Small RNA Modifications: Integral to Functions and Diseases
Molecular Mechanisms of Small RNA Modifications
The Challenges and Solutions of Studying Modified Small RNAs
Introduction
Small RNAs, including microRNAs (miRNAs) and tRNA-derived small RNAs (tsRNAs), harbor a diversity of RNA modifications. RNA modifications such as 5-methylcytidine (m5C), 7-methylguanosine (m7G), 8-oxoguanine(O8G), pseudouridine (Ψ) and m6A-methylation (m6A) modulate the activities of small RNAs in diverse biological processes and play pivotal roles in pathological conditions. High-resolution and high-throughput methods for detecting and quantifying modified small RNA levels provide opportunities to uncover their diagnostic potential as sensitive disease biomarkers.
Biological Functions of Small RNA Modifications
Modifications in small RNAs influence miRNA biogenesis.
pri-miRNAs harboring inosines generated by RNA editing enzyme ADAR1 are resistant to cleavage by miRNA processor Drosha, thus reducing the mature miRNA production [1]. On the other hand, pri-miRNAs harboring m6A enhance Drosha activity, thus increasing the mature miRNA production [2]. Also, m7G modification promotes pre-miRNA maturation [3].
Modifications in small RNAs influence miRNA targeting.
Modifications in the seed regions of miRNAs (e.g. O8G on miR-184 and miR-1) are known to alter the miRNA-mRNA base pairing, targeting specificity, and profound biological consequences [4-6]. Therefore, modifications installed in response to pathophysiological conditions can coordinate the gene expression by influencing miRNA targeting [5, 6].
Modifications in small RNAs influence small RNA stability.
2’-O-methylation (2’-OMe) at the 3’ end of mammalian small RNAs (e.g. siRNAs and piRNAs) protects the small RNAs from uridylation and from degradation by RNA decay pathways [7]. 2’-OMe prolongs plant miRNA half-life when ingested in human body, supporting an intriguing possibility that modified small RNAs derived from plant-based diets may survive long enough in the gastrointestinal tract to modulate human physiology [8, 9].
Discriminating self and foreign RNAs
Lacking inosine modification, foreign dsRNAs are bound by melanoma differentiation-associated protein 5 (MDA5), a RIG-I-like receptor dsRNA helicase, to trigger antiviral responses [10]. Bacterial tRNAs lacking 2’ -O-methylation at position 18 (Gm) are recognized by Toll-like receptor 7 (TLR-7) on the endosomal surface of relevant immune cells, activating downstream innate immunity. [11].
Prominent Small RNA Modifications and Their Functions
8-oxoguanine (O8G)
Reactive Oxygen Species (ROS) can convert guanine (G) to 8-oxoguanine (O8G) in miRNAs. O8G base pairs with adenine (A) instead of unmodified G pairing with C. Thus, O8G modification in the seed region (positions 2–8) of an miRNAs alters the mRNA targeting through O8G•A base pairing. For example, when modified with O8G in rat cardiomyoblast H9c2 cells under oxidative stress, miR-184 binds its new mRNA targets BCL-XL and BCL-W and suppresses their translation, resulting in increased cardiomyocyte cell death [4]. In another example, introducing O8G at the seed region of miR-1 alone is sufficient to cause cardiac hypertrophy in mice [5]. Conversely, the specific inhibition of O8G-miR-1 attenuates cardiac hypertrophy. Thus, O8G oxidation of miRNAs can serve as an epitranscriptional mechanism to coordinate pathophysiological, redox-mediated gene expression [5].
7-methylguanosine(m7G)
m7G methyltransferase METTL1 binds directly to miRNA precursors via the installed m7G and accelerates pre-miRNA processing [3]. After processing, the m7G may still remain in position of their mature miRNAs and modulate the mature miRNA function. For example, m7G in the mature let-7e down regulates the stability and translation efficiency of the target HMGA2 mRNA, inhibiting cell migration and proliferation of lung cancer cells by the reduced HMGA2 level.
Pseudouridine(Ψ)
Pseudouridine (Ψ) is one of the most abundant modifications in the RNA world. Catalyzed by pseudouridine synthase 7 (PUS7), pseudouridylation in the 5’-terminal oligoguanine (TOG) of tsRNA can activate global tsRNA-mediated translation inhibition in human embryonic stem cells [12].
m6A-methylation(m6A)
Well over 200 mature microRNAs are known to be modified by m6A in human embryonic kidney cells HEK293, as evidenced by the m6A immunoprecipitable miRNAs and the enriched presence of m6A modification motif RRACH in these miRNAs [13].
m6A modification affects miRNA functions. For example, in miR-17-5p or let-7a-5p, m6A causes a large structural change around the mRNA recognition site, altering the targeting efficiency [14]. Overall, m6A methylation in miRNAs is significantly increased in cancer tissues as compared with paired normal tissues. In particular, the level of m6A modified miR-17-5p in serum biofluid can distinguish early pancreatic cancer from the healthy controls at extremely high sensitivity and specificity [14]. Therefore, the m6A modification status in miRNAs can be a diagnostic biomarker for early-stage cancer detection. m6A modification is an additional dimension to the understanding of miRNA biology.
5-methylcytidine (m5C)
m5C in miRNAs disrupts the formation of miRNA/mRNA duplex and leads to the loss of gene silencing activity. For example, m5C modification abolishes the tumor suppressor function of miRNA-181a-5p and is associated with a poorer prognosis of glioblastoma multiforme (GBM) [15]. m5C also causes structural changes in the RISC complex. In miR-200c-3p, m5C at position 9 close to the MRE site disrupts the hydrogen bonding of the miRNA with Ser220 of AGO, leading to a positional shift of the guanine at position 8 that interacted with Arg761 of AGO [14].
Small RNA Modifications as Diagnostic Biomarkers
The associations of small RNA modifications with diseases offer opportunities for a new class of epitranscriptional biomarkers for potentially superior diagnostic/prognostic performance [14]. For example, O8G oxidation of miRNAs coordinates redox-mediated gene expression and is correlated with pathophysiological conditions of cardiomyocytes [5]. Overall, m6A modification of miRNA is significantly increased in cancer compared with normal tissues. As mentioned above, m6A modified miR-17-5p level in serum detects early pancreatic cancer with extremely high sensitivity and specificity [14].
Related Service
Small RNA Modification Array Service
References
[1] Kawahara, Y., et al. (2008) "Frequency and fate of microRNA editing in human brain" Nucleic Acids Res 36(16):5270-80 [PMID: 18684997]
[2] Alarcon, C. R., et al. (2015) "N6-methyladenosine marks primary microRNAs for processing" Nature 519(7544):482-5 [PMID: 25799998]
[3] Pandolfini, L., et al. (2019) "METTL1 Promotes let-7 MicroRNA Processing via m7G Methylation" Mol Cell 74(6):1278-1290 e9 [PMID: 31031083]
[4] Wang, J. X., et al. (2015) "Oxidative Modification of miR-184 Enables It to Target Bcl-xL and Bcl-w" Mol Cell 59(1):50-61 [PMID: 26028536]
[5] Seok, H., et al. (2020) "Position-specific oxidation of miR-1 encodes cardiac hypertrophy" Nature 584(7820):279-285 [PMID: 32760005]
[6] Seok, H., et al. (2016) "MicroRNA Target Recognition: Insights from Transcriptome-Wide Non-Canonical Interactions" Mol Cells 39(5):375-81 [PMID: 27117456]
[7] Ji, L. and Chen, X. (2012) "Regulation of small RNA stability: methylation and beyond" Cell Res 22(4):624-36 [PMID: 22410795]
[8] Chin, A. R., et al. (2016) "Cross-kingdom inhibition of breast cancer growth by plant miR159" Cell Res 26(2):217-28 [PMID: 26794868]
[9] Zhang, L., et al. (2012) "Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of cross-kingdom regulation by microRNA" Cell Res 22(1):107-26 [PMID: 21931358]
[10] Liddicoat, B. J., et al. (2015) "RNA editing by ADAR1 prevents MDA5 sensing of endogenous dsRNA as nonself" Science 349(6252):1115-20 [PMID: 26275108]
[11] Jockel, S., et al. (2012) "The 2'-O-methylation status of a single guanosine controls transfer RNA-mediated Toll-like receptor 7 activation or inhibition" J Exp Med 209(2):235-41 [PMID: 22312111]
[12] Guzzi, N., et al. (2018) "Pseudouridylation of tRNA-Derived Fragments Steers Translational Control in Stem Cells" Cell 173(5):1204-1216 e26 [PMID: 29628141]
[13] Berulava, T., et al. (2015) "N6-adenosine methylation in MiRNAs" PLoS One 10(2):e0118438 [PMID: 25723394]
[14] Konno, M., et al. (2019) "Distinct methylation levels of mature microRNAs in gastrointestinal cancers" Nat Commun 10(1):3888 [PMID: 31467274]
[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]