Do long noncoding RNAs (lncRNAs) contribute little or substantively to human biology? To address how lncRNA loci and their transcripts, structures, interactions, and functions contribute to human ...traits and disease, we adopt a genome-wide perspective. We intend to provoke alternative interpretation of questionable evidence and thorough inquiry into unsubstantiated claims. We discuss pitfalls of lncRNA experimental and computational methods as well as opposing interpretations of their results. The majority of evidence, we argue, indicates that most lncRNA transcript models reflect transcriptional noise or provide minor regulatory roles, leaving relatively few human lncRNAs that contribute centrally to human development, physiology, or behavior. These important few tend to be spliced and better conserved but lack a simple syntax relating sequence to structure and mechanism, and so resist simple categorization. This genome-wide view should help investigators prioritize individual lncRNAs based on their likely contribution to human biology.
RNA splicing is a key mechanism linking genetic variation with psychiatric disorders. Splicing profiles are particularly diverse in brain and difficult to accurately identify and quantify. We ...developed a new approach to address this challenge, combining long-range PCR and nanopore sequencing with a novel bioinformatics pipeline. We identify the full-length coding transcripts of CACNA1C in human brain. CACNA1C is a psychiatric risk gene that encodes the voltage-gated calcium channel Ca
1.2. We show that CACNA1C's transcript profile is substantially more complex than appreciated, identifying 38 novel exons and 241 novel transcripts. Importantly, many of the novel variants are abundant, and predicted to encode channels with altered function. The splicing profile varies between brain regions, especially in cerebellum. We demonstrate that human transcript diversity (and thereby protein isoform diversity) remains under-characterised, and provide a feasible and cost-effective methodology to address this. A detailed understanding of isoform diversity will be essential for the translation of psychiatric genomic findings into pathophysiological insights and novel psychopharmacological targets.
Although a small number of the vast array of animal long non-coding RNAs (lncRNAs) have known effects on cellular processes examined in vitro, the extent of their contributions to normal cell ...processes throughout development, differentiation and disease for the most part remains less clear. Phenotypes arising from deletion of an entire genomic locus cannot be unequivocally attributed either to the loss of the lncRNA per se or to the associated loss of other overlapping DNA regulatory elements. The distinction between cis- or trans-effects is also often problematic. We discuss the advantages and challenges associated with the current techniques for studying the in vivo function of lncRNAs in the light of different models of lncRNA molecular mechanism, and reflect on the design of experiments to mutate lncRNA loci. These considerations should assist in the further investigation of these transcriptional products of the genome.
If sequencing was possible only for genomes, and not for RNAs or proteins, then functional protein-coding exons would be recognizable by their unusual patterns of nucleotide composition, specifically ...a high GC content across the body of exons, and an unusual nucleotide content near their edges. RNAs and proteins can, of course, be sequenced but the extent of functionality of intergenic long noncoding RNAs (lncRNAs) remains under question owing to their low nucleotide conservation. Inspired by the nucleotide composition patterns of protein-coding exons, we sought evidence for functionality across lncRNA loci from diverse species. We found that such patterns across multiexonic lncRNA loci mirror those of proteincoding genes, although to a lesser degree: Specifically, compared with introns, lncRNA exons are GC rich. Additionally we report evidence for the action of purifying selection to preserve exonic splicing enhancers within human multiexonic lncRNAs and nucleotide composition in fruit fly lncRNAs. Our findings provide evidence for selection for more efficient rates of transcription and splicing within lncRNA loci. Despite only a minor proportion of their RNA bases being constrained, multiexonic intergenic lncRNAs appear to require accurate splicing of their exons to transact their function.
Ninety-four percent of mammalian protein-coding exons exceed 51 nucleotides (nt) in length. The paucity of micro-exons (≤ 51 nt) suggests that their recognition and correct processing by the splicing ...machinery present greater challenges than for longer exons. Yet, because thousands of human genes harbor processed micro-exons, specialized mechanisms may be in place to promote their splicing. Here, we survey deep genomic data sets to define 13,085 micro-exons and to study their splicing mechanisms and molecular functions. More than 60% of annotated human micro-exons exhibit a high level of sequence conservation, an indicator of functionality. While most human micro-exons require splicing-enhancing genomic features to be processed, the splicing of hundreds of micro-exons is enhanced by the adjacent binding of splice factors in the introns of pre-messenger RNAs. Notably, splicing of a significant number of micro-exons was found to be facilitated by the binding of RBFOX proteins, which promote their inclusion in the brain, muscle, and heart. Our analyses suggest that accurate regulation of micro-exon inclusion by RBFOX proteins and PTBP1 plays an important role in the maintenance of tissue-specific protein-protein interactions.
Parallel sequencing of a single cell's genome and transcriptome provides a powerful tool for dissecting genetic variation and its relationship with gene expression. Here we present a detailed ...protocol for G&T-seq, a method for separation and parallel sequencing of genomic DNA and full-length polyA(+) mRNA from single cells. We provide step-by-step instructions for the isolation and lysis of single cells; the physical separation of polyA(+) mRNA from genomic DNA using a modified oligo-dT bead capture and the respective whole-transcriptome and whole-genome amplifications; and library preparation and sequence analyses of these amplification products. The method allows the detection of thousands of transcripts in parallel with the genetic variants captured by the DNA-seq data from the same single cell. G&T-seq differs from other currently available methods for parallel DNA and RNA sequencing from single cells, as it involves physical separation of the DNA and RNA and does not require bespoke microfluidics platforms. The process can be implemented manually or through automation. When performed manually, paired genome and transcriptome sequencing libraries from eight single cells can be produced in ∼3 d by researchers experienced in molecular laboratory work. For users with experience in the programming and operation of liquid-handling robots, paired DNA and RNA libraries from 96 single cells can be produced in the same time frame. Sequence analysis and integration of single-cell G&T-seq DNA and RNA data requires a high level of bioinformatics expertise and familiarity with a wide range of informatics tools.
Genome analyses reveal that several plants have lost a major part of the immune system.
Abstract
Plant innate immunity relies on nucleotide binding leucine-rich repeat receptors (NLRs) that recognize ...pathogen-derived molecules and activate downstream signaling pathways. We analyzed the variation in NLR gene copy number and identified plants with a low number of NLR genes relative to sister species. We specifically focused on four plants from two distinct lineages, one monocot lineage (Alismatales) and one eudicot lineage (Lentibulariaceae). In these lineages, the loss of NLR genes coincides with loss of the well-known downstream immune signaling complex ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1)/PHYTOALEXIN DEFICIENT 4 (PAD4). We expanded our analysis across whole proteomes and found that other characterized immune genes were absent only in Lentibulariaceae and Alismatales. Additionally, we identified genes of unknown function that were convergently lost together with EDS1/PAD4 in five plant species. Gene expression analyses in Arabidopsis (Arabidopsis thaliana) and Oryza sativa revealed that several homologs of the candidates are differentially expressed during pathogen infection, drought, and abscisic acid treatment. Our analysis provides evolutionary evidence for the rewiring of plant immunity in some plant lineages, as well as the coevolution of the EDS1/PAD4 pathway and drought responses.
The plant immune system is innate and encoded in the germline. Using it efficiently, plants are capable of recognizing a diverse range of rapidly evolving pathogens. A recently described phenomenon ...shows that plant immune receptors are able to recognize pathogen effectors through the acquisition of exogenous protein domains from other plant genes.
We show that plant immune receptors with integrated domains are distributed unevenly across their phylogeny in grasses. Using phylogenetic analysis, we uncover a major integration clade, whose members underwent repeated independent integration events producing diverse fusions. This clade is ancestral in grasses with members often found on syntenic chromosomes. Analyses of these fusion events reveals that homologous receptors can be fused to diverse domains. Furthermore, we discover a 43 amino acid long motif associated with this dominant integration clade which is located immediately upstream of the fusion site. Sequence analysis reveals that DNA transposition and/or ectopic recombination are the most likely mechanisms of formation for nucleotide binding leucine rich repeat proteins with integrated domains.
The identification of this subclass of plant immune receptors that is naturally adapted to new domain integration will inform biotechnological approaches for generating synthetic receptors with novel pathogen "baits."
During the splicing reaction, the 5' intron end is joined to the branchpoint nucleotide, selecting the next exon to incorporate into the mature RNA and forming an intron lariat, which is excised. ...Despite a critical role in gene splicing, the locations and features of human splicing branchpoints are largely unknown. We use exoribonuclease digestion and targeted RNA-sequencing to enrich for sequences that traverse the lariat junction and, by split and inverted alignment, reveal the branchpoint. We identify 59,359 high-confidence human branchpoints in >10,000 genes, providing a first map of splicing branchpoints in the human genome. Branchpoints are predominantly adenosine, highly conserved, and closely distributed to the 3' splice site. Analysis of human branchpoints reveals numerous novel features, including distinct features of branchpoints for alternatively spliced exons and a family of conserved sequence motifs overlapping branchpoints we term B-boxes, which exhibit maximal nucleotide diversity while maintaining interactions with the keto-rich U2 snRNA. Different B-box motifs exhibit divergent usage in vertebrate lineages and associate with other splicing elements and distinct intron-exon architectures, suggesting integration within a broader regulatory splicing code. Lastly, although branchpoints are refractory to common mutational processes and genetic variation, mutations occurring at branchpoint nucleotides are enriched for disease associations.
To generate energy efficiently, the cell is uniquely challenged to co-ordinate the abundance of electron transport chain protein subunits expressed from both nuclear and mitochondrial genomes. How an ...effective stoichiometry of this many constituent subunits is co-ordinated post-transcriptionally remains poorly understood. Here we show that
, an unusually abundant cytoplasmic long noncoding RNA (lncRNA), modulates the levels of mitochondrial complex I subunit transcripts in a manner that requires binding to microRNA-488-3p. Increased abundance of
cooperatively elevates complex I subunit protein abundance and enzymatic activity, decreases reactive oxygen species production, and protects against the complex I inhibitor rotenone.
function is conserved across placental mammals: human and mouse orthologues effectively modulate complex I enzymatic activity in mouse and human cells, respectively.
is the first lncRNA demonstrated, to our knowledge, to regulate mitochondrial oxidative phosphorylation and, with miR-488-3p, represent novel targets for the modulation of complex I activity.
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