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Title: Usb1 controls U6 snRNP assembly through evolutionarily divergent cyclic phosphodiesterase activities

Authors:
; ; ; ; ; ; ; ; ORCiD logo
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
National Institutes of Health (NIH)
OSTI Identifier:
1406616
Resource Type:
Journal Article
Resource Relation:
Journal Name: Nature Communications; Journal Volume: 8; Journal Issue: 1
Country of Publication:
United States
Language:
ENGLISH

Citation Formats

Didychuk, Allison L., Montemayor, Eric J., Carrocci, Tucker J., DeLaitsch, Andrew T., Lucarelli, Stefani E., Westler, William M., Brow, David A., Hoskins, Aaron A., and Butcher, Samuel E. Usb1 controls U6 snRNP assembly through evolutionarily divergent cyclic phosphodiesterase activities. United States: N. p., 2017. Web. doi:10.1038/s41467-017-00484-w.
Didychuk, Allison L., Montemayor, Eric J., Carrocci, Tucker J., DeLaitsch, Andrew T., Lucarelli, Stefani E., Westler, William M., Brow, David A., Hoskins, Aaron A., & Butcher, Samuel E. Usb1 controls U6 snRNP assembly through evolutionarily divergent cyclic phosphodiesterase activities. United States. doi:10.1038/s41467-017-00484-w.
Didychuk, Allison L., Montemayor, Eric J., Carrocci, Tucker J., DeLaitsch, Andrew T., Lucarelli, Stefani E., Westler, William M., Brow, David A., Hoskins, Aaron A., and Butcher, Samuel E. Fri . "Usb1 controls U6 snRNP assembly through evolutionarily divergent cyclic phosphodiesterase activities". United States. doi:10.1038/s41467-017-00484-w.
@article{osti_1406616,
title = {Usb1 controls U6 snRNP assembly through evolutionarily divergent cyclic phosphodiesterase activities},
author = {Didychuk, Allison L. and Montemayor, Eric J. and Carrocci, Tucker J. and DeLaitsch, Andrew T. and Lucarelli, Stefani E. and Westler, William M. and Brow, David A. and Hoskins, Aaron A. and Butcher, Samuel E.},
abstractNote = {},
doi = {10.1038/s41467-017-00484-w},
journal = {Nature Communications},
number = 1,
volume = 8,
place = {United States},
year = {Fri Sep 08 00:00:00 EDT 2017},
month = {Fri Sep 08 00:00:00 EDT 2017}
}
  • The inositol phosphate products formed during the cleavage of phosphatidylinositol by phosphatidylinositol-specific phospholipase C from Bacillus cereus were analyzed by {sup 31}P NMR. {sup 31}P NMR spectroscopy can distinguish between the inositol phosphate species and phosphatidylinositol. Chemical shift values (with reference to phosphoric acid) observed are {minus}0.41, 3.62, 4.45, and 16.30 ppm for phosphatidylinositol, myo-inositol 1-monophosphate, myo-inositol 2-monophosphate, and myo-inositol 1,2-cyclic monophosphate, respectively. It is shown that under a variety of experimental conditions this phospholipase C cleaves phosphatidylinositol via an intramolecular phosphotransfer reaction producing diacylglycerol and D-myo-inositol 1,2-cyclic monophosphate. The authors also report the new and unexpected observation that themore » phosphatidylinositol-specific phospholipase C from B. cereus is able to hydrolyze the inositol cyclic phosphate to form D-myo-inositol 1-monophosphate. The enzyme, therefore, possesses phosphotransferase and cyclic phosphodiesterase activities. The second reaction requires thousandfold higher enzyme concentrations to be observed by {sup 31}P NMR. This reaction was shown to be regiospecific in that only the 1-phosphate was produced and stereospecific in that only D-myo-inositol 1,2-cyclic monophosphate was hydrolyzed. Inhibition with a monoclonal antibody specific for the B.cereus phospholipase C showed that the cyclic phosphodiesterase activity is intrinsic to the bacterial enzyme. They propose a two-step mechanism for the phosphatidyl-inositol-specific phospholipase C from B. cereus involving sequential phosphotransferase and cyclic phosphodiesterase activities. This mechanism bears a resemblance to the well-known two-step mechanism of pancreatic ribonuclease, RNase A.« less
  • Radiation inactivation of the two high affinity cyclic AMP phosphodiesterases (PDE) found in liver plasma membranes afforded an estimation of their molecular target sizes in situ. The activity of the peripheral plasma membrane PDE decayed as a single exponential with a target size corresponding to a monomer of circa 54 kDa. The integral, cyclic GMP-stimulated PDE decayed as a dimer of circa 125 kDa. Preincubation of plasma membranes with insulin (10nM), prior to irradiation, caused the target size of only the peripheral plasma membrane PDE to increase. We suggest that insulin addition causes the peripheral plasma membrane PDE to altermore » its coupling to an integral plasma membrane protein with a target size of circa 90 kDa.« less
  • Newly synthesized snRNAs appear transiently in the cytoplasm where they assemble into ribonucleoprotein particles, the snRNP particles, before returning permanently to the interphase nucleus. In this report, bona fide cytoplasmic fractions, prepared by cell enucleation, are used for a quantitative analysis of snRNP assembly in growing mouse fibroblasts. The half-lives and abundances of the snRNP precursors in the cytoplasm and the rates of snRNP assembly are calculated in L929 cells. With the exception of U6, the major snRNAs are stable RNA species; U1 is almost totally stable while U2 has a half-life of about two cell cycles. In contrast, themore » majority of newly synthesized U6 decays with a half-life of about 15 h. The relative abundances of the newly synthesized snRNA species U1, U2, U3, U4 and U6 in the cytoplasm are determined by Northern hybridization using cloned probes and are approximately 2% of their nuclear abundance. The half-lives of the two major snRNA precursors in the cytoplasm (U1 and U2) are approximately 20 min as determined by labeling to steady state. The relative abundance of the snRNP B protein in the cytoplasm is determined by Western blotting with the Sm class of autoantibodies and is approximately 25% of the nuclear abundance. Kinetic studies, using the Sm antiserum to immunoprecipitate the methionine-labeled snRNP proteins, suggest that the B protein has a half-life of 90 to 120 min in the cytoplasm. These data are discussed and suggest that there is a large pool of more stable snRNP proteins in the cytoplasm available for assembly with the less abundant but more rapidly turning-over snRNAs.« less