Yongfeng Jin

Extensive translation of circular RNAs driven by N 6 -methyladenosine

Extensive pre-mRNA back-splicing generates numerous circular RNAs (circRNAs) in human transcriptome. However, the biological functions of these circRNAs remain largely unclear. Here we report that N6-methyladenosine (m6A), the most abundant base modification of RNA, promotes efficient initiation of protein translation from circRNAs in human cells. We discover that consensus m6A motifs are enriched in circRNAs and a single m6A site is sufficient to drive translation initiation. This m6A-driven translation requires initiation factor eIF4G2 and m6A reader YTHDF3, and is enhanced by methyltransferase METTL3/14, inhibited by demethylase FTO, and upregulated upon heat shock. Further analyses through polysome profiling, computational prediction and mass spectrometry reveal that m6A-driven translation of circRNAs is widespread, with hundreds of endogenous circRNAs having translation potential. Our study expands the coding landscape of human transcriptome, and suggests a role of circRNA-derived proteins in cellular responses to environmental stress.

RNA secondary structure in mutually exclusive splicing

Mutually exclusive splicing is a regulated means to generate protein diversity, but the underlying mechanisms are poorly understood. Here comparative genome analysis revealed the built-in intronic elements for controlling mutually exclusive splicing of the 14-3-3ξ pre-mRNA. These elements are clade specific but are evolutionarily conserved at the secondary structure level. Combined evidence revealed the triple functions of these inter-intronic RNA pairings in synergistically ensuring the selection of only one of multiple exons, through activation of the proximal variable exon outside the loop by the approximation of cis elements, and simultaneous repression of the exon within the loop, in combination with the physical competition of RNA pairing. Additionally, under this model, we also deciphered a similar structural code in exon clusters 4 and 9 of Dscam (38,016 isoforms) and Mhc (480 isoforms). Our findings suggest a broadly applicable mechanism to ensure mutually exclusive splicing.

New insights into RNA secondary structure in the alternative splicing of pre-mRNAs

Alternative splicing is an important mechanism in generating proteomic diversity, and RNA secondary structure is an important element in splicing regulation. The use of high-throughput sequencing and other approaches has increased the number of known pre-mRNA secondary structures by several orders of magnitude, and we now have new insights into the role of RNA secondary structure in alternative splicing and the mechanisms involved (e.g., physical competition, long-range RNA pairing, the structural splicing code, and co-transcriptional splicing). Furthermore, an RNA pairing-based mechanism ensures the selection of only one of several available exons (e.g., Dscam splicing). Here we review several recent discoveries related to the role of RNA secondary structure in alternative splicing and the underlying mechanisms.

Genetic Variation of Chinese PRRSV Strains Based on ORF5 Sequence

Thirteen isolates of porcine reproductive and respiratory syndrome virus (PRRSV) from different provinces of China were studied and compared with several PRRSV isolates from other countries. Phylogenetic analysis shows that all Chinese isolates of PRRSV in this study belong to the American genotype, except for one strain, B13, which clustered as a European genotype. Sequence analysis revealed that PRRSV Chinese isolates of the American genotype were highly similar in the ORF5 sequence and could be classified into two subclades. One contains PRRSV isolates that are more closely related to the American vaccine strain MLV Resp and its parent strain VR-2332, and the other contains ones only distantly related to them. Within the Chinese isolates slight genetic variation occurred, and some strains may originate directly from the vaccine virus.

PmiRKB: a plant microRNA knowledge base

MicroRNAs (miRNAs), one type of small RNAs (sRNAs) in plants, play an essential role in gene regulation. Several miRNA databases were established; however, successively generated new datasets need to be collected, organized and analyzed. To this end, we have constructed a plant miRNA knowledge base (PmiRKB) that provides four major functional modules. In the ‘SNP’ module, single nucleotide polymorphism (SNP) data of seven Arabidopsis (Arabidopsis thaliana) accessions and 21 rice (Oryza sativa) subspecies were collected to inspect the SNPs within pre-miRNAs (precursor microRNAs) and miRNA—target RNA duplexes. Depending on their locations, SNPs can affect the secondary structures of pre-miRNAs, or interactions between miRNAs and their targets. A second module, ‘Pri-miR’, can be used to investigate the tissue-specific, transcriptional contexts of pre- and pri-miRNAs (primary microRNAs), based on massively parallel signature sequencing data. The third module, ‘MiR–Tar’, was designed to validate thousands of miRNA—target pairs by using parallel analysis of RNA end (PARE) data. Correspondingly, the fourth module, ‘Self-reg’, also used PARE data to investigate the metabolism of miRNA precursors, including precursor processing and miRNA- or miRNA*-mediated self-regulation effects on their host precursors. PmiRKB can be freely accessed at http://bis.zju.edu.cn/pmirkb/.

Construction of microRNA- and microRNA*-mediated regulatory networks in plants.

The critical biological roles of microRNAs (miRNAs) have been well recognized. However, knowledge on the regulatory activities of miRNA*s is limited. Although several studies point to the capacity of this small RNA species to repress target genes in animals, few related analyses were performed in plants. Here, we set out to uncover the repressive effects of miRNA*s on their targets in both Arabidopsis and rice. Systemic identification of miRNA*s was performed through secondary structure-based prediction and expression level-based verification. The targets of the miRNA*s were predicted and further filtered based on degradome sequencing data, resulting in comprehensive miRNA*—target lists with high reliability. Comprehensive miRNA—target lists were also obtained. The phenomenon that one transcript was targeted by two or more miRNA(*)s was observed, which was defined as co-regulation. Finally, comprehensive miRNA- and miRNA*-mediated regulatory networks were constructed. Further investigation of some specific subnetworks implied the utility of these networks for biologists. This study could broaden the current understanding of miRNA-mediated regulation in plants.

Target mimics: an embedded layer of microRNA-involved gene regulatory networks in plants

BackgroundMicroRNAs (miRNAs) play an essential role in gene regulation in plants. At the same time, the expression of miRNA genes is also tightly controlled. Recently, a novel mechanism called “target mimicry” was discovered, providing another layer for modulating miRNA activities. However, except for the artificial target mimics manipulated for functional studies on certain miRNA genes, only one example, IPS1 (Induced by Phosphate Starvation 1)—miR399 was experimentally confirmed in planta. To date, few analyses for comprehensive identification of natural target mimics have been performed in plants. Thus, limited evidences are available to provide detailed information for interrogating the questionable issue whether target mimicry was widespread in planta, and implicated in certain biological processes.ResultsIn this study, genome-wide computational prediction of endogenous miRNA mimics was performed in Arabidopsis and rice, and dozens of target mimics were identified. In contrast to a recent report, the densities of target mimic sites were found to be much higher within the untranslated regions (UTRs) when compared to those within the coding sequences (CDSs) in both plants. Some novel sequence characteristics were observed for the miRNAs that were potentially regulated by the target mimics. GO (Gene Ontology) term enrichment analysis revealed some functional insights into the predicted mimics. After degradome sequencing data-based identification of miRNA targets, the regulatory networks constituted by target mimics, miRNAs and their downstream targets were constructed, and some intriguing subnetworks were further exploited.ConclusionsThese results together suggest that target mimicry may be widely implicated in regulating miRNA activities in planta, and we hope this study could expand the current understanding of miRNA-involved regulatory networks.

A-to-I RNA editing alters less-conserved residues of highly conserved coding regions: implications for dual functions in evolution.

The molecular mechanism and physiological function of recoding by A-to-I RNA editing is well known, but its evolutionary significance remains a mystery. We analyzed the RNA editing of the Kv2 K(+) channel from different insects spanning more than 300 million years of evolution: Drosophila melanogaster, Culex pipiens (Diptera), Pulex irritans (Siphonaptera), Bombyx mori (Lepidoptera), Tribolium castaneum (Coleoptera), Apis mellifera (Hymenoptera), Pediculus humanus (Phthiraptera), and Myzus persicae (Homoptera). RNA editing was detected across all Kv2 orthologs, representing the most highly conserved RNA editing event yet reported in invertebrates. Surprisingly, five of these editing sites were conserved in squid (Mollusca) and were possibly of independent origin, suggesting phylogenetic conservation of editing between mollusks and insects. Based on this result, we predicted and experimentally verified two novel A-to-I editing sites in squid synaptotagmin I transcript. In addition, comparative analysis indicated that RNA editing usually occurred within highly conserved coding regions, but mostly altered less-conserved coding positions of these regions. Moreover, more than half of these edited amino acids are genomically encoded in the orthologs of other species; an example of a conversion model of the nonconservative edited site is addressed. Therefore, these data imply that RNA editing might play dual roles in evolution by extending protein diversity and maintaining phylogenetic conservation.

An RNA architectural locus control region involved in Dscam mutually exclusive splicing

The most striking example of alternative splicing in a Drosophila melanogaster gene is observed in the Down syndrome cell adhesion molecule, which can generate 38,016 different isoforms. RNA secondary structures are thought to direct the mutually exclusive splicing of Down syndrome cell adhesion molecule, but the underlying mechanisms are poorly understood. Here we describe a locus control region that can activate the exon 6 cluster and specifically allow for the selection of only one exon variant in combination with docking site selector sequence interactions. Combining comparative genomic studies of 63 species with mutational analysis reveals that intricate, tandem multi-‘subunit’ RNA structures within the locus control region activate species-appropriate alternative variants. Importantly, strengthening the weak splice sites of the target exon can remove the locus control region dependence. Our findings not only provide a locus control region-dependent mechanism for mutually exclusive splicing, but also suggest a model for the evolution of increased complexity in a long-range RNA molecular machine.

RNA editing of nuclear transcripts in Arabidopsis thaliana

BackgroundRNA editing is a transcript-based layer of gene regulation. To date, no systemic study on RNA editing of plant nuclear genes has been reported. Here, a transcriptome-wide search for editing sites in nuclear transcripts of Arabidopsis (Arabidopsis thaliana) was performed.ResultsMPSS (massively parallel signature sequencing) and PARE (parallel analysis of RNA ends) data retrieved from public databases were utilized, focusing on one-base-conversion editing. Besides cytidine (C)-to-uridine (U) editing in mitochondrial transcripts, many nuclear transcripts were found to be diversely edited. Interestingly, a sizable portion of these nuclear genes are involved in chloroplast- or mitochondrion-related functions, and many editing events are tissue-specific. Some editing sites, such as adenosine (A)-to-U editing loci, were found to be surrounded by peculiar elements. The editing events of some nuclear transcripts are highly enriched surrounding the borders between coding sequences (CDSs) and 3′ untranslated regions (UTRs), suggesting site-specific editing. Furthermore, RNA editing is potentially implicated in new start or stop codon generation, and may affect alternative splicing of certain protein-coding transcripts. RNA editing in the precursor microRNAs (pre-miRNAs) of ath-miR854 family, resulting in secondary structure transformation, implies its potential role in microRNA (miRNA) maturation.ConclusionsTo our knowledge, the results provide the first global view of RNA editing in plant nuclear transcripts.