MicroRNAs (miRNAs) represent an extensive class of small endogenous RNAs which regulate gene expression at the post-transcriptional level by messenger RNA (mRNA) cleavage or translation inhibition. A majority of multi-cellular organisms encode dozens to hundreds of miRNA genes which play important roles in the control of biologic processes.
Since a large fraction of protein-coding genes are under direct control of miRNA, appropriate production of specific miRNAs at the right time and in the right place is pivotal to most gene regulatory pathways. Recent research has revealed regulation of mature miRNA turnover in the immune system as well.
Biogenesis of miRNAs is subject to complex regulation at both the transcriptional and post-transcriptional levels. As small deviations in miRNA levels can disturb the regulation of different target genes, adequate control of miRNA biogenesis is essential for the maintenance of normal cell homeostasis.
Overview of miRNA biogenesis
The biogenesis of miRNAs represents a series of sequential processes to generate mature miRNAs. Primary miRNAs (pri-miRNAs) are initially transcribed from the intergenic or intragenic regions by RNA polymerase II. These pri-miRNAs are subsequently excised by double-stranded RNA-specific ribonuclease in the nucleus to produce pre-miRNAs with specific hair-pin structures (also called hairpin precursors).
Hairpin precursors are exported out of the nucleus by Exportin 5, where pre-miRNAs are additionally processed by Dicer (RNase III endonuclease) into 21-14 nucleotide long duplex miRNA. The strand designated to be the mature sequence is then loaded onto Argonaute proteins, forming the miRNA induced silencing complex (RISC).
miRNAs then guide such formed complexes to specific mRNAs using imperfect base pairing in order to down-regulate their expression via mRNA destabilization or translational repression. It is important to note that accessory proteins can regulate miRNA biogenesis at each of these steps, thus proper miRNA homeostasis can be ensured.
Although plant miRNAs are also produced from long primary transcripts, their maturation differs from miRNA maturation in animals, as no plant homologue of double-stranded RNA-specific ribonuclease was discovered. The function is carried out by one or more specialized Dicer enzymes in plants.
Valid and efficient methods to detect miRNAs and evaluate their biogenesis are essential to determine their possible roles in various biological or pathological processes, but also to accelerate clinical application of miRNA-based therapy. Northern blot, real-time polymerase chain reaction (PCR) and microarray have been widely used to detect the endogenous production of miRNAs.
Mechanism of miRNA gene regulation
The exact mechanism of miRNA-mediated gene regulation is still a subject of intense research endeavors. miRNAs have been shown to repress the translation of target mRNA by interfering with translation initiation or elongation. On the other hand, targets of miRNA may be secluded from the translation machinery and conveyed to processing-bodies where mRNAs are degraded.
Early work with let-7 (one of the first miRNAs discovered in the nematode Caenorhabditis elegans) suggested that miRNAs act essentially at the level of translation without changing mRNA levels. However, recent genome-wide profiling indicates that most highly repressed miRNA targets exhibit a certain degree of mRNA degradation. miRNA interaction can also result in the destabilization of target mRNAs by recruiting the deadenylation and decapping complexes.
Alternative mechanisms of mRNA repression can also be exploited depending on the RNA secondary structure or effector proteins associated with specific pairs of miRNA and mRNA. Further elucidation of the mechanisms of miRNA-mediated silencing and the requirements of functional miRNA target sites are areas of active research, which will be crucial in our understanding of the physiological role of miRNA in different areas.