Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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IsomiRage: from functional classification to differential expression of miRNAs and their isoforms

ABSTRACT: Characterization and analysis of miRNAs and their variants (called "IsomiRs") in next-generation sequencing datasets in breast epithelium. Our pipeline, called IsomiRage, allows distinguishing canonical miRNAs from templated and non-templated isomiRs by alignment to a custom database, which comprises all possible 3’-, 5’- and trimmed variants. Functionally equivalent isomiRs can be grouped together according to the type of modification (e.g. uridylation, adenylation, trimming...) to assess which miRNAs are more intensively modified in a given biological context. When applied to the analysis of primary epithelial breast cancer cells, our methodology provided a 40% increase in the number of detected miRNA species and allowed to easily identify and classify more than 1000 variants. Most modifications were compatible with templated IsomiRs, as a consequence of imprecise Drosha or Dicer cleavage. However, some non-templated variants were consistently found either in the normal or in the cancer cells, with the 3’-end adenylation and uridylation as the most frequent events, suggesting that miRNA post- transcriptional modification frequently occurs. Our analytical tool permits the deconvolution of miRNA heterogeneity and could be used to explore the functional role of miRNA isoforms. A matched normal/tumor primary culture from breast epithelium

ORGANISM(S): Homo sapiens

SUBMITTER: Heiko Muller

PROVIDER: E-GEOD-61165 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Modification of microRNA sequences by the 3' addition of nucleotides to generate so-called “isomiRs” adds to the complexity of miRNA function, with recent reports showing that 3' modifications can influence miRNA stability and efficiency of target repression. Here we show that the 3' modification of miRNAs is a physiological and common post-transcriptional event that shows selectivity for specific miRNAs and is observed across species ranging from C. elegans to human. The modifications result predominantly from adenylation and uridylation, and are seen across tissue types, disease states, and developmental stages. To quantitatively profile 3' nucleotide additions, we developed and validated a novel assay based on NanoString Technologies' nCounter platform. For certain miRNAs, the frequency of modification was altered by processes such as cell differentiation, indicating that 3' modification is a biologically regulated process. To investigate the mechanism of 3' nucleotide additions, we used RNA interference to screen a panel of eight candidate miRNA nucleotidyl transferases for 3' miRNA modification activity in human cells. Multiple enzymes, including PAPD1, PAPD4, PAPD5, ZCCHC6, ZCCHC11, and TUT1, were found to govern 3' nucleotide addition to miRNAs in a miRNA-specific manner. Three of these enzymes–PAPD1, ZCCHC6 and TUT1–have not previously been known to modify miRNAs. Collectively, our results indicate that 3' modification observed in next generation small RNA sequencing data is a biologically relevant process, and identify enzymatic mechanisms that may lead to new approaches for modulating miRNA activity in vivo. Total RNA was isolated from HCT-116 cells 48 hours post transfection with siRNAs targeting a panel of nucleotidyl transferases, and profiled with a version of the NanoString nCounter miRNA expression assay adapted to discriminate miRNA 3' variants. Four biological replicates of cells treated with the negative control siRNA directed against Cyclophilin B were profiled. Two biological replicates of cells treated individually with siRNAs targeting each of eight nucleotidyl transferases were profiled.

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IsomiRage: from functional classification to differential expression of miRNAs and their isoforms

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