Post-transcriptional generation of miRNA variants by multiple nucleotidyl transferases contributes to miRNA transcriptome complexity

Abstract

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 MTPAP, PAPD4, PAPD5, ZCCHC6, ZCCHC11, and TUT1, were found to govern 3′ nucleotide addition to miRNAs in a miRNA-specific manner. Three of these enzymes–MTPAP, 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.

PAPD5-mediated 3′ adenylation and subsequent degradation of miR-21 is disrupted in proliferative disease

“…Next-generation sequencing of miRNAs has revealed a wide variety of miRNA variants that may be produced by differential excision by DICER1 or nucleotide additions to the 3′ ends of miRNAs or their precursors (11,45). Multiple nucleotidyl transferases, including PAPD4 and PAPD5, can mediate 3′ adenylation of mature miRNAs in human (17,18); these 3′ additions have been shown to be a physiological phenomenon rather than an artifact of library preparation and are specific for different miRNAs (18). However, the biogenesis and biological significance of miRNA 3′ modifications as well as isomiRs with distinct 3′ ends are generally poorly understood.…”

“…Although several enzymes have been found to govern 3′ nucleotide addition to mature miRNAs (18), for most mature miRNAs 3′ adenylation is mediated by PAP associated domain containing 4 (PAPD4, also known as GLD2) (17). Whereas mature miR-122 is selectively stabilized by PAPD4-mediated adenylation (19), it remains unclear whether in general adenylation of the 3′ end of mature miRNAs increases their stability (17).…”

Lin28-mediated control of let-7 microRNA expression by alternative TUTases Zcchc11 (TUT4) and Zcchc6 (TUT7)

“…Zcchc6 has previously been shown to have poly(U) activity in vitro (Kwak and Wickens 2007;Rissland et al 2007), and depletion of Zcchc6 in colon cancer cells led to reduced levels of uridylated mature let-7e (Wyman et al 2011). Zcchc6 is also a homolog of Caenorhabditis elegans CDE-1, which uridylates a subset of siRNAs bound by the Argonaute protein CSR-1, and loss of CDE-1 leads to aberrant chromosomal segregation and dysregulation of CSR-1-bound siRNAs (van Wolfswinkel et al 2009).…”

A selective screening platform reveals unique global expression patterns of microRNAs in a cohort of human soft-tissue sarcomas

“…The nCounter Human v2 miRNA Expression Assay Kit (NanoString Technologies, WA, USA) 20,21 was used to profile 800 human miRNAs in human sarcoma and normal tissue cells. Total RNA (100 ng) was used as input material.…”