skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Loss of Pnn expression attenuates expression levels of SR family splicing factors and modulates alternative pre-mRNA splicing in vivo

Abstract

SR and SR-related proteins have been implicated as trans-acting factors that play an important role in splice selection and are involved at specific stages of spliceosome formation. A well-established property of SR protein splicing factors is their ability to influence selection of alternative splice sites in a concentration-dependent manner. Identification of molecules that regulate SR family protein expression is therefore of vital importance in RNA biology. Here we report that depletion of Pnn expression, a SR-related protein with functions involved in pre-mRNA splicing and mRNA export, induces reduced expression of a subset of cellular proteins, especially that of SR family proteins, including SC35, SRm300, SRp55, and SRp40, but not that of other nuclear proteins, such as p53, Mdm2, and ki67. Knocking down Pnn expression was achieved in vitro by siRNA transfection. Expression levels of SR and SR-related proteins in Pnn-depleted cells as compared to those in control cells were evaluated by immunofluorescent staining and Western blot with specific antibodies. In addition, we also demonstrate that loss of Pnn expression could modulate splice site selection of model reporter gene in vivo. Our finding is significant in terms of regulation of SR protein cellular concentration because it reveals that Pnn may playmore » a general role in the control of the cellular amount of family SR proteins through down-regulation of its own expression, thereby providing us with a better understanding of the cellular mechanism by which Pnn fulfills its biological function.« less

Authors:
 [1];  [2]
  1. Epithelial Biology Laboratory, Department of Anatomy, Chang Gung University Medical College, Taoyuan 333, Taiwan (China)
  2. Epithelial Biology Laboratory, Department of Anatomy, Chang Gung University Medical College, Taoyuan 333, Taiwan (China) and Transgenic Mice Core-Laboratory, Chang Gung University Medical College, Taoyuan 333, Taiwan (China). E-mail: ouyang@mail.cgu.edu.tw
Publication Date:
OSTI Identifier:
20798840
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 341; Journal Issue: 2; Other Information: DOI: 10.1016/j.bbrc.2005.12.218; PII: S0006-291X(06)00052-0; Copyright (c) 2006 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ANTIBODIES; BIOLOGICAL FUNCTIONS; GENE REGULATION; IN VITRO; IN VIVO; PROTEINS; RNA; SPLICING

Citation Formats

Chiu Yali, and Ouyang Pin. Loss of Pnn expression attenuates expression levels of SR family splicing factors and modulates alternative pre-mRNA splicing in vivo. United States: N. p., 2006. Web. doi:10.1016/j.bbrc.2005.12.218.
Chiu Yali, & Ouyang Pin. Loss of Pnn expression attenuates expression levels of SR family splicing factors and modulates alternative pre-mRNA splicing in vivo. United States. doi:10.1016/j.bbrc.2005.12.218.
Chiu Yali, and Ouyang Pin. Fri . "Loss of Pnn expression attenuates expression levels of SR family splicing factors and modulates alternative pre-mRNA splicing in vivo". United States. doi:10.1016/j.bbrc.2005.12.218.
@article{osti_20798840,
title = {Loss of Pnn expression attenuates expression levels of SR family splicing factors and modulates alternative pre-mRNA splicing in vivo},
author = {Chiu Yali and Ouyang Pin},
abstractNote = {SR and SR-related proteins have been implicated as trans-acting factors that play an important role in splice selection and are involved at specific stages of spliceosome formation. A well-established property of SR protein splicing factors is their ability to influence selection of alternative splice sites in a concentration-dependent manner. Identification of molecules that regulate SR family protein expression is therefore of vital importance in RNA biology. Here we report that depletion of Pnn expression, a SR-related protein with functions involved in pre-mRNA splicing and mRNA export, induces reduced expression of a subset of cellular proteins, especially that of SR family proteins, including SC35, SRm300, SRp55, and SRp40, but not that of other nuclear proteins, such as p53, Mdm2, and ki67. Knocking down Pnn expression was achieved in vitro by siRNA transfection. Expression levels of SR and SR-related proteins in Pnn-depleted cells as compared to those in control cells were evaluated by immunofluorescent staining and Western blot with specific antibodies. In addition, we also demonstrate that loss of Pnn expression could modulate splice site selection of model reporter gene in vivo. Our finding is significant in terms of regulation of SR protein cellular concentration because it reveals that Pnn may play a general role in the control of the cellular amount of family SR proteins through down-regulation of its own expression, thereby providing us with a better understanding of the cellular mechanism by which Pnn fulfills its biological function.},
doi = {10.1016/j.bbrc.2005.12.218},
journal = {Biochemical and Biophysical Research Communications},
number = 2,
volume = 341,
place = {United States},
year = {Fri Mar 10 00:00:00 EST 2006},
month = {Fri Mar 10 00:00:00 EST 2006}
}
  • Differentiating erythroid cells execute a unique gene expression program that insures synthesis of the appropriate proteome at each stage of maturation. Standard expression microarrays provide important insight into erythroid gene expression but cannot detect qualitative changes in transcript structure, mediated by RNA processing, that alter structure and function of encoded proteins. We analyzed stage-specific changes in the late erythroid transcriptome via use of high-resolution microarrays that detect altered expression of individual exons. Ten differentiation-associated changes in erythroblast splicing patterns were identified, including the previously known activation of protein 4.1R exon 16 splicing. Six new alternative splicing switches involving enhanced inclusionmore » of internal cassette exons were discovered, as well as 3 changes in use of alternative first exons. All of these erythroid stage-specific splicing events represent activated inclusion of authentic annotated exons, suggesting they represent an active regulatory process rather than a general loss of splicing fidelity. The observation that 3 of the regulated transcripts encode RNA binding proteins (SNRP70, HNRPLL, MBNL2) may indicate significant changes in the RNA processing machinery of late erythroblasts. Together, these results support the existence of a regulated alternative pre-mRNA splicing program that is critical for late erythroid differentiation.« less
  • The spliced form of MuSVts110 viral RNA is approximately 20-fold more abundant at growth temperatures of 33/sup 0/C or lower than at 37 to 41/sup 0/C. This difference is due to changes in the efficiency of MuSVts110 RNA splicing rather than selective thermolability of the spliced species at 37 to 41/sup 0/C or general thermosensitivity of RNA splicing in MuSVts110-infected cells. Moreover, RNA transcribed from MuSVts110 DNA introduced into a variety of cell lines is spliced in a temperature-sensitive fashion, suggesting that the structure of the viral RNA controls the efficiency of the event. The authors exploited this novel splicingmore » event to study the cleavage and ligation events during splicing in vivo. No spliced viral mRNA or splicing intermediates were observed in MuSVts110-infected cells (6m2 cells) at 39/sup 0/C. However, after a short (about 30-min) lag following a shift to 33/sup 0/C, viral pre-mRNA cleaved at the 5' splice site began to accumulate. Ligated exons were not detected until about 60 min following the initial detection of cleavage at the 5' splice site, suggesting that these two splicing reactions did not occur concurrently. Splicing of viral RNA in the MuSVts110 revertant 54-5A4, which lacks the sequence -AG/TGT- at the usual 3' splice site, was studied. Cleavage at the 5' splice site in the revertant viral RNA proceeded in a temperature-sensitive fashion. No novel cryptic 3' splice sites were activated; however, splicing at an alternate upstream 3' splice site used at low efficiency in normal MuSVts110 RNA was increased to a level close to that of 5'-splice-site cleavage in the revertant viral RNA.« less
  • A physiologically important alternative pre-mRNA splicing switch, involving activation of protein 4.1R exon 16 (E16) splicing, is required for establishing proper mechanical integrity of the erythrocyte membrane during erythropoiesis. Here we identify a conserved exonic splicing silencer element (CE16) in E16 that interacts with hnRNP A/B proteins and plays a role in repression of E16 splicing during early erythropoiesis. Experiments with model pre-mRNAs showed that CE16 can repress splicing of upstream introns, and that mutagenesis or replacement of CE16 can relieve this inhibition. An affinity selection assay with biotinylated CE16 RNA demonstrated specific binding of hnRNP A/B proteins. Depletion ofmore » hnRNP A/B proteins from nuclear extract significantly increased E16 inclusion, while repletion with recombinant hnRNP A/B restored E16 silencing. Most importantly, differentiating mouse erythroblasts exhibited a stage-specific activation of the E16 splicing switch in concert with a drama tic and specific down-regulation of hnRNP A/B protein expression. These findings demonstrate that natural developmental changes in hnRNP A/B proteins can effect physiologically important switches in pre-mRNA splicing.« less
  • Protein 4.1 is a globular 80-kDa component of the erythrocyte membrane skeleton that enhances spectrin-actin interaction via its internal 10-kDa domain. Previous studies have shown that protein 4.1 mRNA is expressed as multiple alternatively spliced isoforms, resulting from the inclusion or exclusion of small cassette sequences called motifs. By tissue screening for protein 4.1 isoforms, we have observed new features of an already complex pattern of alternative splicing within the spectrin/actin binding domain. In particular, we found a new 51-nt exon that is present almost exclusively in muscle tissue. In addition, we have isolated multiple genomic clones spanning over 200more » kb, containing the entire erythroid and nonerythroid coding sequence of the human locus. The exon/intron structure has now been characterized; with the exception of a 17-nt motif, all of the alternatively spliced motifs correspond to individual exons. The 3{prime}-untranslated region (UTR) has also been completely sequenced using various PCR and genomic-sequencing methods. The 3{prime} UTR, over 3 kb, accounts for one-half of the mature mRNA. 83 refs., 8 figs., 1 tab.« less
  • No abstract prepared.