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E-GEOD-26802 - Post-transcriptional generation of miRNA variants by multiple nucleotidyl transferases contributes to miRNA transcriptome complexity (hESC)

Status
Released on 15 June 2011, last updated on 27 June 2011
Organism
Homo sapiens
Samples (30)
Array (1)
Protocols (7)
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. RNA was isolated from undifferentiated or differentiated H1 human embryonic cells as previously described (Bar et al, 2008). Two biological replicates of undifferentiated and differentiated cell RNA was profiled on the NanoString nCounter miRNA expression assay adapted to discriminate miRNA 3' variants.
Experiment type
other 
Contacts
MIAME
PlatformsProtocolsVariablesProcessedRaw
Files
Investigation descriptionE-GEOD-26802.idf.txt
Sample and data relationshipE-GEOD-26802.sdrf.txt
Raw data (1)E-GEOD-26802.raw.1.zip
Processed data (1)E-GEOD-26802.processed.1.zip
Array designA-GEOD-11642.adf.txt
Links