Nucleic acid modifications in DNA and RNA ubiquitously exist among all the three kingdoms of life. This trait significantly broadens the genome diversity and works as an important means of gene transcription regulation. Although mammalian systems have limited types of DNA modifications, over 150 different RNA modification types have been identified, with a wide variety of chemical diversities. Most modifications occur on transfer RNA and ribosomal RNA, however many of the modifications also occur on other types of RNA species including mammalian mRNA and small nuclear RNA, where they are essential for many biological roles, including developmental processes and stem cell differentiation. These post-transcriptional modifications are enzymatically installed and removed in a site-specific manner by writer and eraser proteins respectively, while reader proteins can interpret modifications and transduce the signal for downstream functions. Dysregulation of mRNA modifications manifests as disease states, including multiple types of human cancer. In this review, we will introduce the chemical features and biological functions of these modifications in the coding and non-coding RNA species.

The posttranscriptional modification of messenger RNA (mRNA) and transfer RNA (tRNA) provides an additional layer of regulatory complexity during gene expression. Here, we show that a tRNA methyltransferase, TRMT10A, interacts with an mRNA demethylase FTO (ALKBH9), both in vitro and inside cells. TRMT10A installsN1-methylguanosine (m1G) in tRNA, and FTO performs demethylation onN6-methyladenosine (m6A) andN6,2′-O-dimethyladenosine (m6Am) in mRNA. We show that TRMT10A ablation not only leads to decreased m1G in tRNA but also significantly increases m6A levels in mRNA. Cross-linking and immunoprecipitation, followed by high-throughput sequencing results show that TRMT10A shares a significant overlap of associated mRNAs with FTO, and these mRNAs have accelerated decay rates potentially through the regulation by a specific m6A reader, YTHDF2. Furthermore, transcripts with increased m6A upon TRMT10A ablation contain an overrepresentation of m1G9-containing tRNAs codons read by tRNAGln(TTG), tRNAArg(CCG), and tRNAThr(CGT). These findings collectively reveal the presence of coordinated mRNA and tRNA methylations and demonstrate a mechanism for regulating gene expression through the interactions between mRNA and tRNA modifying enzymes.

Oxidation of 5-methylcytosine (5mC) in DNA by the Ten-eleven translocation (TET) family of enzymes is indispensable for gene regulation in mammals. More recently, evidence has emerged to support a biological function for TET-mediated m5C oxidation in messenger RNA. Here, we describe a previously uncharacterized role of TET-mediated m5C oxidation in transfer RNA (tRNAs). We found that the TET-mediated oxidation product 5-hydroxylmethylcytosine (hm5C) is specifically enriched in tRNA inside cells and that the oxidation activity of TET2 on m5C in tRNAs can be readily observed in vitro. We further observed that hm5C levels in tRNA were significantly decreased in Tet2 KO mouse embryonic stem cells (mESCs) in comparison to wild type mESCs. Reciprocally, induced expression of the catalytic domain of TET2 led to an obvious increase in hm5C and a decrease in m5C in tRNAs relative to uninduced cells. Strikingly, we also show that TET2-mediated m5C oxidation in tRNA promotes translation in vitro. These results suggest TET2 may influence translation through impacting tRNA methylation and reveal an unexpected role for TET enzymes in regulating multiple nodes of the central dogma.

RNA binding proteins (RBPs) frequently regulate the expression of other RBPs in mammalian cells. Such cross-regulation has been proposed to be important to control networks of coordinated gene expression; however, much remains to be understood about how such networks of cross-regulation are established and what the functional consequence is of coordinated or reciprocal expression of RBPs. Here we demonstrate that the RBPs CELF2 and hnRNP C regulate the expression of each other, such that depletion of one results in reduced expression of the other. Specifically, we show that loss of hnRNP C reduces the transcription of CELF2 mRNA, while loss of CELF2 results in decreased efficiency of hnRNP C translation. We further demonstrate that this reciprocal regulation serves to fine tune the splicing patterns of many downstream target genes. Together, this work reveals new activities of hnRNP C and CELF2, provides insight into a previously unrecognized gene regulatory network, and demonstrates how cross-regulation of RBPs functions to shape the cellular transcriptome.

Polypyrimidine binding tract proteins (PTBPs) are members of the hnRNP family and are involved in the modulation of alternative splicing, serving as both repressors and activators of cassette exon inclusion in mature mRNA (Kafasla et al, 2012). The human genome encodes for three different PTBP paralogs, PTBP1, PTBP2 and PTPB3. These homologous proteins are highly similar in primary structure and domain organization, which is characterized by a N‐terminal domain and four RNA recognition motifs (RRMs) connected via linker regions. PTBPs share >70% sequence identity and differ in their tissue expression with PTBP1 being expressed nearly ubiquitously while PTBP2 and PTBP3 show more tissue specific expression (Keppetipola et al, 2012; Tan et al, 2015). Changes in the expression of PTBP1 and PTBP2 have been found to be critical in the process of neuronal differentiation and maturation (Li et al, 2014). PTBP1 has been observed to be overexpressed in ovarian cancer cells (He et al, 2007) and PTBP1 and PTBP2 have been implicated in the splicing of transcripts that promote proliferation of glioma (Cheung et al, 2009). Inhibition of PTBP3 expression in gastric cancer cells induced apoptosis (Liang et al, 2017). Therefore, further understanding of PTBPs may provide insight to how related proteins exert differential splicing outcomes and may provide therapeutic targets for drug design. In this study, we sought to characterize PTBP by elucidating its evolutionary history via sequence and phylogenetic analyses. Homologous sequences, including vertebrate, invertebrate, plant and fungi sequences, were identified by using BLAST search tools across various species databases. Multiple sequencing alignments (MSAs) and phylogenetic analyses were performed using different methods in MEGA6. Our current results reveal that the three human PTBP paralogs have orthologs found ubiquitously amongst jawed vertebrate species. In contrast, jawless vertebrate and invertebrate species contain only one homologous protein. This finding suggests that the gene duplication events that produced the three human PTBPs occurred within the ancestor of all jawed vertebrates. Plant species also have two or three PTBP homologs; however, these do not cluster with their animal homologs. This suggests that in both animal and plant species the genes multiplied independently. Fungi, which are unicellular organisms, contain one or no PTBP homologs. MSAs have also revealed a variety of residues within the RRMs that are highly conserved across the various PTBP orthologs surveyed, which suggests their functional and structural importance. We are conducting analysis of intron and synteny conservation as well as 3D structure comparisons to further develop the tentative conclusions posited.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.