Single-nucleus multiomic mapping of m6A methylomes and transcriptomes in native populations of cells with sn-m6A-CT.

N6-methyladenosine (m6A) RNA modification plays important roles in the governance of gene expression and is temporally regulated in different cell states. In contrast to global m6A profiling in bulk sequencing, single-cell technologies for analyzing m6A heterogeneity are not extensively established. Here, we developed single-nucleus m6A-CUT&Tag (sn-m6A-CT) for simultaneous profiling of m6A methylomes and transcriptomes within a single nucleus using mouse embryonic stem cells (mESCs). m6A-CT is capable of enriching m6A-marked RNA molecules in situ, without isolating RNAs from cells. We adapted m6A-CT to the droplet-based single-cell omics platform and demonstrated high-throughput performance in analyzing nuclei isolated from thousands of cells from various cell types. We show that sn-m6A-CT profiling is sufficient to determine cell identity and allows the generation of cell-type-specific m6A methylome landscapes from heterogeneous populations. These indicate that sn-m6A-CT provides additional dimensions to multimodal datasets and insights into epitranscriptomic landscape in defining cell fate identity and states.

Ribosomal proteins regulate 2-cell-stage transcriptome in mouse embryonic stem cells.

A rare sub-population of mouse embryonic stem cells (mESCs), the 2-cell-like cell, is defined by the expression of MERVL and 2-cell-stage-specific transcript (2C transcript). Here, we report that the ribosomal proteins (RPs) RPL14, RPL18, and RPL23 maintain the identity of mESCs and regulate the expression of 2C transcripts. Disregulation of the RPs induces DUX-dependent expression of 2C transcripts and alters the chromatin landscape. Mechanically, knockdown (KD) of RPs triggers the binding of RPL11 to MDM2, an interaction known to prevent P53 protein degradation. Increased P53 protein upon RP KD further activates its downstream pathways, including DUX. Our study delineates the critical roles of RPs in 2C transcript activation, ascribing a novel function to these essential proteins.

Multi-species single-cell transcriptomic analysis of ocular compartment regulons.

The retina is a widely profiled tissue in multiple species by single-cell RNA sequencing studies. However, integrative research of the retina across species is lacking. Here, we construct the first single-cell atlas of the human and porcine ocular compartments and study inter-species differences in the retina. In addition to that, we identify putative adult stem cells present in the iris tissue. We also create a disease map of genes involved in eye disorders across compartments of the eye. Furthermore, we probe the regulons of different cell populations, which include transcription factors and receptor-ligand interactions and reveal unique directional signalling between ocular cell types. In addition, we study conservation of regulons across vertebrates and zebrafish to identify common core factors. Here, we show perturbation of KLF7 gene expression during retinal ganglion cells differentiation and conclude that it plays a significant role in the maturation of retinal ganglion cells.

Unraveling Heterogeneity in Transcriptome and Its Regulation Through Single-Cell Multi-Omics Technologies.

Cellular heterogeneity plays a pivotal role in tissue homeostasis and the disease development of multicellular organisms. To deconstruct the heterogeneity, a multitude of single-cell toolkits measuring various cellular contents, including genome, transcriptome, epigenome, and proteome, have been developed. More recently, multi-omics single-cell techniques enable the capture of molecular footprints with a higher resolution by simultaneously profiling various cellular contents within an individual cell. Integrative analysis of multi-omics datasets unravels the relationships between cellular modalities, builds sophisticated regulatory networks, and provides a holistic view of the cell state. In this review, we summarize the major developments in the single-cell field and review the current state-of-the-art single-cell multi-omic techniques and the bioinformatic tools for integrative analysis.

DNA Sequencing Method Including Unnatural Bases for DNA Aptamer Generation by Genetic Alphabet Expansion.

The creation of unnatural base pairs (UBPs) has rapidly advanced the genetic alphabet expansion technology of DNA, requiring a new sequencing method for UB-containing DNAs with five or more letters. The hydrophobic UBP, Ds-Px, exhibits high fidelity in PCR and has been applied to DNA aptamer generation involving Ds as a fifth base. Here, we present a sequencing method for Ds-containing DNAs, in which Ds bases are replaced with natural bases by PCR using intermediate UB substrates (replacement PCR) for conventional deep sequencing. The composition rates of the natural bases converted from Ds significantly varied, depending on the sequence contexts around Ds and two different intermediate substrates. Therefore, we made an encyclopedia of the natural-base composition rates for all sequence contexts in each replacement PCR using different intermediate substrates. The Ds positions in DNAs can be determined by comparing the natural-base composition rates in both the actual and encyclopedia data, at each position of the DNAs obtained by deep sequencing after replacement PCR. We demonstrated the sequence determination of DNA aptamers in the enriched Ds-containing DNA libraries isolated by aptamer generation procedures targeting proteins. This study also provides valuable information about the fidelity of the Ds-Px pair in replication.

Creation of unnatural base pairs for genetic alphabet expansion toward synthetic xenobiology.

Artificial extra base pairs (unnatural base pairs, UBPs) expand the genetic alphabet of DNA, thus broadening entire biological systems in the central dogma. UBPs function as third base pairs in replication, transcription, and/or translation, and have created a new research area, synthetic xenobiology, providing genetic engineering tools to generate novel DNAs, RNAs, and proteins with increased functionalities. Several UBPs have been developed and applied to PCR technology, DNA aptamer generation, and semi-synthetic organism creation. Among them, we developed a series of UBPs and demonstrated unique quantitative PCR and high-affinity DNA aptamer generation methods.

Genetic alphabet expansion biotechnology by creating unnatural base pairs.

Recent studies have made it possible to expand the genetic alphabet of DNA, which is originally composed of the four-letter alphabet with A-T and G-C pairs, by introducing an unnatural base pair (UBP). Several types of UBPs function as a third base pair in replication, transcription, and/or translation. Through the UBP formation, new components with different physicochemical properties from those of the natural ones can be introduced into nucleic acids and proteins site-specifically, providing their increased functionalities. Here, we describe the genetic alphabet expansion technology by focusing on three types of UBPs, which were recently applied to the creations of DNA aptamers that bind to proteins and cells and semi-synthetic organisms containing DNAs with a six-letter alphabet.

Expansion of Noncanonical V-Arm-Containing tRNAs in Eukaryotes.

Transfer RNA (tRNA) is essential for the translation of genetic information into proteins, and understanding its molecular evolution is important if we are to understand the genetic code. In general, long variable-arm (V-arm) structures form in tRNA(Leu), tRNA(Ser), and bacterial and organellar tRNA(Tyr). However, as we have previously reported, noncanonical V-arms occur in nematode tRNA(Gly) and tRNA(Ile), and potentially affect translational fidelity. Here, we comprehensively analyzed 69 eukaryotic genome sequences and examined the evolutionary divergence of the V-arm-containing tRNAs. In total, 253 V-arm-containing tRNAs, with neither leucine nor serine anticodons, were identified in organisms ranging from nematodes to fungi, plants, and vertebrates. We defined them as "noncanonical V-arm-containing tRNAs" (nov-tRNAs). Moreover, 2,415 nov-tRNA-like sequences lacking some of the conserved features of tRNAs were also identified, largely in vertebrate genomes. These nov-tRNA/nov-tRNA-like sequences can be categorized into three types, based on differences in their possible evolutionary origins. The type A nov-tRNAs in nematodes probably evolved not only from tRNA(Leu) but also from tRNA(Ser) and other isotypes on several independent occasions. The type B nov-tRNAs are dispersed abundantly throughout vertebrate genomes, and seem to have originated from retrotransposable elements. The type C nov-tRNAs may have been acquired from plant chloroplasts or from bacteria through horizontal transfer. Our findings provide unexpected insight into the evolution of the tRNA molecule, which was diverse and occurred independently in nematodes, vertebrates, and plants.

Analysis of genetic code ambiguity arising from nematode-specific misacylated tRNAs.

The faithful translation of the genetic code requires the highly accurate aminoacylation of transfer RNAs (tRNAs). However, it has been shown that nematode-specific V-arm-containing tRNAs (nev-tRNAs) are misacylated with leucine in vitro in a manner that transgresses the genetic code. nev-tRNA(Gly) (CCC) and nev-tRNA(Ile) (UAU), which are the major nev-tRNA isotypes, could theoretically decode the glycine (GGG) codon and isoleucine (AUA) codon as leucine, causing GGG and AUA codon ambiguity in nematode cells. To test this hypothesis, we investigated the functionality of nev-tRNAs and their impact on the proteome of Caenorhabditis elegans. Analysis of the nucleotide sequences in the 3' end regions of the nev-tRNAs showed that they had matured correctly, with the addition of CCA, which is a crucial posttranscriptional modification required for tRNA aminoacylation. The nuclear export of nev-tRNAs was confirmed with an analysis of their subcellular localization. These results show that nev-tRNAs are processed to their mature forms like common tRNAs and are available for translation. However, a whole-cell proteome analysis found no detectable level of nev-tRNA-induced mistranslation in C. elegans cells, suggesting that the genetic code is not ambiguous, at least under normal growth conditions. Our findings indicate that the translational fidelity of the nematode genetic code is strictly maintained, contrary to our expectations, although deviant tRNAs with misacylation properties are highly conserved in the nematode genome.

Alternative genetic code for amino acids and transfer RNA revisited.

The genetic code is highly conserved among all organisms and its evolution is thought to be strictly limited. However, an increasing number of studies have reported non-standard codes in prokaryotic and eukaryotic genomes. Most of these deviations from the standard code are attributable to tRNA changes relating to, for example, codon/anticodon base pairing and tRNA/aminoacyl-tRNA synthetase recognition. In this review, we focus on tRNA, a key molecule in the translation of the genetic code, and summarize the most recently published information on the evolutionary divergence of the tRNAs. Surprisingly, although higher eukaryotes, such as the nematode (worm), utilize the standard genetic code, newly identified nematode-specific tRNAs (nev-tRNAs) translate nucleotides in a manner that transgresses the code. Furthermore, a variety of additional functions of tRNAs, beyond their translation of the genetic code, have emerged rapidly. We also review these intriguing new aspects of tRNA, which have potential impacts on translational control, RNA silencing, antibiotic resistance, RNA biosynthesis, and transcriptional regulation.

Nematode-specific tRNAs that decode an alternative genetic code for leucine.

Class II transfer RNAs (tRNAs), including tRNA(Leu) and tRNA(Ser), have an additional stem and loop structure, the long variable arm (V-arm). Here, we describe Class II tRNAs with a unique anticodon corresponding to neither leucine nor serine. Because these tRNAs are specifically conserved among the nematodes, we have called them 'nematode-specific V-arm-containing tRNAs' (nev-tRNAs). The expression of nev-tRNA genes in Caenorhabditis elegans was confirmed experimentally. A comparative sequence analysis suggested that the nev-tRNAs derived phylogenetically from tRNA(Leu). In vitro aminoacylation assays showed that nev-tRNA(Gly) and nev-tRNA(Ile) are only charged with leucine, which is inconsistent with their anticodons. Furthermore, the deletion and mutation of crucial determinants for leucylation in nev-tRNA led to a marked loss of activity. An in vitro translation analysis showed that nev-tRNA(Gly) decodes GGG as leucine instead of the universal glycine code, indicating that nev-tRNAs can be incorporated into ribosomes and participate in protein biosynthesis. Our findings provide the first example of unexpected tRNAs that do not consistently obey the general translation rules for higher eukaryotes.