processing patterns. Using a benchmark simulated dataset, SHARAKU exhibited superior performance to previous methods for correctly clustering the read mapping profiles with respect to 5'-end processing and 3'-end processing from degradation patterns and in detecting similar processing patterns in deriving the shorter RNAs. Further, using experimental data of small RNA sequencing for the common marmoset brain, SHARAKU succeeded in identifying the significant clusters of read mapping profiles for similar processing patterns of small derived RNA families expressed in the brain. AVAILABILITY AND IMPLEMENTATION: The source code of our program SHARAKU is available at http://www.dna.bio.keio.ac.jp/sharaku/, and the simulated dataset used in this work is available at the same link. Accession code: The sequence data from the whole RNA transcripts in the hippocampus of the left brain used in this work is available from the DNA DataBank of Japan (DDBJ) Sequence Read Archive (DRA) under the accession number DRA004502. CONTACT: yasu@bio.keio.ac.jp SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Transcriptional factors, Mafs and their biological roles.

The Maf family of transcription factors is characterized by a typical bZip structure; these transcription factors act as important regulators of the development and differentiation of many organs and tissues, including the kidney. The Maf family consists of two subgroups that are characterized according to their structure: large Maf transcription factors and small Maf transcription factors. The large Maf subgroup consists of four proteins, designated as MAFA, MAFB, c-MAF and neural retina-specific leucine zipper. In particular, MAFA is a distinct molecule that has been attracting the attention of researchers because it acts as a strong transactivator of insulin, suggesting that Maf transcription factors are likely to be involved in systemic energy homeostasis. In this review, we focused on the regulation of glucose/energy balance by Maf transcription factors in various organs.

MafA is a Key Molecule in Glucose and Energy Balance in the Central Nervous System and Peripheral Organs.

MafA is a strong transactivator of insulin in pancreatic ß cells. Elucidating the profile of MafA action in organs other than the pancreas is essential. We established an mRNA interference technique that modifies the level of target mRNAs in mice in vivo. After rapidly injecting MafA-siRNA, the resulting changes in the gene profile were analyzed using a microarray system. Significant suppression of the MafA mRNA levels was observed in the pancreas, liver, adipose tissue, and brain of siRNA-injected mice. As we reported previously, the down-regulation of insulin mRNA and adipocytokines was observed in the pancreas, and MafA siRNA caused alterations in the expressions of genes related to lipid metabolism and cell growth in the liver, and the attenuation of cell differentiation in cultured adipocytes. In addition to the effects on these organs, MafA expression was immunohistochemically detected in the brain in our preliminary data, and the expression level in siRNA-treated mice was significantly suppressed. The expressions of the affected genes were distinct, including growth hormone, vasopressin, hypocretin, and pro-melanin-concentrating hormone, were almost completely down-regulated (to ~1/100). These results suggested that MafA is likely involved in the regulation of hormonal systems related to glucose metabolism, and MafA is likely positioned near the beginning of the cascade or may influence the expressions of the above-mentioned genes in coordination with other factors in brain tissue. Taken together, the findings in this study suggested that MafA functions as a transcription factor with distinct activities in each organ and is cross-linked in several organs.

Suppression of MafA mRNA with siRNA prevents adipose cell differentiation in 3T3-L1 cells.

One of the large Mafs, MafA protein, is a strong transactivator of insulin in pancreatic beta cells. Mafs are also known to play important roles in a variety of developmental and differentiation processes in many organs and tissues. Adipocytes are highly involved in insulin actions and glucose and lipid metabolism, and their proliferation and differentiation is regulated by coordination of several signal transduction and transcriptional factors, including members of the Maf family. To explore the role of MafA in adipocytes, we modified the MafA mRNA level in cultured adipocytes by the RNA interference technique and analyzed the resulting morphological changes and changes in expression of related genes. MafA siRNA was transfected into 3T3-L1 adipocytes. Expression of MafA was confirmed by real-time PCR and Western blotting. Expression of adipocytokines and transcriptional factors was also measured by real-time PCR. Cells were examined for morphological changes and lipid accumulation by microscopy. The MafA expression level in the MafA-siRNA-transfected pre-adipocytes was reduced by approximately 30% on day 0 pre-induction and by approximately 70% on day 3 post-induction, in comparison with stop-siRNA-transfected cells. Cell growth and lipid droplet accumulation were prevented by MafA mRNA suppression, and peroxisome proliferator-activated receptor (PPAR) gamma2 and CCAAT/enhancer-binding proteins (C/EBP)alpha, both of which are transcriptional factors essential for adipocyte differentiation, were down-regulated. Expression of the genes encoding the adipocytokines, adiponectin and adipsin was also suppressed. The results suggested a possible role of the transcriptional factor MafA in regulation of adipocyte function and differentiation.

Potential roles of large mafs in cell lineages and developing pancreas.

OBJECTIVES: Maf is a family of transcription factor proteins characterized by a typical bZip structure, and mafA, a member of the large-maf family, is a strong transactivator of insulin in cell lines. The present study investigated the expression profiles of the large-maf family proteins in porcine pancreatic tissue and in primary culture cells. METHODS: Immunohistochemical staining was performed to localize each maf protein. Messenger RNA expression was quantitated by real-time polymerase chain reaction, and protein expression was assessed by Western blotting. RESULTS: Islet formation was not as clear in newborn pancreatic tissue as in adult pancreatic tissue. MafA- and c-maf-positive cells were more diffusely localized in pancreatic tissue with fewer mafB-positive cell clusters scattered throughout. By contrast, islet formation was clearer, and positive staining for mafA and c-maf tended to be more prominent in the islets of adult pancreatic tissue. Messenger RNA and protein expressions were consistent with the immunohistochemical findings. MafA, mafB, and c-maf coexpressed with insulin-positive cells, and c-maf coexpressed with glucagon-positive cells in adult porcine pancreas based on the results of a double-staining study. CONCLUSIONS: Large mafs were identified in normal porcine and human pancreas, and the expression levels and localizations of the large mafs in newborn and adult pancreatic tissues differed. Mafs may play important roles in establishing endocrine function during pancreatic cell differentiation.

Effect of GLP-1 (glucagon-like peptide 1:7-36 amide) on porcine pancreatic endocrine cell proliferation and insulin secretion.

INTRODUCTION: We have established a method of isolation and primary monolayer culture of porcine pancreatic endocrine (PE) cells using nicotinamide. Recently, several insulin-stimulating factors have been recognized as modifying the function of endocrine cells. Glucagon-like peptide 1 (GLP-1), derived from intestinal cells, is one of the substances that may regulate insulin secretion and cell proliferation. AIM: The aim of the current study was to examine the effect of GLP-1 on PE cell proliferation and insulin secretion in our culture system. METHODOLOGY: The PE cells were prepared by nonenzymatic digestion and cultured in media with or without 10 nmol/L GLP-1 and maintained for 15 days. Basal insulin secretion and the glucose stimulated-response were observed by enzyme assay. PE-cell proliferation was assessed using 5-bromo 2' deoxyuridine (BrdU). beta-Cell specificity was confirmed by double staining with anti-BrdU and insulin antibody. mRNA expression in PE cells for insulin and pancreatic and duodenal homeobox gene 1 (PDX-1), a transcription factor, was semiquantitated by a real-time PCR method. RESULTS: Neither baseline nor glucose-stimulated insulin secretion differed significantly with or without the addition of 10 nmol/L GLP-1. However, significant changes of insulin secretion were observed in doses up to 100 nmol/L of GLP-1. BrdU incorporation increased with 10 nmol/L GLP-1 from 2.35+/- 0.16% to 9.11+/- 0.53% as compared with the control (from 2.17 +/- 0.57% to 6.13 +/- 0.35%). This increase was observed after a 12-day culture period. Double staining identified well-preserved insulin-containing cells, but neither the number of positive cells nor staining intensity differed significantly between the 2 groups during the 15-day culture period. The level of mRNA expression for both insulin and PDX-1 increased slightly but significantly. CONCLUSIONS: GLP-1 has a proliferative effect on PE cells and affects gene expression in short-term culture.

Analysis of gene expression and insulin secretion by monolayer-forming adult porcine pancreatic endocrine cells.

INTRODUCTION: We recently established a method of isolation and primary monolayer culture of porcine pancreatic endocrine cells that involves the use of nicotinamide. AIM: To obtain genetic information on cultured porcine endocrine cells and to examine cell function in relation to insulin secretion during long-term culture. METHODOLOGY: Gene expression of insulin and several transcription factors, including PDX-1, Beta2/NeuroD, Pax6, and Nkx6.1, was assessed by reverse transcription-polymerase chain reaction analysis, and the insulin protein level was estimated by immunohistochemistry and enzyme assay during a 12-week period. RESULTS: During the culture period, insulin accumulation in the medium at 5 weeks had decreased by almost half the level of accumulation in the first week. In contrast to the alteration of secretory function, insulin gene expression was maintained for at least 12 weeks, and regulatory transcription factors were expressed at the same levels until 9 weeks. These observations suggest that gene expression is not involved in the cause of decreased baseline insulin secretion. Moreover, although the insulin response to high glucose and potassium loading was maintained, the magnitude of the responses to both stimuli was attenuated in the late period of culture. Insulin secretion tended to decrease in our culture system, and the secretory response to pharmacological stimulation was attenuated despite maintenance of messenger RNA expression of insulin and other islet-specific genes for at least 9 weeks in vitro. CONCLUSION: These findings indicate that cell integrity is maintained and that the alteration in insulin secretion must be explained by another mechanism.