Molecular and Phenotypic Characterization of Ire1a Gene Deletion in Mouse Embryo Fibroblasts and Pancreatic ß Cells.

IRE1;Diabetes;Splicing;Biological Chemistry;Science;Biological Chemistry

Hassler, Justin RayFuller, Robert S. ;

University of Michigan

关键词: IRE1;    Diabetes;    Splicing;    Biological Chemistry;    Science;    Biological Chemistry;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/96075/hassler_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Pancreatic beta cells have a unique ability to increase insulin synthesis and secretion in response to glucose, although the mechanism(s) is largely unknown.Here, we tested whether the unfolded protein response transducer IRE1a that initiates unconventional splicing of Xbp1 mRNA is required for glucose-stimulated insulin secretion.Beta cell-specific Ire1a deletion in both neonatal and adult mice caused hyperglycemia due to beta cell failure.Ultrastructure analysis demonstrated IRE1a is required for integrity of cellular organelles and for insulin granule biogenesis.We show IRE1a-dependent Xbp1 mRNA splicing is required for glucose-stimulated increases in ER proteins critical for proinsulin translation, signal peptide cleavage and secretion.Inversely, Ire1a deletion increases mRNAs encoding extracellular matrix and plasma membrane proteins associated with oxidative stress, inflammation and fibrosis in islets. Taken together, these findings demonstrate beta cells require IRE1a for glucose-stimulated expansion of the ER to support increased insulin production and prevent islet destruction.The unconventional mRNA splicing of Xbp1 by the stress response kinase-RNase IRE1a was interrogated using a novel algorithm to align previously unalignable sequences from massive-parallel sequencing data. This was accomplished by splitting the sequence reads in half, then mapping each half independently to the mouse genome with simultaneous calculation of the distance between the two halves.As proof of principle the chimeric reads generated by the IRE1a-dependent 26nt intron splicing of Xbp1’s mRNA were found within this data set, therefore we reasoned novel splice targets of IRE1a should also be within this data set.Importantly, the Ire1a-null cells served as an excellent negative control as they would not exhibit IRE1a-dependent intron removal. The read-split walk (RSW) algorithm was applied to the Ire1a-null cell data set and this was then subtracted from the Ire1a heterozygous cell data set to yield the chimeras exclusively present where functional IRE1a exists.RNA folding structure predictions and sequence alignments revealed shared characteristics among these newly discovered spliceoforms.