RNA modification

To identify diverse post-transcriptional modifications of RNA molecules and determine the transcriptome-wide landscape of RNA modifications by means of next generation RNA sequencing, recently many studies have developed conventional[6] or specialised sequencing methods.[1][2][3] Examples of specialised methods are MeRIP-seq,[7] m6A-seq,[8] methylation-iCLIP,[9] m6A-CLIP,[10] Pseudo-seq,[11] Ψ-seq,[12] CeU-seq,[13] Aza-IP[14] and RiboMeth-seq[15]). Application of these methods have identified various modifications (e.g. pseudouridine, m⁶A, m5C, 2′-O-Me) within coding genes and non-coding genes (e.g. tRNA, lncRNAs, microRNAs) at single nucleotide or very high resolution.[4] A novel database, RMBase (http://mirlab.sysu.edu.cn/rmbase/),[4] has provide various web interfaces to show all RNA modification sites identified from above-mentioned sequencing technologies.

Mass spectrometry is a way to qualitatively and (relatively) quantify RNA modifications.[16] More often than not, modifications cause an increase in mass for a given nucleoside. This gives a characteristic readout for the nucleoside and the modified counterpart.[16] Moreover, mass spectrometry allows the investigation of modification dynamics by labeling RNA molecules with stable (non-radioactive) heavy isotopes in vivo. Due to the defined mass increase of heavy isotope labeled nucleosides they can be distinguished from their respective unlabeled isotopomeres by mass spectrometry. This method, called NAIL-MS (nucleic acid isotope labeling coupled mass spectrometry), enables a variety of approaches to investigate RNA modification dynamics.[17][18][19]

Recently, functional experiments have revealed many novel functional roles of RNA modifications. For example, m⁶A has been predicted to affect protein translation and localization,[1][2][3] mRNA stability,[20] alternative polyA choice [10] and stem cell pluripotency.[21] Pseudouridylation of nonsense codons suppresses translation termination both in vitro and in vivo, suggesting that RNA modification may provide a new way to expand the genetic code.[22] Importantly, many modification enzymes are dysregulated and genetically mutated in many disease types.[1] For example, genetic mutations in pseudouridine synthases cause mitochondrial myopathy, sideroblastic anemia (MLASA) [23] and dyskeratosis congenital.[24]

Transfer RNA or tRNA is the most abundantly modified type of RNA.[25] Modifications in tRNA play crucial roles in maintaining translation efficiency through supporting structure, anticodon-codon interactions, and interactions with enzymes.[26]

Anticodon modifications are important for proper decoding of mRNA. Since the genetic code is degenerate, anticodon modifications are necessary to properly decode mRNA. Particularly, the wobble position of the anticodon determines how the codons are read. For example, in eukaryotes an adenosine at position 34 of the anticodon can be converted to inosine. Inosine is a modification that is able to base-pair with cytosine, adenine, and uridine.[27]

Another commonly modified base in tRNA is the position adjacent to the anticodon. Position 37 is often hypermodified with bulky chemical modifications. These modifications prevent frameshifting and increase anticodon-codon binding stability through stacking interactions[28].

Ribosomal RNA modifications are made throughout the ribosome synthesis. Modifications primarily play a role in the structure of the rRNA in order to protect translational efficiency[29].