Deep annotation of mouse iso-miR and iso-moR variation

Abstract

With a dataset of more than 600 million small RNAs deeply sequenced from mouse hippocampal and staged sets of mouse cells that underwent reprogramming to induced pluripotent stem cells, we annotated the stem–loop precursors of the known miRNAs to identify isomoRs (miRNA-offset RNAs), loops, non-preferred strands, and guide strands. Products from both strands were readily detectable for most miRNAs. Changes in the dominant isomiR occurred among the cell types, as did switches of the preferred strand. The terminal nucleotide of the dominant isomiR aligned well with the dominant off-set sequence suggesting that Drosha cleavage generates most miRNA reads without terminal modification. Among the terminal modifications detected, most were non-templated mono- or di-nucleotide additions to the 3′-end. Based on the relative enrichment or depletion of specific nucleotide additions in an Ago-IP fraction there may be differential effects of these modifications on RISC loading. Sequence variation of the two strands at their cleavage sites suggested higher fidelity of Drosha than Dicer. These studies demonstrated multiple patterns of miRNA processing and considerable versatility in miRNA target selection.

Global characterization of the Dicer-like protein DrnB roles in miRNA biogenesis in the social amoeba Dictyostelium discoideum

“…These pairs are situated immediately adjacent to each other and are DrnB dependent, indicating that the same processing machinery is involved. This is similar to the extra single or pair of reads from pre-miRNAs, termed miRNA-offset RNAs (moRs), reported in the simple chordate Ciona intestinalis and later also in human and mouse [32,33,47]. For each pair, we named these mir-1180-1 and −2 and mir-7102–1 and −2 where ‘1’ designates the miRNA pair closest to the base of the predicted stem-loop.…”

“…Interestingly, the two intron sequences generate each two sets of miRNA pairs (mir-5p and mir-3p pairs) where each pair is situated immediately adjacent to each other. This resembles some of the miRNA-like RNAs in plants and the miRNA-offset RNAs (moRs) present in animals, where more than one miRNA derive from the same pre-miRNAs [32,33,47]. Although miRNAs are severely reduced in the drnB knock-out strain, some small RNAs, including miRNAs, derived from the miRNA hairpin precursors are still present (Fig.…”

“…Furthermore, we discovered oligo(A)-tailed processing intermediates which accumulate in the absence of DrnB. Finally, several new miRNAs were identified of which each of the two intronic miRNA loci produce two sets of miRNAs, a set up resembling miRNA-offset RNAs (moRs) found in animals [32,33]. Collectively, our data give new insights into miRNA biogenesis in an unicellular organism, demonstrating that miRNA production is characterized by features found in plants as well as animals, emphasizing the evolutionary position of D. discoideum .…”

Rat mir-155 generated from the lncRNA Bic is ‘hidden’ in the alternate genomic assembly and reveals the existence of novel mammalian miRNAs and clusters

“…To advance the discovery of novel miRNAs and other small RNAs, such as nuclear or nucleolar RNAs (snoRNAs), miRNA-offset RNAs (moRNAs) (Shi et al 2009), and isomiRs (Zhou et al 2012), novel bioinformatics approaches for miRNA prediction coupled to deep-sequencing experiments are emerging continuously (Fasold et al 2011;Guan et al 2011;Oulas and Poirazi 2011;Zhang et al 2012). The predictive strength of these and other tools generally relies on the application of computational constraints derived from the biological knowledge of miRNA biogenesis (i.e., type of Dicer cut, 5…”

A convenient system for highly specific and sensitive detection of miRNA expression

“…The extremely small size of miRNAs leads to low annealing temperature, reducing the stringency of hybridization. The closely similar and identical sequences among miRNAs family members, their ancestral RNAs (pre-miRNAs and pri-miRNAs), and isomiRs present a challenge to detection specificity (Thomas et al 2010;Chugh and Dittmer 2012;Zhou et al 2012). The lack of a poly-A tail and 5 ′ cap in a miRNA sequence inhibits its selective purification, thus affecting detection sensitivity.…”