mikro-RNAs

miRNAs are short, evolutionarily conserved non-coding RNAs (≈20-24 nt) that are formed in a multi-step process via Drosha/DGCR8, Exportin-5, Dicer and Argonaute proteins. They regulate gene expression mainly post-transcriptionally through seed-dependent binding to target mRNAs, leading to translational repression and mRNA degradation.

In addition to the canonical biogenesis pathway, Drosha- and Dicer-independent alternative pathways exist. miRNAs are integral components of almost all cellular regulatory networks. Their dysregulation can lead to numerous diseases (especially cancer, cardiovascular and neurological diseases). At the same time, miRNAs are being intensively studied as stable circulating biomarkers and as targets for therapeutic interventions (see RNA interference; RNA therapeutics below). A single miRNA can regulate dozens to hundreds of target mRNAs, and conversely, one mRNA is often controlled by several miRNAs.

Canonicalbiogenesis: Canonical biogenesis involves two-step processing by RNase III enzymes (Drosha, Dicer):

1. Transcription (nucleus)
   1. miRNA genes are mostly transcribed by RNA polymerase II pri-miRNA (hundreds to thousands of nt, 5'-cap, poly(A)-tail, hairpin structure(s)).
2. Microprocessor complex (Drosha/DGCR8)
   1. DGCR8 recognizes dsRNA hairpins in pri-miRNA
   2. Drosha cuts ~11 nt upstream of hairpin basi pre-miRNA (~60-70 nt hairpin with 2-nt 3' overhang).
3. Export from the nucleus
   1. pre-miRNA is transported into the cytoplasm by exportin-5/Ran-GTP.
4. Dicer processing (cytoplasm)
   1. Dicer (RNase-III) recognizes the pre-miRNA and cleaves near the hairpin loop. The result is a ~22 nt dsRNA duplex with 2-nt 3'-overhangs (miRNA:miRNA*).
5. RISC loading
   1. The duplex is loaded into the Argonaute (AGO) complex. One strand (guide strand, e.g. miR-21-5p) remains in RISC; mature miRNA.
   2. The other strand (passenger / miRNA*) is usually degraded (or in some cases functional).

Non-canonical biogenesis pathways. In addition to the canonical pathway, several non-canonical pathways exist that bypass parts of the standard machinery:

1. Drosha-independent (but Dicer-dependent) pathways.
2. Drosha-independent (but Drosha-dependent) paths
   1. Drosha produces an unusually short pre-miRNA.
   2. Instead of Dicer, AGO2 performs endonucleolytic processing → mature miRNA.
3. Further variants
   1. pri-miRNAs that originate from RNA polymerase III
   2. miRNA-like RNAs from tRNAs, snoRNAs or other structured RNAs. These alternative pathways expand the regulatory potential and explain miRNAs in contexts where classical Drosha/Dicer signaling pathways are disrupted but miRNA-like molecules still occur.

Mechanisms of gene regulation by miRNA. The mature miRNA in miRISC (miRNA-induced silencing complex) recognizes target mRNAs mainly via the seed region (nt 2-8 from the 5'-end). Target recognition:

1. Main binding site: 3'-UTR of mRNAs
2. Binding to CDS and 5'-UTR also described
3. Binding mostly via partial complementarity (animals)

Regulation of miRNA expression. miRNAs are themselves part of complex regulatory networks:

1. Transcriptional control
2. miRNA promoters are regulated by classical transcription factors (e.g. MYC, p53).
3. Epigenetic modifications (DNA methylation, histone marks) alter miRNA gene activity.
4. Post-transcriptional regulation of biogenesis
   1. Modulation of Drosha, DGCR8, Dicer, AGO2 (expression, PTMs)
   2. RNA binding proteins (e.g. hnRNPs, Lin28) influence specific pri- or pre-miRNAs.
5. RNA editing and SNPs
   1. A-to-I editing in pri-/pre-miRNAs can alter seed sequence or structure.
   2. Polymorphisms in miRNA genes or target sequences modify binding and function.
6. ceRNA networks (Competing Endogenous RNAs)
7. lncRNAs, circular RNAs (circRNAs) and mRNAs can bind miRNAs as "sponges". This reduces the effective availability of the miRNA for other targets.

Turnover and degradation of miRNAs. miRNAs are relatively stable (half-lives of hours to days), but are actively degraded to allow dynamic regulation:

1. 3'-tailing and trimming
2. Addition of U or A nucleotides by TUTases or PAPs → destabilization.
3. Exonucleases trim the 3'-end.
4. Exonucleolytic degradation
5. including XRN- and SDN-like nucleases (species-dependent).
6. Target-Directed miRNA Degradation (TDMD)
   1. strongly complementary target RNAs induce specific miRNA degradation.

Biological functions. miRNAs are involved in most fundamental cellular processes:

1. Development and differentiation
2. z. e.g. miR-1 (muscle), miR-124 (neuron), let-7 (developmental timing)
3. Cell cycle, proliferation, apoptosis
4. miR-17-92 cluster, miR-34 family, etc.
5. Stem cell self-renewal & pluripotency
6. Metabolism and energy metabolism
7. Regulation of insulin signaling pathways, lipid and glucose metabolism
8. Immune system and inflammation
9. e.g. miR-155, miR-146a in innate and adaptive immune response
10. Neuronal functions
11. Synaptic plasticity, memory formation, axon guidance
12. Stress responses
13. Hypoxia, DNA damage, oxidative stress

miRNAs in diseases. Misregulated miRNA networks have been demonstrated in many diseases:

1. Cancer
2. OncomiRs (e.g. miR-21, miR-155) promote proliferation, angiogenesis, invasion.
3. Tumor suppressor miRNAs (e.g. let-7, miR-34) are often downregulated.
4. Cardiovascular diseases
5. miR-1, miR-133, miR-208, miR-499 and others in hypertrophy, infarction, arrhythmias.
6. Neurological and neurodegenerative diseases
7. miR-9, miR-124, miR-132 etc. discussed for schizophrenia, Alzheimer's, Parkinson's.
8. Metabolic diseases
9. miRNA profiles altered in diabetes, fatty liver, obesity.
10. Infectious diseases
11. Host and viral miRNAs modulate viral replication (e.g. herpes viruses, hepatitis viruses).

miRNAs as biomarkers

1. miRNAs are detectable in blood, serum, plasma, saliva, urine, often in
2. protected in exosomes, microvesicles or AGO complexes.
3. High stability, relative specificity of patterns, attractive diagnostic and prognostic biomarkers (e.g. in oncology, cardiology).

Therapeutic approaches:

1. miRNA mimetics
2. synthetic miRNAs to replace missing tumor suppressor miRNAs.
3. miRNA inhibitors
4. Antagomirs, LNA-modified oligonucleotides, sponge constructs to inhibit oncomiRs.