Bias-minimized quantification of microRNA reveals widespread alternative processing and 3′ end modification

Abstract

MicroRNAs (miRNAs) modulate diverse biological and pathological processes via post-transcriptional gene silencing. High-throughput small RNA sequencing (sRNA-seq) has been widely adopted to investigate the functions and regulatory mechanisms of miRNAs. However, accurate quantification of miRNAs has been limited owing to the severe ligation bias in conventional sRNA-seq methods. Here, we quantify miRNAs and their variants (known as isomiRs) by an improved sRNA-seq protocol, termed AQ-seq (accurate quantification by sequencing), that utilizes adapters with terminal degenerate sequences and a high concentration of polyethylene glycol (PEG), which minimize the ligation bias during library preparation. Measurement using AQ-seq allows us to correct the previously misannotated 5′ end usage and strand preference in public databases. Importantly, the analysis of 5′ terminal heterogeneity reveals widespread alternative processing events which have been underestimated. We also identify highly uridylated miRNAs originating from the 3p strands, indicating regulations mediated by terminal uridylyl transferases at the pre-miRNA stage. Taken together, our study reveals the complexity of the miRNA isoform landscape, allowing us to refine miRNA annotation and to advance our understanding of miRNA regulation. Furthermore, AQ-seq can be adopted to improve other ligation-based sequencing methods including crosslinking-immunoprecipitation-sequencing (CLIP-seq) and ribosome profiling (Ribo-seq).

Population variation of miRNAs and isomiRs and their impact on human immunity to infection

“…We observed a stronger degree of 3' terminal uridylation at -3p miRNAs (+12% of uridylated miRNAs on 3p arm compared to 5p; Wilcoxon p < 2.8×10 -11 , Supplemental Fig. S2F), consistent with the reported role of uridyl-transferases in pre-miRNA maturation (Heo et al 2009;Kim et al 2015;Kim et al 2019). This increased uridylation was not associated to a higher rate of 3' extensions among miRNAs located on the 3p arm (Wilcoxon p = 0.47), due to a higher rate of template extensions among 5p miRNAs (+7.6% on 5p arm compared to 3p; Wilcoxon p < 0.006, Supplemental Fig.…”

“…Fueled by the advent of deep sequencing technologies, growing evidence has emerged that mature miRNAs undergo important post-transcriptional modifications (Neilsen et al 2012;Ameres and Zamore 2013;Tan et al 2014;Trontti et al 2018;Kim et al 2019). These include nucleotide substitutions (miRNA editing) (de Hoon et al 2010;Li et al 2018), 3' adenylation or urydilation by terminal nucleotidyl transferases (Jones et al 2009;Katoh et al 2009), shortening of their 3' end by poly(A)-specific ribonuclease (Lee et al 2019), and, more rarely, shifts in their 5' start sites (Tan et al 2014).…”

An alternative miRISC targeting a coding mutation site inFOXL2links to granulosa cell tumor

“…The steady state level of miR-1236 expression seems to be more than 300 copies per KGN cell, based on northern blot analysis of small RNAs purified from 2  10 7 miR-1236 +/+ KGN cells, which showed a radioactive intensity similar to 10 fmol of miR-1236 mimics (Appendix Fig S4I). According to a very recent miRNA profiling data by Kim et al (Kim, Kim et al, 2019), in which they developed and adopted bias-minimized accurate quantification by sequencing (AQ-seq) technology, miR-1236 was fairly well expressed as being in the top 50% of abundance among all miRNAs detected in human cells. Cumulative recent data revealed altered various cellular responses after modulating miR-1236 expression levels in diverse cell types (An, Ma et al, 2019, Chen, Teng et al, 2016, Gao et al, 2015, Jones, Li et al, 2012, Ma, Shen et al, 2014, Sato, Yoshimura et al, 2012, Thoma, 2015, Wang, Tang et al, 2016, Wang, Liu et al, 2018, Zhu, Wang et al, 2018.…”

MicroRNA arm switching regulated by uridylation

“…However, the 30 molecular principle underlying such "arm switching" remains to be 31 elucidated. 32 The miRNA duplex is generated by consecutive actions of two RNase 33 III enzymes. The nuclear RNase III DROSHA initiates miRNA 34 maturation by processing the primary transcript (pri-miRNA).…”

“…Our study provides insights into the biological roles and 439 potential applications of miRNA regulation and modification. AQ-seq (bias-minimized sRNA-seq) 442 libraries were constructed using total RNAs from fifteen mouse tissues 443 (Mouse Total RNA Master Panel; Takara) as described in our previous 444 study (Kim et al, 2019). Briefly, we mixed 5 µg of total RNAs with 10 445 fmole of thirty equimolar spike-in RNAs which are miRNA-like 446 non-human/mouse/frog/fish RNAs used for bias evaluation.…”