Parallel gene size and isoform expansion of ancient neuronal genes.

How nervous systems evolved is a central question in biology. A diversity of synaptic proteins is thought to play a central role in the formation of specific synapses leading to nervous system complexity. The largest animal genes, often spanning hundreds of thousands of base pairs, are known to be enriched for expression in neurons at synapses and are frequently mutated or misregulated in neurological disorders and diseases. Although many of these genes have been studied independently in the context of nervous system evolution and disease, general principles underlying their parallel evolution remain unknown. To investigate this, we directly compared orthologous gene sizes across eukaryotes. By comparing relative gene sizes within organisms, we identified a distinct class of large genes with origins predating the diversification of animals and, in many cases, the emergence of neurons as dedicated cell types. We traced this class of ancient large genes through evolution and found orthologs of the large synaptic genes potentially driving the immense complexity of metazoan nervous systems, including in humans and cephalopods. Moreover, we found that while these genes are evolving under strong purifying selection, as demonstrated by low dN/dS ratios, they have simultaneously grown larger and gained the most isoforms in animals. This work provides a new lens through which to view this distinctive class of large and multi-isoform genes and demonstrates how intrinsic genomic properties, such as gene length, can provide flexibility in molecular evolution and allow groups of genes and their host organisms to evolve toward complexity.

Subcytoplasmic location of translation controls protein output.

The cytoplasm is highly compartmentalized, but the extent and consequences of subcytoplasmic mRNA localization in non-polarized cells are largely unknown. We determined mRNA enrichment in TIS granules (TGs) and the rough endoplasmic reticulum (ER) through particle sorting and isolated cytosolic mRNAs by digitonin extraction. When focusing on genes that encode non-membrane proteins, we observed that 52% have transcripts enriched in specific compartments. Compartment enrichment correlates with a combinatorial code based on mRNA length, exon length, and 3' UTR-bound RNA-binding proteins. Compartment-biased mRNAs differ in the functional classes of their encoded proteins: TG-enriched mRNAs encode low-abundance proteins with strong enrichment of transcription factors, whereas ER-enriched mRNAs encode large and highly expressed proteins. Compartment localization is an important determinant of mRNA and protein abundance, which is supported by reporter experiments showing that redirecting cytosolic mRNAs to the ER increases their protein expression. In summary, the cytoplasm is functionally compartmentalized by local translation environments.

Genetic Architecture and Functional Implications of the CSF-Contacting Nucleus.

We previously identified a unique nucleus, the cerebrospinal fluid (CSF)-contacting nucleus. This study aims to understand its gene architecture and preliminarily suggest its functions. The results showed that there were about 19,666 genes in this nucleus, of which 913 were distinct from the dorsal raphe nucleus (non-CSF contacting). The top 40 highly-expressed genes are mainly related to energy metabolism, protein synthesis, transport, secretion, and hydrolysis. The main neurotransmitter is 5-HT. The receptors of 5-HT and GABA are abundant. The channels for Cl-, Na+, K+, and Ca2+ are routinely expressed. The signaling molecules associated with the CaMK, JAK, and MAPK pathways were identified accurately. In particular, the channels of transient receptor potential associated with nociceptors and the solute carrier superfamily members associated with cell membrane transport were significantly expressed. The relationship between the main genes of the nucleus and life activities is preliminarily verified.

PARP1 Regulates Circular RNA Biogenesis though Control of Transcriptional Dynamics.

Circular RNAs (circRNAs) are a recently discovered class of RNAs derived from protein-coding genes that have important biological and pathological roles. They are formed through backsplicing during co-transcriptional alternative splicing; however, the unified mechanism that accounts for backsplicing decisions remains unclear. Factors that regulate the transcriptional timing and spatial organization of pre-mRNA, including RNAPII kinetics, the availability of splicing factors, and features of gene architecture, have been shown to influence backsplicing decisions. Poly (ADP-ribose) polymerase I (PARP1) regulates alternative splicing through both its presence on chromatin as well as its PARylation activity. However, no studies have investigated PARP1's possible role in regulating circRNA biogenesis. Here, we hypothesized that PARP1's role in splicing extends to circRNA biogenesis. Our results identify many unique circRNAs in PARP1 depletion and PARylation-inhibited conditions compared to the wild type. We found that while all genes producing circRNAs share gene architecture features common to circRNA host genes, genes producing circRNAs in PARP1 knockdown conditions had longer upstream introns than downstream introns, whereas flanking introns in wild type host genes were symmetrical. Interestingly, we found that the behavior of PARP1 in regulating RNAPII pausing is distinct between these two classes of host genes. We conclude that the PARP1 pausing of RNAPII works within the context of gene architecture to regulate transcriptional kinetics, and therefore circRNA biogenesis. Furthermore, this regulation of PARP1 within host genes acts to fine tune their transcriptional output with implications in gene function.

Genetic Architecture and Functional Implications of the CSF-Contacting Nucleus / 神经科学通报·英文版

We previously identified a unique nucleus, the cerebrospinal fluid (CSF)-contacting nucleus. This study aims to understand its gene architecture and preliminarily suggest its functions. The results showed that there were about 19,666 genes in this nucleus, of which 913 were distinct from the dorsal raphe nucleus (non-CSF contacting). The top 40 highly-expressed genes are mainly related to energy metabolism, protein synthesis, transport, secretion, and hydrolysis. The main neurotransmitter is 5-HT. The receptors of 5-HT and GABA are abundant. The channels for Cl-, Na+, K+, and Ca2+ are routinely expressed. The signaling molecules associated with the CaMK, JAK, and MAPK pathways were identified accurately. In particular, the channels of transient receptor potential associated with nociceptors and the solute carrier superfamily members associated with cell membrane transport were significantly expressed. The relationship between the main genes of the nucleus and life activities is preliminarily verified.

Intronization Signatures in Coding Exons Reveal the Evolutionary Fluidity of Eukaryotic Gene Architecture.

The conventionally clear distinction between exons and introns in eukaryotic genes is actually blurred. To illustrate this point, consider sequences that are retained in mature mRNAs about 50% of the time: how should they be classified? Moreover, although it is clear that RNA splicing influences gene expression levels and is an integral part of interdependent cellular networks, introns continue to be regarded as accidental insertions; exogenous sequences whose evolutionary origin is independent of mRNA-associated processes and somewhat still elusive. Here, we present evidence that aids to resolve this disconnect between conventional views about introns and current knowledge about the role of RNA splicing in the eukaryotic cell. We first show that coding sequences flanked by cryptic splice sites are negatively selected on a genome-wide scale in Paramecium. Then, we exploit selection intensity to infer splicing-related evolutionary dynamics. Our analyses suggest that intron gain begins as a splicing error, involves a transient phase of alternative splicing, and is preferentially completed at the 5' end of genes, which through intron gain can become highly expressed. We conclude that relaxed selective constraints may promote biological complexity in Paramecium and that the relationship between exons and introns is fluid on an evolutionary scale.

Partitioning gene-mediated disease heritability without eQTLs.

Unknown SNP-to-gene regulatory architecture complicates efforts to link noncoding GWAS associations with genes implicated by sequencing or functional studies. eQTLs are often used to link SNPs to genes, but expression in bulk tissue explains a small fraction of disease heritability. A simple but successful approach has been to link SNPs with nearby genes via base pair windows, but genes may often be regulated by SNPs outside their window. We propose the abstract mediation model (AMM) to estimate (1) the fraction of heritability mediated by the closest or kth-closest gene to each SNP and (2) the mediated heritability enrichment of a gene set (e.g., genes with rare-variant associations). AMM jointly estimates these quantities by matching the decay in SNP enrichment with distance from genes in the gene set. Across 47 complex traits and diseases, we estimate that the closest gene to each SNP mediates 27% (SE: 6%) of heritability and that a substantial fraction is mediated by genes outside the ten closest. Mendelian disease genes are strongly enriched for common-variant heritability; for example, just 21 dyslipidemia genes mediate 25% of LDL heritability (211× enrichment, p = 0.01). Among brain-related traits, genes involved in neurodevelopmental disorders are only about 4× enriched, but gene expression patterns are highly informative, as they have detectable differences in per-gene heritability even among weakly brain-expressed genes.

Gene architecture directs splicing outcome in separate nuclear spatial regions.

How the splicing machinery defines exons or introns as the spliced unit has remained a puzzle for 30 years. Here, we demonstrate that peripheral and central regions of the nucleus harbor genes with two distinct exon-intron GC content architectures that differ in the splicing outcome. Genes with low GC content exons, flanked by long introns with lower GC content, are localized in the periphery, and the exons are defined as the spliced unit. Alternative splicing of these genes results in exon skipping. In contrast, the nuclear center contains genes with a high GC content in the exons and short flanking introns. Most splicing of these genes occurs via intron definition, and aberrant splicing leads to intron retention. We demonstrate that the nuclear periphery and center generate different environments for the regulation of alternative splicing and that two sets of splicing factors form discrete regulatory subnetworks for the two gene architectures. Our study connects 3D genome organization and splicing, thus demonstrating that exon and intron definition modes of splicing occur in different nuclear regions.

Proximity to the Promoter and Terminator Regions Regulates the Transcription Enhancement Potential of an Intron.

An evolutionarily conserved feature of introns is their ability to enhance expression of genes that harbor them. Introns have been shown to regulate gene expression at the transcription and post-transcription level. The general perception is that a promoter-proximal intron is most efficient in enhancing gene expression and the effect diminishes with the increase in distance from the promoter. Here we show that the intron regains its positive influence on gene expression when in proximity to the terminator. We inserted ACT1 intron into different positions within IMD4 and INO1 genes. Transcription Run-On (TRO) analysis revealed that the transcription of both IMD4 and INO1 was maximal in constructs with a promoter-proximal intron and decreased with the increase in distance of the intron from the promoter. However, activation was partially restored when the intron was placed close to the terminator. We previously demonstrated that the promoter-proximal intron stimulates transcription by affecting promoter directionality through gene looping-mediated recruitment of termination factors in the vicinity of the promoter region. Here we show that the terminator-proximal intron also enhances promoter directionality and results in compact gene architecture with the promoter and terminator regions in close physical proximity. Furthermore, we show that both the promoter and terminator-proximal introns facilitate assembly or stabilization of the preinitiation complex (PIC) on the promoter. On the basis of these findings, we propose that proximity to both the promoter and the terminator regions affects the transcription regulatory potential of an intron, and the terminator-proximal intron enhances transcription by affecting both the assembly of preinitiation complex and promoter directionality.

Papain-Like Cysteine Protease Gene Family in Fig (Ficus carica L.): Genome-Wide Analysis and Expression Patterns.

The papain-like cysteine proteases (PLCPs) are the most abundant family of cysteine proteases in plants, with essential roles in biotic/abiotic stress responses, growth and senescence. Papain, bromelain and ficin are widely used in food, medicine and other industries. In this study, 31 PLCP genes (FcPCLPs) were identified in the fig (Ficus carica L.) genome by HMM search and manual screening, and assigned to one of nine subfamilies based on gene structure and conserved motifs. SAG12 and RD21 were the largest subfamilies with 10 and 7 members, respectively. The FcPCLPs ranged from 1,128 to 5,075 bp in length, containing 1-10 introns, and the coding sequence ranged from 624 to 1,518 bp, encoding 207-505 amino acids. Subcellular localization analysis indicated that 24, 2, and 5 PLCP proteins were targeted to the lysosome/vacuole, cytoplasm and extracellular matrix, respectively. Promoter (2,000 bp upstream) analysis of FcPLCPs revealed a high number of plant hormone and low temperature response elements. RNA-seq revealed differential expression of 17 FcPLCPs in the inflorescence and receptacle, and RD21 subfamily members were the major PLCPs expressed in the fruit; 16 and 5 FcPLCPs responded significantly to ethylene and light, respectively. Proteome analyses revealed 18 and 5 PLCPs in the fruit cell soluble proteome and fruit latex, respectively. Ficins were the major PLCP in fig fruit, with decreased abundance in inflorescences, but increased abundance in receptacles of commercial-ripe fruit. FcRD21B/C and FcALP1 were aligned as the genes encoding the main ficin isoforms. Our study provides valuable multi-omics information on the FcPLCP family and lays the foundation for further functional studies.

Gene Architecture Facilitates Intron-Mediated Enhancement of Transcription.

Introns impact several vital aspects of eukaryotic organisms like proteomic plasticity, genomic stability, stress response and gene expression. A role for introns in the regulation of gene expression at the level of transcription has been known for more than thirty years. The molecular basis underlying the phenomenon, however, is still not entirely clear. An important clue came from studies performed in budding yeast that indicate that the presence of an intron within a gene results in formation of a multi-looped gene architecture. When looping is defective, these interactions are abolished, and there is no enhancement of transcription despite normal splicing. In this review, we highlight several potential mechanisms through which looping interactions may enhance transcription. The promoter-5' splice site interaction can facilitate initiation of transcription, the terminator-3' splice site interaction can enable efficient termination of transcription, while the promoter-terminator interaction can enhance promoter directionality and expedite reinitiation of transcription. Like yeast, mammalian genes also exhibit an intragenic interaction of the promoter with the gene body, especially exons. Such promoter-exon interactions may be responsible for splicing-dependent transcriptional regulation. Thus, the splicing-facilitated changes in gene architecture may play a critical role in regulation of transcription in yeast as well as in higher eukaryotes.

A Bioinformatics Tutorial for Comparative Development Genomics in Diverse Meiofauna.

Miniaturization, which is a common feature in animals, is particularly manifest in meiofauna-animals sharing peculiar phenotypic features that evolved as adaptations to the highly specialized aquatic interstitial habitat. While revealing much about the extreme phyletic diversity of meiofauna, the genome structure of meiofaunal species could also characterize the phenotype of ancestral states as well as explain the origin and evolution of miniaturization. Here, we present a practical bioinformatics tutorial for genome assembly, genome comparison, and characterization of Hox clusters in meiofaunal species.

Transcriptome, proteome and draft genome of Euglena gracilis.

BACKGROUND: Photosynthetic euglenids are major contributors to fresh water ecosystems. Euglena gracilis in particular has noted metabolic flexibility, reflected by an ability to thrive in a range of harsh environments. E. gracilis has been a popular model organism and of considerable biotechnological interest, but the absence of a gene catalogue has hampered both basic research and translational efforts. RESULTS: We report a detailed transcriptome and partial genome for E. gracilis Z1. The nuclear genome is estimated to be around 500 Mb in size, and the transcriptome encodes over 36,000 proteins and the genome possesses less than 1% coding sequence. Annotation of coding sequences indicates a highly sophisticated endomembrane system, RNA processing mechanisms and nuclear genome contributions from several photosynthetic lineages. Multiple gene families, including likely signal transduction components, have been massively expanded. Alterations in protein abundance are controlled post-transcriptionally between light and dark conditions, surprisingly similar to trypanosomatids. CONCLUSIONS: Our data provide evidence that a range of photosynthetic eukaryotes contributed to the Euglena nuclear genome, evidence in support of the 'shopping bag' hypothesis for plastid acquisition. We also suggest that euglenids possess unique regulatory mechanisms for achieving extreme adaptability, through mechanisms of paralog expansion and gene acquisition.

Impact of transposable elements in the architecture of genes of the human parasite Schistosoma mansoni.

The parasites belonging to the genus Schistosoma are agents of schistosomiasis, a disease estimated as affecting 235 million people in the world. To better understand the structure of Schistosoma mansoni genome, transposable elements (TEs) distribution and impact on gene structures were investigated. Our analyses indicated a differential distribution of TEs throughout the gene structure. Introns located at the 5' end of the genes are less prone to display TEs and introns lacking TEs tend to be shorter. Therefore, this could be one of the factors explaining previous data showing that S. mansoni displays shorter introns near the 5' end of the genes. Identification of six genes harboring TEs in their coding region suggests a positive contribution for the evolution of proteome repertory of S. mansoni. Taken together, our data suggest significant contributions of TEs to the architecture of genes from S. mansoni.

Origin of exon skipping-rich transcriptomes in animals driven by evolution of gene architecture.

BACKGROUND: Alternative splicing, particularly through intron retention and exon skipping, is a major layer of pre-translational regulation in eukaryotes. While intron retention is believed to be the most prevalent mode across non-animal eukaryotes, animals have unusually high rates of exon skipping. However, when and how this high prevalence of exon skipping evolved is unknown. Since exon skipping can greatly expand proteomes, answering these questions sheds light on the evolution of higher organismal complexity in metazoans. RESULTS: We used RNA-seq data to quantify exon skipping and intron retention frequencies across 65 eukaryotic species, with particular focus on early branching animals and unicellular holozoans. We found that only bilaterians have significantly increased their exon skipping frequencies compared to all other eukaryotic groups. Unlike in other eukaryotes, however, exon skipping in nearly all animals, including non-bilaterians, is strongly enriched for frame-preserving sequences, suggesting that exon skipping involvement in proteome expansion predated the increase in frequency. We also identified architectural features consistently associated with higher exon skipping rates within all studied eukaryotic genomes. Remarkably, these architectures became more prevalent during animal evolution, indicating co-evolution between genome architectures and exon skipping frequencies. CONCLUSION: We suggest that the increase of exon skipping rates in animals followed a two-step process. First, exon skipping in early animals became enriched for frame-preserving events. Second, bilaterian ancestors dramatically increased their exon skipping frequencies, likely driven by the interplay between a shift in their genome architectures towards more exon definition and recruitment of frame-preserving exon skipping events to functionally diversify their cell-specific proteomes.

Integrating Early Transcriptomic Responses to Rhizotoxins in Rice (Oryza sativa. L.) Reveals Key Regulators and a Potential Early Biomarker of Cadmium Toxicity.

As sessile organisms, plants were constantly challenged with biotic and abiotic stresses. Transcriptional activation of stress-responsive genes is a crucial part of the plant adaptation to environmental changes. Here, early response of rice root to eight rhizotoxic stressors: arsenate, copper, cadmium, mercury, chromate, vanadate, ferulic acid and juglone, was analyzed using published microarray data. There were 539 general stress response (GSR) genes up-regulated under all eight treatments, including genes related to carbohydrate metabolism, phytohormone balance, and cell wall structure. Genes related to transcriptional coactivation showed higher Ka/Ks ratio compared to the other GSR genes. Network analysis discovered complicated interaction within GSR genes and the most connected signaling hubs were WRKY53, WRKY71, and MAPK5. Promoter analysis discovers enriched SCGCGCS cis-element in GSR genes. Moreover, GSR genes tend to be intronless and genes with shorter total intron length were induced in a higher level. Among genes uniquely up-regulated by a single stress, a phosphoenolpyruvate carboxylase kinase (PPCK) was identified as a candidate biomarker for detecting cadmium contamination. Our findings provide insights into the transcriptome dynamics of molecular response of rice to different rhizotoxic stress and also demonstrate potential use of comparative transcriptome analysis in identifying a novel potential early biomarker.

Gene Architectures that Minimize Cost of Gene Expression.

Gene expression burdens cells by consuming resources and energy. While numerous studies have investigated regulation of expression level, little is known about gene design elements that govern expression costs. Here, we ask how cells minimize production costs while maintaining a given protein expression level and whether there are gene architectures that optimize this process. We measured fitness of ∼14,000 E. coli strains, each expressing a reporter gene with a unique 5' architecture. By comparing cost-effective and ineffective architectures, we found that cost per protein molecule could be minimized by lowering transcription levels, regulating translation speeds, and utilizing amino acids that are cheap to synthesize and that are less hydrophobic. We then examined natural E. coli genes and found that highly expressed genes have evolved more forcefully to minimize costs associated with their expression. Our study thus elucidates gene design elements that improve the economy of protein expression in natural and heterologous systems.

Matrix Gla protein and osteocalcin: from gene duplication to neofunctionalization.

Osteocalcin (OC or bone Gla protein, BGP) and matrix Gla protein (MGP) are two members of the growing family of vitamin K-dependent (VKD) proteins. They were the first VKD proteins found not to be involved in coagulation and synthesized outside the liver. Both proteins were isolated from bone although it is now known that only OC is synthesized by bone cells under normal physiological conditions, but since both proteins can bind calcium and hydroxyapatite, they can also accumulate in bone. Both OC and MGP share similar structural features, both in terms of protein domains and gene organization. OC gene is likely to have appeared from MGP through a tandem gene duplication that occurred concomitantly with the appearance of the bony vertebrates. Despite their relatively close relationship and the fact that both can bind calcium and affect mineralization, their functions are not redundant and they also have other unrelated functions. Interestingly, these two proteins appear to have followed quite different evolutionary strategies in order to acquire novel functionalities, with OC following a gene duplication strategy while MGP variability was obtained mostly by the use of multiple promoters and alternative splicing, leading to proteins with additional functional characteristics and alternative gene regulatory pathways.

The function of introns.

The intron-exon architecture of many eukaryotic genes raises the intriguing question of whether this unique organization serves any function, or is it simply a result of the spread of functionless introns in eukaryotic genomes. In this review, we show that introns in contemporary species fulfill a broad spectrum of functions, and are involved in virtually every step of mRNA processing. We propose that this great diversity of intronic functions supports the notion that introns were indeed selfish elements in early eukaryotes, but then independently gained numerous functions in different eukaryotic lineages. We suggest a novel criterion of evolutionary conservation, dubbed intron positional conservation, which can identify functional introns.

A universal nonmonotonic relationship between gene compactness and expression levels in multicellular eukaryotes.

Analysis of gene architecture and expression levels of four organisms, Homo sapiens, Caenorhabditis elegans, Drosophila melanogaster, and Arabidopsis thaliana, reveals a surprising, nonmonotonic, universal relationship between expression level and gene compactness. With increasing expression level, the genes tend at first to become longer but, from a certain level of expression, they become more and more compact, resulting in an approximate bell-shaped dependence. There are two leading hypotheses to explain the compactness of highly expressed genes. The selection hypothesis predicts that gene compactness is predominantly driven by the level of expression, whereas the genomic design hypothesis predicts that expression breadth across tissues is the driving force. We observed the connection between gene expression breadth in humans and gene compactness to be significantly weaker than the connection between expression level and compactness, a result that is compatible with the selection hypothesis but not the genome design hypothesis. The initial gene elongation with increasing expression level could be explained, at least in part, by accumulation of regulatory elements enhancing expression, in particular, in introns. This explanation is compatible with the observed positive correlation between intron density and expression level of a gene. Conversely, the trend toward increasing compactness for highly expressed genes could be caused by selection for minimization of energy and time expenditure during transcription and splicing and for increased fidelity of transcription, splicing, and/or translation that is likely to be particularly critical for highly expressed genes. Regardless of the exact nature of the forces that shape the gene architecture, we present evidence that, at least, in animals, coding and noncoding parts of genes show similar architectonic trends.