ABSTRACT
The initial step in microRNA (miRNA) biogenesis requires processing of the precursor miRNA (pre-miRNA) from a longer primary transcript. Many pre-miRNAs originate from introns, and both a mature miRNA and a spliced RNA can be generated from the same transcription unit. We have identified a mechanism in which RNA splicing negatively regulates the processing of pre-miRNAs that overlap exon-intron junctions. Computational analysis identified dozens of such pre-miRNAs, and experimental validation demonstrated competitive interaction between the Microprocessor complex and the splicing machinery. Tissue-specific alternative splicing regulates maturation of one such miRNA, miR-412, resulting in effects on its targets that code a protein network involved in neuronal cell death processes. This mode of regulation specifically controls maturation of splice-site-overlapping pre-miRNAs but not pre-miRNAs located completely within introns or exons of the same transcript. Our data present a biological role of alternative splicing in regulation of miRNA biogenesis.
Subject(s)
Alternative Splicing , Exons , Introns , MicroRNAs/biosynthesis , Animals , Base Sequence , Cell Death/genetics , Gene Regulatory Networks , HEK293 Cells , High-Throughput Nucleotide Sequencing , Humans , Inverted Repeat Sequences , Mice , MicroRNAs/genetics , Molecular Sequence Data , Multigene Family , Neurons/physiology , Nucleic Acid Conformation , Proteins/metabolism , RNA Interference , RNA Splice Sites , RNA-Binding Proteins , Ribonuclease III/genetics , Ribonuclease III/metabolismABSTRACT
Splicing aberrations are prominent drivers of cancer, yet the regulatory pathways controlling them are mostly unknown. Here we develop a method that integrates physical interaction, gene expression, and alternative splicing data to construct the largest map of transcriptomic and proteomic interactions leading to cancerous splicing aberrations defined to date, and identify driver pathways therein. We apply our method to colon adenocarcinoma and non-small-cell lung carcinoma. By focusing on colon cancer, we reveal a novel tumor-favoring regulatory pathway involving the induction of the transcription factor MYC by the transcription factor ELK1, as well as the subsequent induction of the alternative splicing factor PTBP1 by both. We show that PTBP1 promotes specific RAC1,NUMB, and PKM splicing isoforms that are major triggers of colon tumorigenesis. By testing the pathway's activity in patient tumor samples, we find ELK1,MYC, and PTBP1 to be overexpressed in conjunction with oncogenic KRAS mutations, and show that these mutations increase ELK1 levels via the RAS-MAPK pathway. We thus illuminate, for the first time, a full regulatory pathway connecting prevalent cancerous mutations to functional tumor-inducing splicing aberrations. Our results demonstrate our method is applicable to different cancers to reveal regulatory pathways promoting splicing aberrations.
Subject(s)
Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , RNA Splicing , Signal Transduction , ets-Domain Protein Elk-1/metabolism , Cluster Analysis , Computational Biology , Gene Expression Profiling , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Humans , Mitogen-Activated Protein Kinases/metabolism , Polypyrimidine Tract-Binding Protein/genetics , Polypyrimidine Tract-Binding Protein/metabolism , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins p21(ras)/metabolismABSTRACT
MicroRNA (miRNA) biogenesis initiates co-transcriptionally, but how the Microprocessor machinery pinpoints the locations of short precursor miRNA sequences within long flanking regions of the transcript is not known. Here we show that miRNA biogenesis depends on DNA methylation. When the regions flanking the miRNA coding sequence are highly methylated, the miRNAs are more highly expressed, have greater sequence conservation, and are more likely to drive cancer-related phenotypes than miRNAs encoded by unmethylated loci. We show that the removal of DNA methylation from miRNA loci leads to their downregulation. Further, we found that MeCP2 binding to methylated miRNA loci halts RNA polymerase II elongation, leading to enhanced processing of the primary miRNA by Drosha. Taken together, our data reveal that DNA methylation directly affects miRNA biogenesis.