Abstract
Heterogeneous nuclear ribonucleoparticule A1/A2 (hnRNP A1/A2) and splicing factor 2/alternative splicing factor (SF2/ASF) are pivotal for precursor messenger RNA (pre-mRNA) splicing. Interferon regulatory factor-3 (IRF-3) plays critical roles in host defense against viral and microbial infection. Truncated IRF-3 proteins resulting from alternative splicing have been identified and characterized as functional antagonists to full-length IRF-3. In this study, we examined the molecular mechanism for splicing regulation of IRF-3 pre-mRNA and first reported the regulatory effect of hnRNP A1/A2 and SF2/ASF on IRF-3 splicing and activation. RNA interference-mediated depletion of hnRNP A1/A2 or SF2/ASF in human non-small cell lung cancer (NSCLC) cells increased exclusion of exons 2 and 3 of IRF-3 gene and reduced expression levels of IRF-3 protein and IRF-3 downstream effector molecules interferon-beta and CXCL10/IP-10. In addition, direct binding of hnRNP A1 and SF2/ASF to specific binding motifs in IRF-3 intron 1 was confirmed by RNA electrophoretic mobility shift assay. Subsequent minigene splicing assay showed that IRF-3 minigenes with mutated hnRNPA 1/A2 or SF2/ASF binding motifs increased exclusion of exons 2 and 3. Moreover, knockdown of hnRNP A1/A2 or SF2/ASF in NSCLC cells reinforced phytohemagglutinin-induced tumor necrosis factor-alpha release by peripheral blood mononuclear cells (PBMC) but suppressed that of interleukin-10 in NSCLC/PBMC co-cultures. Taken together, our results suggest that specific knockdown for hnRNP A1/A2 or SF2/ASF increase exclusion of exons 2 and 3 of IRF-3 pre-mRNA and influence immunomodulatory functions of human NSCLC cells.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Alternative Splicing
-
Base Sequence
-
Carcinoma, Non-Small-Cell Lung / genetics
-
Carcinoma, Non-Small-Cell Lung / immunology*
-
Carcinoma, Non-Small-Cell Lung / pathology
-
Cell Line, Tumor
-
Chemokine CXCL10 / genetics
-
Chemokine CXCL10 / immunology
-
Coculture Techniques
-
Exons
-
Gene Expression Regulation
-
Heterogeneous Nuclear Ribonucleoprotein A1
-
Heterogeneous-Nuclear Ribonucleoprotein Group A-B / antagonists & inhibitors
-
Heterogeneous-Nuclear Ribonucleoprotein Group A-B / genetics
-
Heterogeneous-Nuclear Ribonucleoprotein Group A-B / immunology*
-
Humans
-
Immunomodulation
-
Interferon Regulatory Factor-3 / genetics
-
Interferon Regulatory Factor-3 / immunology*
-
Interferon-beta / genetics
-
Interferon-beta / immunology
-
Introns
-
Leukocytes, Mononuclear / immunology
-
Leukocytes, Mononuclear / pathology
-
Lung Neoplasms / genetics
-
Lung Neoplasms / immunology*
-
Lung Neoplasms / pathology
-
Molecular Sequence Data
-
Nuclear Proteins / antagonists & inhibitors
-
Nuclear Proteins / genetics
-
Nuclear Proteins / immunology*
-
RNA Precursors / genetics
-
RNA Precursors / immunology*
-
RNA, Small Interfering / genetics
-
RNA-Binding Proteins / antagonists & inhibitors
-
RNA-Binding Proteins / genetics
-
RNA-Binding Proteins / immunology*
-
Serine-Arginine Splicing Factors
-
Signal Transduction
Substances
-
CXCL10 protein, human
-
Chemokine CXCL10
-
Heterogeneous Nuclear Ribonucleoprotein A1
-
Heterogeneous-Nuclear Ribonucleoprotein Group A-B
-
Interferon Regulatory Factor-3
-
Nuclear Proteins
-
RNA Precursors
-
RNA, Small Interfering
-
RNA-Binding Proteins
-
hnRNP A2
-
Serine-Arginine Splicing Factors
-
Interferon-beta
Grants and funding
This study was supported by the National Natural Science Foundation of China (Grant No. 30572072) to XL and by the Beijing Municipal Natural Science Foundation (Grant No. 5092009) and the National Natural Science Foundation of China (Grant No. 30871366) to YL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.