Figures
Figure 1
Chemical structures of RNA modifications discussed in this review. Additional chemical groups are shown in red. Panel A – Cap-related mRNA modifications, the structure of m7 GpppN cap as well as cap0/cap1 and cap2 are shown in the middle, R stands for ribose moiety in RNA, terminal guanosine can be additionally singly or doubly methylated at N2 atom (m27 G and m227 G, left). The first (and the second) encoded nucleotide in mRNA may be methylated at the 2’-OH (Am/Gm, cap1/cap2 structures) and/or at the N6 of adenosine m6 Am (right). Panel B – Internal RNA modifications reported in cellular and viral mRNAs. Ribose moiety in pseudouridine structure is red colored since it is connected to the base by unusual C-C glycosidic bond.
Figure 1
Figure 2
Cytoplasmic and nuclear cycles for viral replication and interplay with the RNA modification machinery. Depending on the virus type, viral DNA, directly or after the cDNA synthesis by viral RT, can be transported into the nucleus and integrated (or not) into host genomic DNA (left). Viral mRNAs produced in the nucleus may be exposed to the cellular RNA modification machinery located in this compartment (shown in violet). After maturation and export to cytoplasm, viral mRNAs (and gRNA) may be modified by the cytoplasmic host RNA modification machinery (shown in yellow and blue). For the viruses with cytoplasmic only replication cycle (right), viral mRNAs are exposed to cytoplasmic RNA modification machinery and, if present to viral RNA modification enzymes encoded by the virus (in red). Only ‘RNA-related’ steps of viral replication are shown on the figure, other events of the viral cycle are omitted for clarity.
Figure 2
Figure 3
Overview of detection approaches used for RNA modification analyses .
Panel A – Analysis of RNA modifications can be performed by total digestion of purified RNA to nucleosides, their separation by C18 reverse phase HPLC followed by MS (or MS/MS) analysis of the substance of interest (top). Alternatively, RNA can be fragmented by specific endonuclease (example of RNase T1 shown), the resulting RNA fragments separated by HPLC followed by MS analysis of their masses (bottom).
Panel B – Most popular deep sequencing-based approaches for RNA modification mapping. Antibody (Ab)-directed enrichment of modified RNA fragments (left), direct reverse transcription (RT) signature by the RNA modification unable to base-pair with any partner during primer extension (middle), chemically-induced RT-signature due to RNA treatment with specific chemical reagent (right). Expected RNA sequencing coverage profiles of mapped reads are shown at the bottom.
Panel C – Single-molecule direct RNA sequencing by Oxford nanopore technology. RNA strand (red) is pushed through the nanopore by the motor protein and variations of the ionic current are recorded (middle). Since the modification in RNA affects its physico-chemical properties, the ion current trace is somehow different for the modified residue. Identification requires the use of training sets and deep learning algorithms.
Figure 3
Figure 4
RNA modification influences RNA structure and properties. Modified RNA nucleotides affect RNA stability towards nucleases and unspecific RNA degradation (top left), improve RNA thermostability under high temperatures (top middle) and contribute to antibiotic resistance by altering target sites in rRNA (top right). The central panel illustrates modulation of RNA recognition (here tRNA) by cognate proteins. Bottom panels show the importance of RNA modification in decoding (codon-anticodon interactions, left) and in modulation of translational frameshift (middle). The right bottom panel illustrates modulation of tRNA recognition by TLR7 endosomal receptor.
Figure 4
Figure 5
Modulation of innate immunity by RNA modifications. Non-self RNA is recognized both by endosomal membrane receptors TLR7/TLR8 (and TLR13 in mouse). TLR7 and TLR8 are sensitive to the modification status of tRNAs, namely to the Gm18 presence, which serves as immunosuppressor. rRNA modification m6 2 A plays the same role for TLR13. In the cytoplasm, non-host RNA is sensed by RIG-I and MDA5 receptors, which are modulated by Nm and inosine in RNAs. Both pathways activate Type I IFN and inflammatory cytokine secretion by transcriptional regulation in the nucleus.
Figure 5
Authors
1 Université de Lorraine, CNRS, INSERM, IBSLor (UMS2008/US40), Epitranscriptomics and RNA Sequencing Core Facility, F54000 Nancy, France
2 Université de Lorraine, CNRS, IMoPA (UMR7365), F54000 Nancy, France
Viral RNAs (either derived from DNA viruses and genomic/mRNAs of RNA viruses) produced and replicated in eukaryotic cells are exposed to the activity of host cell RNA modification machinery. Moreover, some complex viruses encode their own RNA modification enzymes, generally cap-related m7 G-and 2’-O-methyltransferases whose expression allows specific modification of viral transcripts and modulation of viral RNA recognition by host restriction systems. Here we review current achievements in the detection of viral RNA modifications by liquid chromatography coupled to mass spectrometry (LC-MS) and deep sequencing-based approaches. The presence, origin and characterized functions of RNA modifications in viral RNAs are discussed.