The MicroRNA Family Gets Wider: The IsomiRs Classification and Role

Abstract

MicroRNAs (miRNAs or miRs) are the most characterized class of non-coding RNAs and are engaged in many cellular processes, including cell differentiation, development, and homeostasis. MicroRNA dysregulation was observed in several diseases, cancer included. Epitranscriptomics is a branch of epigenomics that embraces all RNA modifications occurring after DNA transcription and RNA synthesis and involving coding and non-coding RNAs. The development of new high-throughput technologies, especially deep RNA sequencing, has facilitated the discovery of miRNA isoforms (named isomiRs) resulting from RNA modifications mediated by enzymes, such as deaminases and exonucleases, and differing from the canonical ones in length, sequence, or both. In this review, we summarize the distinct classes of isomiRs, their regulation and biogenesis, and the active role of these newly discovered molecules in cancer and other diseases.

Keywords: isomiRs; microRNA; microRNA biogenesis; novel microRNA in cancer; variants.

FIGURE 1

Examples of isomiRs. MicroRNA isoforms can variate for length, sequence, or both. The current classification identified five classes of variants: (1) canonical microRNAs; (2) 5′ isomiRs; (3) 3′ isomiRs; (4) polymorphic isomiRs; (5) mixed type isomiRs.

FIGURE 2

IsomiR biogenesis. (A) The RNAse III DROSHA, aided by DGCR8, processes pri-miRNA in the nucleus and produces the first cut in correspondence of the 5′ end of the 5p arm and 3′ end of the 3p arm. DGCR8 acts as a molecular meter and identifies the cleavage site 11 bp far away from the junction point between the lower stem and the basal unpaired sequences. (B) The secondary structure of the lower stem of pri-miRNA affects the DROSHA cleavage precision: a perfect or bulged lower stem leads to a homogeneous cleavage site in more than 97% of the cases. On the contrary, a distorted and flexible lower stem creates three potential cleavage sites. (C) The RNase III DICER processes the short hairpin RNA (shRNA) by eliminating the terminal loop and forming a double-strand miRNA/miRNA*. Different lengths and the presence of bulges can affect the PAZ domain-mediated “measurement” of the lower stem leading to the selection of multiple cleavage sites.

FIGURE 3

Three different qPCR techniques for the detection of isomiRs. (A) The stem-loop qRT-PCR, with the employment of hydrolysis-based probes (Taqman), has become the most commonly used commercial technique. However, this method has significant limitations in detecting and quantifying isomiRs accurately. The discrimination of two sequences that differ by only one or a few nucleotides is not guaranteed by this protocol and must be established empirically for each molecule using customized probes and appropriate controls. (B) Dumbbell-PCR employs a 3′-stem-loop adapter, which acts as the reverse transcription trigger, and a 5′-stem-loop adapter, which contains a stop signal for reverse transcription. IsomiR gaps or overlaps strongly impact the efficacy of the ligation process and the annealing of a Taqman probe partially complementary to the microRNA and partially to the 3′ adapter sequences. (C) Two-tailed RT-qPCR is characterized by the design of a long-structured primer (∼50 nucleotides) holding two hemiprobes complementary to the 5′ and 3′ ends of the microRNA. The reverse transcription starts from the 3′ end, extending the primer sequence with the complementary sequence of the target microRNA and simultaneously detaching the 5′ end. The amplification step uses two specific primers, one annealing the microRNA sequence and the other the 5′ hemiprobe. The use of two short hemiprobes increases the sensitivity and specificity of this technique: the brevity of these two sequences makes them more susceptible to possible isomiR mismatches.