Modification on RNA molecules, such as 5-methylcytidine(m5C), 7-methylguanosine(m7G), Pseudouridine(Ψ) and N6- methyladenosine(m6A) can regulate the activity of the corresponding small RNA in different biological processes and play a key role in pathological processes. Small RNA may store unstable second layer genetic information in the form of modified nucleotides. Research data show that small RNA, including microRNA (miRNA), piwi interacting RNA (piRNA) and tRNA derived small RNA (tsRNA), has a variety of RNA modifications. These findings emphasize the importance of RNA modification in regulating basic properties, such as RNA stability and other complex physiological processes, which involve stress response, metabolism, immunity and epigenetics caused by environmental factors. The high-resolution and high-throughput methods used to detect, localize and screen these small RNA modifications are expected to make them a new class of high-sensitivity disease biomarkers in clinical diagnosis.

Ⅰ.Biological functions of small RNA modification

1. Modification on small RNA affects miRNA generation

Pri-miRNAs containing inosine produced by the RNA editing enzyme ADAR1 on the RNA strand can resist the cleavage of the miRNA cleavage enzyme Drosha, thereby reducing the production of mature miRNAs [1] . On the other hand, pri-miRNAs with m6A modification can promote the cleavage of Drosha, resulting in the increase of mature miRNAs[2] . In addition, m7G modification was also found to promote the maturation of pre-miRNAs (such as m7G modification on let-7e) [3] .

2. Modification on small RNA affects miRNA recognition of target genes

It is known that miRNA seed region modifications (such as the o8G modification on mir-184 and miR-1) can change the base pairing of miRNA-mRNA, affect the specificity of miRNA targeting target genes, and then have significant biological effects [4-6] . When these modifications are added to small RNA under pathological conditions, they can serve as an apparent transcription mechanism to regulate gene expression [5,6] .

3. Modification on small RNA affects its stability

The 3 '- Terminal 2'- O-methylation (2 '- OME) of mammalian small RNAs (such as siRNA and piRNA) can protect small RNA from degradation by uridylation and RNA degradation pathways [7] . 2 '- OMe can prolong the half-life of plant miRNA in the human body after ingestion, which creates an interesting possibility that modified small RNA from plant-based diet can exist in the human gastrointestinal tract for a long enough time to regulate human physiological processes[8,9] .

4. Distinguish between self and foreign RNA

Exogenous dsRNA lacking inosine modification can bind to the RIG-I-like receptor protein MDA5, a type of dsRNA helicase, and trigger the antiviral innate immune response[10] . Some studies have shown that bacterial tRNAs lacking 2'- O methylation (Gm) at position 18 can be recognized by toll like receptor 7 (TLR-7) on the endosomal surface of relevant immune cells and activate downstream innate immune responses[11] .

Ⅱ.Important small RNA modifications and their functions

7-methylguanosine (m7G)

The m7G methyltransferase mettl1 directly binds to miRNA precursors through m7G and accelerates pre-miRNA maturation[3] . After pre-miRNA processing, m7G may still be on mature miRNA and regulate the function of mature miRNA. For example, mature let-7e with m7G modification can downregulate the stability and translation efficiency of target HMGA2 mRNA, which can inhibit the migration and proliferation of lung cancer cells by reducing the level of HMGA2.

Pseudouridine(Ψ)

Pseudouridine (Ψ) It is the most abundant modified nucleoside on RNA, also known as the "fifth nucleoside" of RNA. Under the catalysis of pseudouridine synthetase 7 (PUS7), pseudouridylation in tsrna 5'- terminal oligoguanine (TOG) can activate tsRNA mediated global translation inhibition in human embryonic stem cells [12] .

N6-methyladenosine (m6A)

On the one hand, through the analysis of m6A antibody enriched miRNAs and their m6A modification motif RRACH, it was found that more than 200 mature miRNA were modified by m6A in human embryonic kidney cell HEK293[13] .

On the other hand, m6A modification can affect miRNA function. For example, in mir-17-5p or let-7a-5p, m6A causes a wide range of conformational changes around the mRNA recognition site, changing the targeting efficiency of miRNA[14] . Overall, m6A methylation on miRNA was significantly increased in cancer tissues compared with paired normal tissues. It is particularly noteworthy that the level of m6A modified mir-17-5p in serum has extremely high sensitivity and specificity in distinguishing patients with early-stage pancreatic cancer from healthy people[14] . Therefore, the m6A modification status in miRNA can be used as a diagnostic biomarker for early cancer. The above results indicate that the study of m6A modification is another important angle for us to understand the biological functions of miRNA.

5-methylcytidine (m5C)

m5C modification on miRNA can interfere with the formation of miRNA / mRNA duplex, resulting in the loss of gene silencing activity of miRNA itself. For example, m5C modification abolished the tumor suppressive function of mirna-181a-5p and was associated with poor prognosis of glioblastoma multiforme (GBM) [15] . In addition, m5C modification can also cause structural changes of RISC silencing complex. For example, in miR-200c-3p, the m5C modification at the 9th base near the MRE site disrupted the hydrogen bond formed between miRNA and Ser220 of AGO, resulting in the displacement of guanine at the 8th position of miRNA interacting with Arg761 of ago[14] .

Ⅲ.Small RNA modification as a biomarker for disease diagnosis

Several different methods have been used for high-throughput analysis of RNA modification. Each method has different advantages in monitoring specific RNA modifications, characterizing global modification profiles in biological samples, or revealing the identity of modified RNA with sequence level specificity. Antibody based detection of modified small RNA has shown promise as a sensitive diagnosis for identifying early markers of disease. For example, O8G oxidation of miRNA affects redox mediated gene expression and is associated with diseases occurring on cardiomyocytes[5] . Moreover, m6A modified miRNA were significantly increased in cancer compared with normal tissues. For example, the detection of the level of m6A modified miR-17-5p in serum has high sensitivity and specificity in the diagnosis of early pancreatic cancer[14] .

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References

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[15] Cheray, M., et al. (2020) "Cytosine methylation of mature microRNAs inhibits their functions and is associated with poor prognosis in glioblastoma multiforme" Mol Cancer 19(1):36 [PMID: 32098627]