A novel source for miR-21 expression through the alternative polyadenylation of VMP1 gene transcripts

miR-21 is the most commonly over-expressed microRNA (miRNA) in cancer and a proven oncogene. Hsa-miR-21 is located on chromosome 17q23.2, immediately downstream of the vacuole membrane protein-1 (VMP1) gene, also known as TMEM49. VMP1 transcripts initiate ∼130 kb upstream of miR-21, are spliced, and polyadenylated only a few hundred base pairs upstream of the miR-21 hairpin. On the other hand, primary miR-21 transcripts (pri-miR-21) originate within the last introns of VMP1, but bypass VMP1 polyadenylation signals to include the miR-21 hairpin. Here, we report that VMP1 transcripts can also bypass these polyadenylation signals to include miR-21, thus providing a novel and independently regulated source of miR-21, termed VMP1–miR-21. Northern blotting, gene-specific RT-PCR, RNA pull-down and DNA branching assays support that VMP1–miR-21 is expressed at significant levels in a number of cancer cell lines and that it is processed by the Microprocessor complex to produce mature miR-21. VMP1 and pri-miR-21 are induced by common stimuli, such as phorbol-12-myristate-13-acetate (PMA) and androgens, but show differential responses to some stimuli such as epigenetic modifying agents. Collectively, these results indicate that miR-21 is a unique miRNA capable of being regulated by alternative polyadenylation and two independent gene promoters.

[1]  C. Klinge,et al.  Estradiol downregulates miR-21 expression and increases miR-21 target gene expression in MCF-7 breast cancer cells , 2009, Nucleic acids research.

[2]  V. Kim,et al.  Processing of intronic microRNAs , 2007, The EMBO journal.

[3]  J. Steitz,et al.  Nuclear networking fashions pre-messenger RNA and primary microRNA transcripts for function. , 2010, Trends in cell biology.

[4]  J. Ribas,et al.  The transcriptional regulation of miR-21, its multiple transcripts and their implication in prostate cancer , 2010, Cell cycle.

[5]  Sam Griffiths-Jones,et al.  The microRNA Registry , 2004, Nucleic Acids Res..

[6]  J. Steitz,et al.  Primary microRNA transcript retention at sites of transcription leads to enhanced microRNA production , 2008, The Journal of cell biology.

[7]  Yoshikazu Nakamura,et al.  Altered expression profiles of microRNAs during TPA-induced differentiation of HL-60 cells. , 2004, Biochemical and biophysical research communications.

[8]  G. Hannon,et al.  Processing of primary microRNAs by the Microprocessor complex , 2004, Nature.

[9]  Eric T. Wang,et al.  Alternative Isoform Regulation in Human Tissue Transcriptomes , 2008, Nature.

[10]  E. Olson,et al.  Modulation of K-Ras-dependent lung tumorigenesis by MicroRNA-21. , 2010, Cancer cell.

[11]  C. Croce,et al.  MicroRNA signatures in human ovarian cancer. , 2007, Cancer research.

[12]  V. Kim,et al.  The Drosha-DGCR8 complex in primary microRNA processing. , 2004, Genes & development.

[13]  F. Couch,et al.  Structural analysis of the 17q22-23 amplicon identifies several independent targets of amplification in breast cancer cell lines and tumors. , 2001, Cancer research.

[14]  Nicholas T. Ingolia,et al.  Mammalian microRNAs predominantly act to decrease target mRNA levels , 2010, Nature.

[15]  D. Bartel.  MicroRNAs: Target Recognition and Regulatory Functions , 2009, Cell.

[16]  Li Yu,et al.  C. elegans Screen Identifies Autophagy Genes Specific to Multicellular Organisms , 2010, Cell.

[17]  C. Mayr,et al.  Widespread Shortening of 3′UTRs by Alternative Cleavage and Polyadenylation Activates Oncogenes in Cancer Cells , 2009, Cell.

[18]  Megan F. Cole,et al.  Connecting microRNA Genes to the Core Transcriptional Regulatory Circuitry of Embryonic Stem Cells , 2008, Cell.

[19]  D. Corcoran,et al.  Features of Mammalian microRNA Promoters Emerge from Polymerase II Chromatin Immunoprecipitation Data , 2009, PloS one.

[20]  Catalin C. Barbacioru,et al.  Evaluation of DNA microarray results with quantitative gene expression platforms , 2006, Nature Biotechnology.

[21]  Michael A. Beer,et al.  Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis. , 2007, Molecular cell.

[22]  Robert M. Miura,et al.  Prediction of mRNA polyadenylation sites by support vector machine , 2006, Bioinform..

[23]  V. Ambros.  The functions of animal microRNAs , 2004, Nature.

[24]  T. Shuin,et al.  Detection of genetic alterations in advanced prostate cancer by comparative genomic hybridization. , 2002, Cancer genetics and cytogenetics.

[25]  Ji Wan,et al.  Structure and activity of putative intronic miRNA promoters. , 2010, RNA.

[26]  J. Iovanna,et al.  A novel mammalian trans-membrane protein reveals an alternative initiation pathway for autophagy , 2008, Autophagy.

[27]  Shuji Fujita,et al.  miR-21 Gene expression triggered by AP-1 is sustained through a double-negative feedback mechanism. , 2008, Journal of molecular biology.

[28]  L. Rorke,et al.  Chromosome abnormalities in pediatric brain tumors. , 1988, Cancer research.

[29]  J. Iovanna,et al.  Zymophagy, a Novel Selective Autophagy Pathway Mediated by VMP1-USP9x-p62, Prevents Pancreatic Cell Death*♦ , 2010, The Journal of Biological Chemistry.

[30]  J. Iovanna,et al.  Autophagy and VMP1 Expression Are Early Cellular Events in Experimental Diabetes , 2008, Pancreatology.

[31]  Guohua Wang,et al.  Estradiol-regulated microRNAs control estradiol response in breast cancer cells , 2009, Nucleic acids research.

[32]  Munish Kumar,et al.  Loss of the miR-21 allele elevates the expression of its target genes and reduces tumorigenesis , 2011, Proceedings of the National Academy of Sciences.

[33]  Ana Kozomara,et al.  miRBase: integrating microRNA annotation and deep-sequencing data , 2010, Nucleic Acids Res..

[34]  D. Bartel,et al.  Microarray profiling of microRNAs reveals frequent coexpression with neighboring miRNAs and host genes. , 2005, RNA.

[35]  J. Iovanna,et al.  VMP1 Expression Correlates with Acinar Cell Cytoplasmic Vacuolization in Arginine-Induced Acute Pancreatitis , 2003, Pancreatology.

[36]  F. Slack,et al.  OncomiR addiction in an in vivo model of microRNA-21-induced pre-B-cell lymphoma , 2010, Nature.

[37]  Jörg Hackermüller,et al.  Interleukin-6 dependent survival of multiple myeloma cells involves the Stat3-mediated induction of microRNA-21 through a highly conserved enhancer. , 2007, Blood.

[38]  N. Mizushima,et al.  Characterization of autophagosome formation site by a hierarchical analysis of mammalian Atg proteins , 2010, Autophagy.

[39]  Gregory J. Hannon,et al.  Diverse endonucleolytic cleavage sites in the mammalian transcriptome depend upon microRNAs, Drosha, and additional nucleases. , 2010, Molecular cell.

[40]  E. Wentzel,et al.  miR-21: an androgen receptor-regulated microRNA that promotes hormone-dependent and hormone-independent prostate cancer growth. , 2009, Cancer research.

[41]  R. Shiekhattar,et al.  The Microprocessor complex mediates the genesis of microRNAs , 2004, Nature.

[42]  D. Cacchiarelli,et al.  Coupled RNA Processing and Transcription of Intergenic Primary MicroRNAs , 2009, Molecular and Cellular Biology.

[43]  Jun S. Song,et al.  Chromatin structure analyses identify miRNA promoters , 2008 .

[44]  M. Robinson,et al.  Discovery pipeline for epigenetically deregulated miRNAs in cancer: integration of primary miRNA transcription , 2011, BMC Genomics.

[45]  Stijn van Dongen,et al.  miRBase: tools for microRNA genomics , 2007, Nucleic Acids Res..

[46]  Sanghyuk Lee,et al.  ECgene: an alternative splicing database update , 2006, Nucleic Acids Res..

[47]  P. Sharp,et al.  Proliferating Cells Express mRNAs with Shortened 3' Untranslated Regions and Fewer MicroRNA Target Sites , 2008, Science.

[48]  J. Iovanna,et al.  The Pancreatitis-induced Vacuole Membrane Protein 1 Triggers Autophagy in Mammalian Cells* , 2007, Journal of Biological Chemistry.

[49]  C. Croce,et al.  MicroRNA gene expression deregulation in human breast cancer. , 2005, Cancer research.

[50]  D. Haussler,et al.  Posttranscriptional Crossregulation between Drosha and DGCR8 , 2009, Cell.

[51]  B. Cullen,et al.  Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs. , 2004, RNA.

[52]  C. Croce,et al.  A microRNA expression signature of human solid tumors defines cancer gene targets , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[53]  J. Iovanna,et al.  Gemcitabine Induces the VMP1-Mediated Autophagy Pathway to Promote Apoptotic Death in Human Pancreatic Cancer Cells , 2010, Pancreatology.

[54]  Anna M. Krichevsky,et al.  miR-21: a small multi-faceted RNA , 2008, Journal of cellular and molecular medicine.