Small RNA Modifications: Integral to Function and Disease

doi:10.1016/j.molmed.2016.10.009

Trends in Molecular Medicine

Volume 22, Issue 12, December 2016, Pages 1025-1034

https://doi.org/10.1016/j.molmed.2016.10.009 Get rights and content

Trends

RNA modifications such as 2′-O-methylation, 5-methylcytidine (m⁵C), and adenosine-to-inosine (A-to-I) editing modulate the activities of small RNAs in diverse biological processes and play pivotal roles in pathological conditions.

Antibody-based detection of modified RNAs and high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) are promising diagnostic approaches for distinguishing normal versus diseased conditions.

Novel methods for RNA sequencing facilitate the identification of modified small RNAs that are recalcitrant to sequencing using standard approaches, expanding the diversity of small RNAs that can be detected as potential diagnostic markers of disease.

Small RNAs have the potential to store a secondary layer of labile biological information in the form of modified nucleotides. Emerging evidence has shown that small RNAs including microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs) and tRNA-derived small RNAs (tsRNAs) harbor a diversity of RNA modifications. These findings highlight the importance of RNA modifications in the modulation of basic properties such as RNA stability and other complex physiological processes involved in stress responses, metabolism, immunity, and epigenetic inheritance of environmentally acquired traits, among others. High-resolution, high-throughput methods for detecting, mapping and screening these small RNA modifications now provide opportunities to uncover their diagnostic potential as sensitive disease markers.

Section snippets

A Glimpse into RNA Modifications

RNAs are post-transcriptionally altered with a variety of chemical modifications in all domains of life. More than 100 types of RNA modification have been identified to date, in many cases showing context-dependent functions that are only beginning to become clear [1]. Post-transcriptional modifications are known to be especially prevalent in stable, structured RNAs such as tRNA and rRNA where they play important roles in biogenesis, correct structural folding, and function [2]. Emerging

2′-O-Methylation in Small RNAs: Stability and Beyond

miRNAs are key players in RNA silencing pathways inducing mRNA degradation, translational repression, and transcriptional silencing [17]. 2′-O-Methylation, mediated by HUA enhancer 1 (HEN1), was first recognized in plants [18], where it protects miRNAs from small RNA decay pathways that are triggered by 3′-uridylation 19, 20, a well-characterized RNA decay pathway [21] (Figure 2A). Although mature miRNAs in animals lack 2′-O-methylation, other mammalian small RNAs such as siRNAs and

m⁵C: A Stress Sensor and Transgenerational Mark in tRNA and tsRNAs

m⁵Cs are frequently detected in various non-coding RNAs such as tRNA, rRNA, and, more recently, mRNAs. While the full range of potential functions for m⁵C in RNA is not completely understood, bisulfite RNA sequencing, which pinpoints m⁵C residues [31], has helped reveal the importance of these residues in tRNAs 32, 33, 34, 35, 36. One group has shown that m⁵C modifications of tRNAs by the methyltransferases DNA methyltransferase 2 (Dnmt2) and NOP2/Sun RNA methyltransferase family member 2

RNA Editing Events in Small RNAs: Biology and Disease Associations

RNA editing is a process that alters the primary nucleotide sequence of RNA by specific enzymes. The chief distinction between RNA editing and RNA modification is that editing is usually irreversible and generally understood to produce altered base-pairing behavior. RNA editing events are widely observed in mRNA, tRNA, rRNA, and miRNA in all kingdoms of life. The most frequent editing events are deaminations that convert adenosine to inosine (A to I) and cytidine to uridine (C to U) [52].

Profiling Small RNA Modifications As Biomarkers of Disease

Several different approaches have been used for high-throughput analysis of RNA modifications; each has different strengths in terms of its ability to monitor specific RNA modifications, characterize global modification profiles in biological samples, or reveal the identity of modified RNAs with sequence-level specificity. Antibody-based detection of modified small RNAs has shown promise as a sensitive diagnostic for identifying early markers of disease. For example, m¹A antibodies can detect

Emerging Methods for Profiling and Mapping RNA Modifications in Small RNAs

While LC-MS/MS is a powerful approach for quantifying the compositional changes of RNA modifications, this method requires digestion of RNAs into short segments or single nucleosides before detection, therefore making it difficult to unambiguously assign modifications to specific transcripts (Figure 4A) 51, 66, 67. Thus, complementary approaches to map RNA modifications to specific RNAs are required to gain a more comprehensive view of modified small RNAs.

Sequencing-based protocols have been

Concluding Remarks: How Many New Bits of Information?

Compared with the accelerating rate of discovery of many types of small RNAs [75], the study of small RNA modifications in biology and disease remains in its infancy. Emerging evidence indicates that RNA modifications are important molecular markers that can alter the biogenesis, function, and informational capacity of small RNAs. New techniques including high-throughput LC-MS/MS and sequencing-based approaches that specifically target modified RNAs using antibodies, enzymatic treatments, or

Acknowledgments

This research was partially supported by the NIH/NHGRI (HG006753-02 to T.M.L, P30GM110767-03 to Q.C.) and start-up support from the University of Nevada, Reno School of Medicine (to Q.C.).

Glossary

Bisulfite RNA sequencing: method to detect m⁵C methylation patterns in RNAs by bisulfite treatment of RNAs.
cDNA library preparation: synthesis of single-strand complementary DNAs from RNA samples by reverse transcription.
Eraser: a demethylase or associated enzymatic complex that removes RNA modifications.
P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs): the largest class of small non-coding RNAs expressed in animal cells; form RNA–protein complexes with PIWI proteins.
Reader: proteins that

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Small noncoding RNAs (sncRNAs) play diverse roles in numerous biological processes. While the widely used RNA sequencing (RNA-Seq) method has advanced sncRNA discovery, RNA modifications can interfere with the complementary DNA library construction process, preventing the discovery of highly modified sncRNAs including transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs) that may have important functions in disease development. To address this technical obstacle, we recently developed a novel PANDORA-Seq (Panoramic RNA Display by Overcoming RNA Modification Aborted Sequencing) method to overcome RNA modification-elicited sequence interferences. To identify novel sncRNAs associated with atherosclerosis development, LDL receptor-deficient (LDLR^−/−) mice were fed a low-cholesterol diet or high-cholesterol diet (HCD) for 9 weeks. Total RNAs isolated from the intima were subjected to PANDORA-Seq and traditional RNA-Seq. By overcoming RNA modification-elicited limitations, PANDORA-Seq unveiled an rsRNA/tsRNA-enriched sncRNA landscape in the atherosclerotic intima of LDLR^−/− mice, which was strikingly different from that detected by traditional RNA-Seq. While microRNAs were the dominant sncRNAs detected by traditional RNA-Seq, PANDORA-Seq substantially increased the reads of rsRNAs and tsRNAs. PANDORA-Seq also detected 1,383 differentially expressed sncRNAs induced by HCD feeding, including 1,160 rsRNAs and 195 tsRNAs. One of HCD-induced intimal tsRNAs, tsRNA-Arg-CCG, may contribute to atherosclerosis development by regulating the proatherogenic gene expression in endothelial cells. Overall, PANDORA-Seq revealed a hidden rsRNA and tsRNA population associated with atherosclerosis development. These understudied tsRNAs and rsRNAs, which are much more abundant than microRNAs in the atherosclerotic intima of LDLR^−/− mice, warrant further investigations.
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Fresh tumor tissues of patients that were diagnosed as GBMs (4 cases) and LGs (5 cases) at the First Affiliated Hospital of Wenzhou Medical University from 2019.05 to 2021.01 were collected. The tsRNAs’ levels were analyzed and compared through high-throughput sequencing, candidate tsRNAs were chosen according to the expression level, and the expression of the candidate tsRNAs was validated through qPCR. Finally, the potential targets were imputed using the Miranda and TargetScan databases, and possible biological functions of the differentially expressed (DE) tsRNAs' targets were enriched based on GO and KEGG databases.
A total of 4 GBMs and 5 LGs patients were enrolled in the current study. High-throughput sequencing showed that 186 tsRNAs were expressed in two groups, over them, 43 tsRNAs were unique to GBMs, and 24 tsRNAs were unique to LGs. A total of 9 tsRNAs were selected as candidate tsRNAs according to the tsRNA expression level, among which 6 tsRNAs were highly expressed in GBMs and 3 tsRNAs were low expressed in GBMs. qPCR verification further demonstrated that 5 tsRNAs were significantly up-regulated and 1 tsRNA was significantly down-regulated in GBMs: tRF-1-32-chrM.Lys-TTT (p=0.00118), tiRNA-1-33-Gly-GCC-1 (p=0.00203), tiRNA-1-33-Gly-CCC-1 (p=0.00460), tRF-1-31-His-GTG-1 (p=0.00819), tiRNA-1-33-Gly-GCC-2-M3 (p=0.01032), and tiRNA-1-34-Lys-CTT-1-M2 (p=0.03569). Enrichment analysis of the qPCR verified DE tsRNAs showed that the 5 up-regulated tsRNAs seemed to be associated with axon guidance, pluripotent stem cells regulation, nucleotide excision repair, Hippo signaling pathway, and cancer-related pathways, while the down-regulated tsRNA (tRF-1-32-chrM.Lys-TTT) was associated with oocyte meiosis and renin secretion.
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³: These authors contributed equally to this work.

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Trends in Molecular Medicine

ReviewSmall RNA Modifications: Integral to Function and Disease

Trends

Section snippets

A Glimpse into RNA Modifications

2′-O-Methylation in Small RNAs: Stability and Beyond

m5C: A Stress Sensor and Transgenerational Mark in tRNA and tsRNAs

RNA Editing Events in Small RNAs: Biology and Disease Associations

Profiling Small RNA Modifications As Biomarkers of Disease

Emerging Methods for Profiling and Mapping RNA Modifications in Small RNAs

Concluding Remarks: How Many New Bits of Information?

Acknowledgments

Glossary

Cell

Mol. Cell

Cell

Cell

Trends Biotechnol.

Mol. Cell

Cell

Trends Biochem. Sci.

FEBS Lett.

Trends Mol. Med.

Trends Mol. Med.

Methods Enzymol.

Cell Chem. Biol.

Trends Biochem. Sci.

RNA modifications: what have we learned and where are we headed?

Nat. Rev. Genet.

The pivotal regulatory landscape of RNA modifications

Annu. Rev. Genomics Hum. Genet.

Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq

Nature

Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase

Cell Res.

A METTL3–METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation

Nat. Chem. Biol.

N6-Methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO

Nat. Chem. Biol.

N6-Methyladenosine-dependent regulation of messenger RNA stability

Nature

Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells

Nature

Chemical pulldown reveals dynamic pseudouridylation of the mammalian transcriptome

Nat. Chem. Biol.

Characterizing 5-methylcytosine in the mammalian epitranscriptome

Genome Biol.

Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA

Nucleic Acids Res.

Transcriptome-wide mapping reveals reversible and dynamic N1-methyladenosine methylome

Nat. Chem. Biol.

The dynamic N1-methyladenosine methylome in eukaryotic messenger RNA

Nature

Towards a molecular understanding of microRNA-mediated gene silencing

Nat. Rev. Genet.

Methylation as a crucial step in plant microRNA biogenesis

Science

Regulation of small RNA stability: methylation and beyond

Cell Res.

Methylation protects microRNAs from an AGO1-associated activity that uridylates 5′ RNA fragments generated by AGO1 cleavage

Proc. Natl Acad. Sci. U. S. A.

Companies in footrace to deliver RNAi

Nat. Biotechnol.

Cross-kingdom inhibition of breast cancer growth by plant miR159

Cell Res.

Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of cross-kingdom regulation by microRNA

Cell Res.

Detection of dietary plant-based small RNAs in animals

Cell Res.

A novel chemopreventive strategy based on therapeutic microRNAs produced in plants

Cell Res.

Honeysuckle-encoded atypical microRNA2911 directly targets influenza A viruses

Cell Res.

Review
Small RNA Modifications: Integral to Function and Disease

m⁵C: A Stress Sensor and Transgenerational Mark in tRNA and tsRNAs

Topology of the human and mouse m⁶A RNA methylomes revealed by m⁶A-seq

Mammalian WTAP is a regulatory subunit of the RNA N⁶-methyladenosine methyltransferase

A METTL3–METTL14 complex mediates mammalian nuclear RNA N⁶-adenosine methylation

N⁶-Methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO

N⁶-Methyladenosine-dependent regulation of messenger RNA stability

Transcriptome-wide mapping reveals reversible and dynamic N¹-methyladenosine methylome

The dynamic N¹-methyladenosine methylome in eukaryotic messenger RNA