miRNA – An Introduction

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For many years, it was believed that all of the information needed to understand the human genome was contained within the coding regions. The discovery of small, non-coding RNA species has proven that idea false. These small RNAs include microRNAs (miRNAs), small interfering RNAs, and piwi-interacting RNAs (piRNAs).

miRNA Biogenesis

A miRNA in its final form is a non-coding RNA molecule ~22 nucleotides in length. However, it is initially transcribed as a longer precursor molecule (>1000 nucleotides long) called a primary miRNA transcript (pri-miRNA). Pri-miRNAs have hairpin structures that are processed by the Drosha enzyme (as part of the microprocessor complex). The microprocessor complex functions by recognizing and cleaving near the junction between hairpin structure and ssRNA. After Drosha processing, the pri-miRNAs are only 60-100 nucleotides long, and are called precursor miRNAs (pre-miRNAs). At this point, the pre-miRNA is exported to the cytoplasm, where it encounters the Dicer enzyme. Dicer cuts the miRNA in two, resulting in duplexed miRNA strands.

Traditionally, only one of these miRNA arms was considered important in gene regulation: the arm that is destined to be loaded into the RNA-induced silencing complex (RISC), and occurs at a higher concentration in the cell. This is often called the ‘guide’ strand and is designated as miR. The other arm is called the ‘minor miRNA’ or ‘passenger miRNA’, and is often designated as miR*. It was thought that passenger miRNAs were completely degraded, but deep sequencing studies have found that some minor miRNAs persist and in fact have a functional role in gene regulation (1)(2)(3).

Due to these developments, the naming convention has shifted. Instead of the miR/miR* name scheme, a miR-5p/miR-3p nomenclature has been adopted. By the new system, the 5’ arm of the miRNA is always designated miR-5p and the 3’ arm is miR-3p. However, as this is a recent change, literature will often refer to the original miR/miR* names. For more information on miRNA names, see the section on miRNA Nomenclature.

After processing, the duplexed miRNA strands are loaded onto an Argonaute (AGO) protein to form a precursor to the RISC. The complex causes the duplex to unwind and the passenger RNA strand is discarded, leaving behind a mature RISC carrying the mature, single stranded miRNA. The miRNA remains part of the RISC as it silences the expression of its target genes.

While this is the canonical pathway for miRNA biogenesis, a variety of others have been discovered. These include Drosha-independent pathways (such as the mirtron pathway, snoRNA-derived pathway, and shRNA-derived pathway) and Dicer-independent pathways (such as one that relies on AGO for cleavage, and another which is dependent on tRNaseZ) (4).

miRNA Biogenesis - Canonical Pathway

Figure 1 – The canonical pathway of miRNA biogenesis. The miRNA is transcribed, creating primary miRNA (pri-miRNA), which is cleaved by Drosha into precursor-miRNA (pre-miRNA). Pre-miRNAs are exported via exportin-5 to the cytoplasm, where they are further processed by Dicer to create a duplex of two mature miRNAs. The duplex associates with the protein AGO to form the precursor RNA-Induced Silencing Complex (pre-RISC). The duplex unwinds and the passenger strand is lost, leaving the mature miRNA as part of the RISC, which mediates gene regulation.

Once fully mature, the miRNA within the RISC regulates the expression of genes by binding to complementary sequences in the 3’ untranslated region (UTR) of mRNAs (5). In animals, the mRNA is bound by nucleotides 2 through 8 of the miRNA’s 5’ end. This region is called the seed region, and complementarity to this region is critical for regulation of a given gene.

Perfect or near perfect binding of the seed sequence to the mRNA leads to complete mRNA degradation, while imperfect binding leads to the inhibition of protein synthesis. Either method causes gene regulation via an overall reduction in protein levels.

miRNA Seed Region Binding

Figure 2 – The seed region consists of nucleotides 2 to 8 on the 5’ end of the miRNA. The complementarity of the seed region to the mRNA determines how the gene will be silenced. If there is perfect complementarity between miRNA and mRNA it leads to degradation of the mRNA. If there is imperfect complementarity, it leads to inhibition of protein synthesis.

miRNAs are able to regulate genes in other ways as well (6). For example, some miRNAs bind to a sequence in the 5’UTR of a gene, instead of the 3’UTR. Binding in the 5’UTR can lead to either transcriptional gene activation or silencing. Some miRNAs can regulate other small RNA species. They may induce either post-transcriptional degradation or increased processing of small RNA molecules, leading to either reduced or increased expression of the small RNA.

We carry reporter constructs with the 3'UTR or 5'UTR of any gene, specifically designed for studying miRNA binding and gene regulation.
miRNA Nomenclature

For people new to the field, miRNA naming conventions can be difficult to understand. For a summary of miRNA nomenclature (7), see the table below:

miRNA Names Notes on Nomenclature
hsa-miR-XX vs. mmu-miR-XX vs. rno-miR-XX The first three letters indicate the organism the miRNA is found in. hsa = human, mmu = mouse, rno = rat.
hsa-mir-XX vs. hsa-miR-XX Capitalization indicates whether it is the mature or precursor miRNA. mir = precursor sequence, miR = mature sequence.
hsa-miR-XX-5p vs. hsa-miR-XX-3p The guide and passenger miRNAs processed from the pre-miRNA. The guide miRNA is found in higher abundance than the passenger miRNA. Older alternative to the -5p and -3p nomenclature.
hsa-miR-XX-1 vs. hsa-miR-XX-2 vs. hsa-miR-XX Mature miRNA sequences that are identical, but are originally transcribed from different genes and have distinct precursor sequences. As the sequences are identical, they may be referred to without the numerical suffix.
hsa-mir-XX-1 vs. hsa-mir-XX-2 Precursor miRNAs that are different, but are processed into an identical miRNA.
hsa-miR-XXa vs. hsa-miR-XXb Closely related mature miRNAs (differ by only one or two nucleotides).

Adapted from: Table 2 from Bernardo, et al. (8)

The first three letters of a miRNA name indicate the species that it is found in. For example, three of the most popular organisms to study miRNAs in are human, mouse, and rat. miRNAs from these species will be named with the prefixes hsa-, mmu-, and rno-, respectively.

Unlike the descriptive names that genes have, miRNAs are simply named sequentially. For example, if the last published novel human miRNA was hsa-miR-400, then next published will be named hsa-miR-401. However, if a miRNA from mouse is found with the same sequence as hsa-miR-400, it should be called mmu-miR-400, even if only 200 mouse miRNAs have been assigned so far.

Information may be encoded in the capitalization of the ‘r’ in ‘mir’ or ‘miR’. A sequence labelled with a lowercase ‘r’, such as hsa-mir-XX, refers to the miRNA gene and/or predicted stem-loop portion of a primary transcript. The corresponding sequence with a capital ‘R”, hsa-miR-XX, would be that of one of the mature miRNAs released after processing. hsa-miR-XX may or may not be followed by further suffixes, which contain more information about the mature miRNA sequence.

Mature miRNAs are created by processing a pre-miRNA into two arms. According to traditional nomenclature, if one arm is found in much higher abundance than the other, it would be called the guide strand, and would be labelled hsa-miR-XX. The other passenger strand would be called hsa-miR-XX*.

However, as increasing importance has been ascribed to the passenger strand, naming conventions have changed. Now they would be named according to which end of the miRNA they are found on. The 3’ miRNA would be hsa-miR-XX-3p and the 5’ miRNA would be hsa-miR-XX-5p. No information regarding importance is encoded in these names. To further confuse matters, an even older naming convention used ‘–s’ and ‘–as’ to differentiate between the 5’ and 3’ arms of each miRNA. Because of these changes, it’s possible that the same miRNA could have been called at different times by as many as three different names.

Even more suffixes may be attached to the name to indicate closely related miRNAs. For example, hsa-miR-XXa and hsa-miR-XXb would indicate two miRNAs with very similar sequences (only one or two nucleotides different). They would be expressed from the precursors hsa-mir-XXa and hsa-mir-XXb. A numbered suffix is assigned to show mature miRNA sequences that are identical, but are transcribed from different genes and thus have a different precursor sequence. An example of this would be the precursor miRNAs hsa-mir-XX-1 and hsa-mir-XX-2, which would be processed into identical mature miRNAs called hsa-miR-XX-1 and hsa-miR-XX-2.

There are exceptions to the above rules. For example, plants and viral miRNAs have slightly different naming conventions. As well, certain miRNAs have retained non-numerical names for historical reasons, such as let-7 and lin-4.

For these reasons, it is always best to rely on the literature for information on miRNAs, rather than trying to extrapolate from their names. Remember that your miRNA of interest may have been assigned a different ID in the past. Unlike miRNA names, miRNA accession numbers always remain stable, so they may serve as a better reference.

miRNA Clustering

miRNA genes may be encoded in various places in the genome, including both coding and non-coding regions. However, it has become obvious that miRNA genes do not occur randomly; they have a tendency to cluster near one another (9). A typical miRNA cluster will consist of two or three miRNA genes. These clusters are transcribed as polycistrons, which are then processed into multiple pre-miRNAs. Clustered miRNAs sometimes, but not always, consist of miRNAs with similar sequences and/or functions in the cell.

Small RNAs - A Comparison

There are many different small RNA species, including miRNA (microRNA), siRNA (small interfering RNA), and piRNA (Piwi-interacting RNA). piRNA, miRNA, and siRNA have in common their short length and ability to silence genes. However, their functions and processing are different. See below for a summary.

  miRNA siRNA piRNA
Length ~22 nucleotides ~21 nucleotides 24-30 nucleotides
Processng Enzymes Drosha and Dicer Dicer Zucchini
Derived From ssRNA hairpin ssRNA duplex Long ssRNA
Argonaute Subfamily AGO AGO PIWI
Mechanism of Action Translational repression, mRNA degradation RNA cleavage Repression of transposons, epigenetic changes
Function Regulation of protein-coding genes Regulation of protein-coding genes and transposons, antiviral defense Transposon silencing, other unknown functions

Adapted from: Table 1 from Ha, et al. (10)

What about shRNAs? shRNA stands for “short hairpin” RNA, as they consist of a ssRNA forming a tight hairpin structure. shRNAs are artificial constructs and are not found endogenously in the human body. However, they can be used to silence gene expression via RNA interference just like miRNAs.

A shRNA is typically expressed in the cell from a viral vector, at which point its hairpin structure mimics that of a pri-miRNA. From that point on, the shRNA is processed and functions like a normal miRNA, including the ability to silence genes.

We offer a comprehensive collection of miRNA expressed from lenti-, AAV, and adenovirus.
Experimental Workflow

What follows is a general experimental workflow for miRNA studies.

  • Identify phenotype of interest. This is often a disease state.
  • Screen for candidate miRNAs that may have an influence on the phenotype of interest. Use a high throughput technique like microarray analysis or RNA-seq. Determine which miRNAs to study in more detail.
  • Confirm results of screening using another miRNA detection technique (RT-qPCR, Northern Blotting, or in situ hybridization).
  • Identify candidate target genes for the miRNAs of interest. Use a computational approach.
  • Confirm target genes. Use a UTR reporter assay in combination with overexpression or inhibition of miRNA.
  • Confirm phenotypic effects. Overexpress, inhibit, or knockout miRNA. Monitor the effects using proteomic/transcriptomic methods, or other detection methods (qPCR, Western Blotting, etc.).
  • Widespread regulatory activity of vertebrate microRNA* species. Yang, Jr-Shiuan, et al. 2010, RNA, pp. 312-326.
  • Small RNA sorting: matchmaking for Argonautes. Czech, Benjamin and Hannon, Gregory J. 2011, Nature Reviews, Vol. 12, pp. 19-31.
  • MicroRNA evolution by arm switching. Griffiths-Jones, Sam, et al. 2011, Scientific Report, Vol. 12, pp. 172-177.
  • Many ways to generate microRNA-like small RNAs: Non-canonical pathways for microRNA production. Miyoshi, Keita, Miyoshi, Tomohiro and Siomi, Haruhiko. 2, Molecular & General Genetics, Vol. 284, pp. 95-103.
  • Mechanisms of microRNA-mediated gene regulation in animal cells. Nilsen, Timothy W. 5, 2007, Trends in Genetics, Vol. 23, pp. 243-249.
  • MicroRNA in Control of Gene Expression: An Overview of Nuclear Functions. Catalanotto, Caterina, Cogoni, Carlo and Zardo, Giuseppe. 10, 2016, International Journal of Molecular Sciences, Vol. 17, p. 1712.
  • A uniform system for microRNA annotation. Ambros, Victor, et al. 3, 2003, RNA, Vol. 9, pp. 277-279.
  • A MicroRNA Guide for Clinicians and Basic Scientists: Background and Experimental Techniques. Bernardo, Bianca C, et al. 3, 2012, Heart Lung and Circulation, Vol. 21, pp. 131-142.
  • Clustering and conservation patterns of human microRNAs. Altuvia, Yael, et al. 8, 2005, Nucleic Acids Research, Vol. 33, pp. 2697-2706.
  • Regulation of microRNA biogenesis. Ha, Minju and Kim, Narry V. 2014, Nature Reviews Molecular Cell Biology, pp. 509-524.