Posttranscriptional regulation comprises those mechanisms occurring after the initial copy of the DNA sequence is transcribed into an intermediate RNA molecule (i.e., messenger RNA) until such a molecule is used as a template to generate a protein. A subset of these posttranscriptional regulatory mechanisms essentially are destined to process the immature mRNA toward its mature form, conferring the adequate mRNA stability, providing the means for pertinent introns excision, and controlling mRNA turnover rate and quality control check. An additional layer of complexity is added in certain cases, since discrete nucleotide modifications in the mature RNA molecule are added by RNA editing, a process that provides large mature mRNA diversity. Moreover, a number of posttranscriptional regulatory mechanisms occur in a cell- and tissue-specific manner, such as alternative splicing and noncoding RNA-mediated regulation. In this chapter, we will briefly summarize current state-of-the-art knowledge of general posttranscriptional mechanisms, while major emphases will be devoted to those tissue-specific posttranscriptional modifications that impact on cardiac development and congenital heart disease.

Methylation of proteins and nucleic acids plays a fundamental role in epigenetic regulation, and discovery of methyltransferase (MT) inhibitors is an area of intense activity. Because of the diversity of MTs and their products, assay methods that detect S-adenosylhomocysteine (SAH) - the invariant product of S-adenosylmethionine (SAM)-dependent methylation reactions - offer some advantages over methods that detect specific methylation events. However, direct, homogenous detection of SAH requires a reagent capable of discriminating between SAH and SAM, which differ by a single methyl group. Moreover, MTs are slow enzymes and many have submicromolar affinities for SAM; these properties translate to a need for detection of SAH at low nanomolar concentrations in the presence of excess SAM. To meet these needs, we leveraged the exquisite molecular recognition properties of a naturally occurring SAH-sensing RNA aptamer, or riboswitch. By splitting the riboswitch into two fragments, such that SAH binding induces assembly of a trimeric complex, we engineered sensors that transduce binding of SAH into positive fluorescence polarization (FP) and time resolved Förster resonance energy transfer (TR-FRET) signals. The split riboswitch configuration, called the AptaFluor™ SAH Methyltransferase Assay, allows robust detection of SAH (Z\' > 0.7) at concentrations below 10 nM, with overnight signal stability in the presence of typical MT assay components. The AptaFluor assay tolerates diverse MT substrates, including histones, nucleosomes, DNA and RNA, and we demonstrated its utility as a robust, enzymatic assay method for several methyltransferases with SAM Km values < 1 µM. The assay was validated for HTS by performing a pilot screen of 1,280 compounds against the SARS-CoV-2 RNA capping enzyme, nsp14. By enabling direct, homogenous detection of SAH at low nanomolar concentrations, the AptaFluor assay provides a universal platform for screening and profiling MTs at physiologically relevant SAM concentrations.

Background: KIAA1429, a member of the RNA methyltransferase complex, is involved in cancer progression; however, the clinical significance and underlying mechanism of KIAA1429 in osteosarcoma (OS) remains to be reported. Methods: We evaluated the clinical significance of KIAA1429 in OS by performing RT-qPCR, microarray, and RNA sequencing and using published data as a reference. Two KIAA1429-targeting siRNA constructs were transfected into SW1353 cells. CCK-8 assay, colony formation assays, flow cytometry and the xenograft mouse model were conducted to investigate the biological function of KIAA1429 in OS. Results: The mRNA expression of KIAA1429 was markedly upregulated in 250 OS samples as compared to that in 71 non-cancer samples (standardized mean difference = 0.67). Summary receiver operating characteristic curve analysis revealed that KIAA1429 exhibited reliable diagnostic capacity to differentiate OS samples from non-cancer samples (area under the curve = 0.83). Further, survival analysis indicated that KIAA1429 overexpression was associated with shorter overall survival time. Knocking down KIAA1429 reduced m6A methylation levels, inhibited proliferation, prevented the growth of tumors in vivo and accelerated apoptosis of OS cells. In total, 395 KIAA1429-related genes were identified among co-expressed genes and differentially expressed genes, which were enriched in the cell cycle pathway. Protein-protein interaction network analysis showed that CDK1, CCNA2, and CCNB1 were KIAA1429-related genes, serving as major network hubs in OS. Conclusions: Our findings indicate that KIAA1429 plays an oncogenic role in OS and potentially facilitates OS progression via a mechanism that involves regulating CDK1, CCNA2, and CCNB1.

PM2.5 exposure leads to a variety of respiratory diseases, including pulmonary fibrosis, metastatic lung cancer, etc. Exposure to PM2.5 results in the alteration of epigenetic modification. M6A RNA methylation is an essential epigenetic modification that regulates gene expression at the post-transcriptional level. Our previous study found that PM2.5 exposure up-regulated m6A RNA methylation and TGF-β expression level in the lung, but the mechanisms and pathways of PM2.5 regulation of m6A RNA methylation are still unclear. Moreover, a previous study reported that the TGF-β signal pathway could regulate m6A RNA methylation. Based on this evidence, we investigate the role of the TGF-β signaling pathway in PM2.5-induced m6A RNA methylation with the A549 cell line. Our results showed that PM2.5 could induce upregulation of m6A RNA methylation, accompanied by increased expression of TGF-β, Smad3, methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14). Furthermore, these alterations induced by PM2.5 exposure could be reversed by treatment with TGF-β inhibitor. Therefore, we speculated that the TGF-β signal pathway plays an indispensable role in regulating m6A RNA methylation after PM2.5 exposure. Our study demonstrates that PM2.5 exposure influences m6A RNA methylation by inducing the alteration of the TGF-β signal pathway, which could be an essential mechanism for lung-related diseases induced by PM2.5 exposure.

Therapeutic mRNAs are generated using modified nucleotides, namely N1-methylpseudouridine (m1Ψ) triphosphate, so that the mRNA evades detection by the immune system. RNA modifications, even at a single-nucleotide position, perturb RNA structure, although it is not well understood how structure and function is impacted by globally modified RNAs. Therefore, we examined the metastasis-associated lung adenocarcinoma transcript 1 triple helix, a highly structured stability element that includes single-, double-, and triple-stranded RNA, globally modified with N6-methyladenosine (m6A), pseudouridine (Ψ), or m1Ψ. UV thermal denaturation assays showed that m6A destabilizes both the Hoogsteen and Watson-Crick faces of the RNA by ∼20 °C, Ψ stabilizes the Hoogsteen and Watson-Crick faces of the RNA by ∼12 °C, and m1Ψ has minimal effect on the stability of the Hoogsteen face of the RNA but increases the stability of the Watson-Crick face by ∼9 °C. Native gel-shift assays revealed that binding of the methyltransferase-like protein 16 to the metastasis-associated lung adenocarcinoma transcript 1 triple helix was weakened by at least 8-, 99-, and 23-fold, respectively, when RNA is globally modified with m6A, Ψ, or m1Ψ. These results demonstrate that a more thermostable RNA structure does not lead to tighter RNA-protein interactions, thereby highlighting the regulatory power of RNA modifications by multiple means.

Transfer RNA (tRNA) delivers amino acids to the ribosome and functions as an essential adapter molecule for decoding codons on the messenger RNA (mRNA) during protein synthesis. Before attaining their proper activity, tRNAs undergo multiple post-transcriptional modifications with highly diversified roles such as stabilization of the tRNA structure, recognition of aminoacyl tRNA synthetases, precise codon-anticodon recognition, support of viral replication and onset of immune responses. The synthesis of the majority of modified nucleosides is catalyzed by a site-specific tRNA modification enzyme. This chapter provides a detailed protocol for using mutational profiling to analyze the enzymatic function of a tRNA methyltransferase in a high-throughput manner. In a previous study, we took tRNA m1A22 methyltransferase TrmK from Geobacillus stearothermophilus as a model tRNA methyltransferase and applied this protocol to gain mechanistic insights into how TrmK recognizes the substrate tRNAs. In theory, this protocol can be used unaltered for studying enzymes that catalyze modifications at the Watson-Crick face such as 1-methyladenosine (m1A), 3-methylcytosine (m3C), 3-methyluridine (m3U), 1-methylguanosine (m1G), and N2,N2-dimethylguanosine (m22G).

Despite the discovery of modified nucleic acids nearly 75 years ago, their biological functions are still being elucidated. N6 -methyladenosine (m6 A) is the most abundant modification in eukaryotic messenger RNA (mRNA) and has also been detected in non-coding RNAs, including long non-coding RNA, ribosomal RNA, and small nuclear RNA. In general, m6 A marks can alter RNA secondary structure and initiate unique RNA-protein interactions that can alter splicing, mRNA turnover, and translation, just to name a few. Although m6 A marks in human RNAs have been known to exist since 1974, the structures and functions of methyltransferases responsible for writing m6 A marks have been established only recently. Thus far, there are four confirmed human methyltransferases that catalyze the transfer of a methyl group from S-adenosylmethionine (SAM) to the N6 position of adenosine, producing m6 A: methyltransferase-like protein (METTL) 3/METTL14 complex, METTL16, METTL5, and zinc-finger CCHC-domain-containing protein 4. Though the methyltransferases have unique RNA targets, all human m6 A RNA methyltransferases contain a Rossmann fold with a conserved SAM-binding pocket, suggesting that they utilize a similar catalytic mechanism for methyl transfer. For each of the human m6 A RNA methyltransferases, we present the biological functions and links to human disease, RNA targets, catalytic and kinetic mechanisms, and macromolecular structures. We also discuss m6 A marks in human viruses and parasites, assigning m6 A marks in the transcriptome to specific methyltransferases, small molecules targeting m6 A methyltransferases, and the enzymes responsible for hypermodified m6 A marks and their biological functions in humans. Understanding m6 A methyltransferases is a critical steppingstone toward establishing the m6 A epitranscriptome and more broadly the RNome. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Depression is caused by a variety of factors such as genetic factors, biological factors, and psychosocial factors, and the pathogenesis is complex. RNA methylations and related downstream signaling pathways influence a variety of biological mechanisms, including cell differentiation, tumorigenesis, sex determination, and stress response. In this work, we searched the PubMed, Web of Science, National Library of Science and Technology (NSTL), and ScienceDirect Online (SDOL) databases to summarize the biological roles of RNA methylations and their impact on the pathological mechanisms of depression. RNA methylations play a key role in the development of many diseases, and current research shows that RNA methylations are also closely linked to depression. RNA methylations in depression mainly involve \"writers\" (mediating the methylation modification process of RNAs), \"erasers\" (mediating the demethylation modification process of RNA methylation). Fat Mass and Obesity Associated (FTO) influences the development of depression by increasing body mass index (BMI), decreases the dopamine level, inhibits the adrenoceptor beta 2 (ADRB2)-c-Myc-sirt1 pathway, results in the m6A/m6Am dysregulation in brain, and may be involved in the pathogenesis of depression. The study of RNA methylations in depression has further deepened our understanding of the pathogenesis and development process of depression, provides new perspectives for the study of the pathological mechanism of depression, and provides new targets for the prevention and treatment of this disease.

Here, we report the characterization of a plant RNA methyltransferase, orthologous to yeast trimethylguanosine synthase1 (Tgs1p) and whose downregulation was associated with apomixis in Paspalum grasses. Using phylogenetic analyses and yeast complementation, we determined that land plant genomes all encode a conserved, specific TGS1 protein. Next, we studied the role of TGS1 in female reproduction using reporter lines and loss-of-function mutants in Arabidopsis thaliana. pAtTGS1:AtTGS1 reporters showed a dynamic expression pattern. They were highly active in the placenta and ovule primordia at emergence but, subsequently, showed weak signals in the nucellus. Although expressed throughout gametophyte development, activity became restricted to the female gamete and was also detected after fertilization during embryogenesis. TGS1 depletion altered the specification of the precursor cells that give rise to the female gametophytic generation and to the sporophyte, resulting in the formation of a functional aposporous-like lineage. Our results indicate that TGS1 participates in the mechanisms restricting cell fate acquisition to a single cell at critical transitions throughout the female reproductive lineage and, thus, expand our current knowledge of the mechanisms governing female reproductive fate in plants.

5-methylcytosine RNA modifications are driven by NSUN methyltransferases. Although variants in NSUN2 and NSUN3 were associated with neurodevelopmental diseases, the physiological role of NSUN6 modifications on transfer RNAs and messenger RNAs remained elusive.
We combined exome sequencing of consanguineous families with functional characterization to identify a new neurodevelopmental disorder gene.
We identified 3 unrelated consanguineous families with deleterious homozygous variants in NSUN6. Two of these variants are predicted to be loss-of-function. One maps to the first exon and is predicted to lead to the absence of NSUN6 via nonsense-mediated decay, whereas we showed that the other maps to the last exon and encodes a protein that does not fold correctly. Likewise, we demonstrated that the missense variant identified in the third family has lost its enzymatic activity and is unable to bind the methyl donor S-adenosyl-L-methionine. The affected individuals present with developmental delay, intellectual disability, motor delay, and behavioral anomalies. Homozygous ablation of the NSUN6 ortholog in Drosophila led to locomotion and learning impairment.
Our data provide evidence that biallelic pathogenic variants in NSUN6 cause one form of autosomal recessive intellectual disability, establishing another link between RNA modification and cognition.