A comprehensive survey of 3′ animal miRNA modification events and a possible role for 3′ adenylation in modulating miRNA targeting effectiveness

Abstract

Animal microRNA sequences are subject to 39 nucleotide addition. Through detailed analysis of deep-sequenced short RNA data sets, we show adenylation and uridylation of miRNA is globally present and conserved across Drosophila and vertebrates. To better understand 39 adenylation function, we deep-sequenced RNA after knockdown of nucleotidyltransferase enzymes. The PAPD4 nucleotidyltransferase adenylates a wide range of miRNA loci, but adenylation does not appear to affect miRNA stability on a genome-wide scale. Adenine addition appears to reduce effectiveness of miRNA targeting of mRNA transcripts while deep-sequencing of RNA bound to immunoprecipitated Argonaute (AGO) subfamily proteins EIF2C1-EIF2C3 revealed substantial reduction of adenine addition in miRNA associated with EIF2C2 and EIF2C3. Our findings show 39 addition events are widespread and conserved across animals, PAPD4 is a primary miRNA adenylating enzyme, and suggest a role for 39 adenine addition in modulating miRNA effectiveness, possibly through interfering with incorporation into the RNA-induced silencing complex (RISC), a regulatory role that would complement the role of miRNA uridylation in blocking DICER1 uptake.

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

“…2A). This finding surprised us, as previously most miRNA 3′ adenylation events appeared to be mediated by PAPD4 and few, if any, by PAPD5 (17). We therefore repeated the PAPD5 knockdown experiment, which again showed a significant decrease in the miR-21+C adenylation ratio (by 71%; P = 2.6e-18; Fig.…”

“…Previously, we investigated nontemplated 3′ nucleotide additions to miRNAs by analyzing sequencing data from THP1 cultures in which the nucleotidyl transferases ZFR, PAPD4, PAPD5, PAPD7, and ILF3 were knocked down by RNAi (17). We reanalyzed these data and found that knocking down PAPD5 dramatically decreases the adenylation ratio of miR-21+C (by 74%, P = 2.1e-11; Fig.…”

“…IsomiRs can also be generated by nontemplated addition of A and U nucleotides to their 3′ end (11,17). 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).…”

“…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). miRNAs are involved in the regulation of many cellular processes including developmental and oncogenic pathways (3,20).…”

Testing hypotheses on the rate of molecular evolution in relation to gene expression using microRNAs

“…First, the extra nucleotide in the 5′ or 3′ end usually corresponds to the one in the genomic sequence, hence arguing against the addition of a random nucleotide. (We estimated ≈20% of the reads have a nontemplated extranucleotide, mostly A or U, because it is known that some miRs are naturally modified at their 5′ or 3′ end with a uridyl or adenyl residue (41,42). The modification of miRNA ends is a known biological mechanism rather than experimental artifacts.…”