Evolution of the ancestral mammalian karyotype and syntenic regions.

Decrypting the rearrangements that drive mammalian chromosome evolution is critical to understanding the molecular bases of speciation, adaptation, and disease susceptibility. Using 8 scaffolded and 26 chromosome-scale genome assemblies representing 23/26 mammal orders, we computationally reconstructed ancestral karyotypes and syntenic relationships at 16 nodes along the mammalian phylogeny. Three different reference genomes (human, sloth, and cattle) representing phylogenetically distinct mammalian superorders were used to assess reference bias in the reconstructed ancestral karyotypes and to expand the number of clades with reconstructed genomes. The mammalian ancestor likely had 19 pairs of autosomes, with nine of the smallest chromosomes shared with the common ancestor of all amniotes (three still conserved in extant mammals), demonstrating a striking conservation of synteny for ∼320 My of vertebrate evolution. The numbers and types of chromosome rearrangements were classified for transitions between the ancestral mammalian karyotype, descendent ancestors, and extant species. For example, 94 inversions, 16 fissions, and 14 fusions that occurred over 53 My differentiated the therian from the descendent eutherian ancestor. The highest breakpoint rate was observed between the mammalian and therian ancestors (3.9 breakpoints/My). Reconstructed mammalian ancestor chromosomes were found to have distinct evolutionary histories reflected in their rates and types of rearrangements. The distributions of genes, repetitive elements, topologically associating domains, and actively transcribed regions in multispecies homologous synteny blocks and evolutionary breakpoint regions indicate that purifying selection acted over millions of years of vertebrate evolution to maintain syntenic relationships of developmentally important genes and regulatory landscapes of gene-dense chromosomes.

Autogenous cross-regulation of Quaking mRNA processing and translation balances Quaking functions in splicing and translation.

Quaking protein isoforms arise from a single Quaking gene and bind the same RNA motif to regulate splicing, translation, decay, and localization of a large set of RNAs. However, the mechanisms by which Quaking expression is controlled to ensure that appropriate amounts of each isoform are available for such disparate gene expression processes are unknown. Here we explore how levels of two isoforms, nuclear Quaking-5 (Qk5) and cytoplasmic Qk6, are regulated in mouse myoblasts. We found that Qk5 and Qk6 proteins have distinct functions in splicing and translation, respectively, enforced through differential subcellular localization. We show that Qk5 and Qk6 regulate distinct target mRNAs in the cell and act in distinct ways on their own and each other's transcripts to create a network of autoregulatory and cross-regulatory feedback controls. Morpholino-mediated inhibition of Qk translation confirms that Qk5 controls Qk RNA levels by promoting accumulation and alternative splicing of Qk RNA, whereas Qk6 promotes its own translation while repressing Qk5. This Qk isoform cross-regulatory network responds to additional cell type and developmental controls to generate a spectrum of Qk5/Qk6 ratios, where they likely contribute to the wide range of functions of Quaking in development and cancer.

Correction: Rapidly evolving protointrons in Saccharomyces genomes revealed by a hungry spliceosome.

[This corrects the article DOI: 10.1371/journal.pgen.1008249.].

Rapidly evolving protointrons in Saccharomyces genomes revealed by a hungry spliceosome.

Introns are a prevalent feature of eukaryotic genomes, yet their origins and contributions to genome function and evolution remain mysterious. In budding yeast, repression of the highly transcribed intron-containing ribosomal protein genes (RPGs) globally increases splicing of non-RPG transcripts through reduced competition for the spliceosome. We show that under these "hungry spliceosome" conditions, splicing occurs at more than 150 previously unannotated locations we call protointrons that do not overlap known introns. Protointrons use a less constrained set of splice sites and branchpoints than standard introns, including in one case AT-AC in place of GT-AG. Protointrons are not conserved in all closely related species, suggesting that most are not under positive selection and are fated to disappear. Some are found in non-coding RNAs (e. g. CUTs and SUTs), where they may contribute to the creation of new genes. Others are found across boundaries between noncoding and coding sequences, or within coding sequences, where they offer pathways to the creation of new protein variants, or new regulatory controls for existing genes. We define protointrons as (1) nonconserved intron-like sequences that are (2) infrequently spliced, and importantly (3) are not currently understood to contribute to gene expression or regulation in the way that standard introns function. A very few protointrons in S. cerevisiae challenge this classification by their increased splicing frequency and potential function, consistent with the proposed evolutionary process of "intronization", whereby new standard introns are created. This snapshot of intron evolution highlights the important role of the spliceosome in the expansion of transcribed genomic sequence space, providing a pathway for the rare events that may lead to the birth of new eukaryotic genes and the refinement of existing gene function.

Competition between pre-mRNAs for the splicing machinery drives global regulation of splicing.

During meiosis in yeast, global splicing efficiency increases and then decreases. Here we provide evidence that splicing improves due to reduced competition for the splicing machinery. The timing of this regulation corresponds to repression and reactivation of ribosomal protein genes (RPGs) during meiosis. In vegetative cells, RPG repression by rapamycin treatment also increases splicing efficiency. Downregulation of the RPG-dedicated transcription factor gene IFH1 genetically suppresses two spliceosome mutations, prp11-1 and prp4-1, and globally restores splicing efficiency in prp4-1 cells. We conclude that the splicing apparatus is limiting and that pre-messenger RNAs compete. Splicing efficiency of a pre-mRNA therefore depends not just on its own concentration and affinity for limiting splicing factor(s), but also on those of competing pre-mRNAs. Competition between RNAs for limiting processing factors appears to be a general condition in eukaryotes for a variety of posttranscriptional control mechanisms including microRNA (miRNA) repression, polyadenylation, and splicing.

Genome-wide analysis reveals SR protein cooperation and competition in regulated splicing.

SR proteins are well-characterized RNA binding proteins that promote exon inclusion by binding to exonic splicing enhancers (ESEs). However, it has been unclear whether regulatory rules deduced on model genes apply generally to activities of SR proteins in the cell. Here, we report global analyses of two prototypical SR proteins, SRSF1 (SF2/ASF) and SRSF2 (SC35), using splicing-sensitive arrays and CLIP-seq on mouse embryo fibroblasts (MEFs). Unexpectedly, we find that these SR proteins promote both inclusion and skipping of exons in vivo, but their binding patterns do not explain such opposite responses. Further analyses reveal that loss of one SR protein is accompanied by coordinated loss or compensatory gain in the interaction of other SR proteins at the affected exons. Therefore, specific effects on regulated splicing by one SR protein actually depend on a complex set of relationships with multiple other SR proteins in mammalian genomes.

Evidence for convergent evolution of SINE-directed Staufen-mediated mRNA decay.

Primate-specific Alu short interspersed elements (SINEs) as well as rodent-specific B and ID (B/ID) SINEs can promote Staufen-mediated decay (SMD) when present in mRNA 3'-untranslated regions (3'-UTRs). The transposable nature of SINEs, their presence in long noncoding RNAs, their interactions with Staufen, and their rapid divergence in different evolutionary lineages suggest they could have generated substantial modification of posttranscriptional gene-control networks during mammalian evolution. Some of the variation in SMD regulation produced by SINE insertion might have had a similar regulatory effect in separate mammalian lineages, leading to parallel evolution of the Staufen network by independent expansion of lineage-specific SINEs. To explore this possibility, we searched for orthologous gene pairs, each carrying a species-specific 3'-UTR SINE and each regulated by SMD, by measuring changes in mRNA abundance after individual depletion of two SMD factors, Staufen1 (STAU1) and UPF1, in both human and mouse myoblasts. We identified and confirmed orthologous gene pairs with 3'-UTR SINEs that independently function in SMD control of myoblast metabolism. Expanding to other species, we demonstrated that SINE-directed SMD likely emerged in both primate and rodent lineages >20-25 million years ago. Our work reveals a mechanism for the convergent evolution of posttranscriptional gene regulatory networks in mammals by species-specific SINE transposition and SMD.

The splicing regulator Rbfox2 is required for both cerebellar development and mature motor function.

The Rbfox proteins (Rbfox1, Rbfox2, and Rbfox3) regulate the alternative splicing of many important neuronal transcripts and have been implicated in a variety of neurological disorders. However, their roles in brain development and function are not well understood, in part due to redundancy in their activities. Here we show that, unlike Rbfox1 deletion, the CNS-specific deletion of Rbfox2 disrupts cerebellar development. Genome-wide analysis of Rbfox2(-/-) brain RNA identifies numerous splicing changes altering proteins important both for brain development and mature neuronal function. To separate developmental defects from alterations in the physiology of mature cells, Rbfox1 and Rbfox2 were deleted from mature Purkinje cells, resulting in highly irregular firing. Notably, the Scn8a mRNA encoding the Na(v)1.6 sodium channel, a key mediator of Purkinje cell pacemaking, is improperly spliced in RbFox2 and Rbfox1 mutant brains, leading to highly reduced protein expression. Thus, Rbfox2 protein controls a post-transcriptional program required for proper brain development. Rbfox2 is subsequently required with Rbfox1 to maintain mature neuronal physiology, specifically Purkinje cell pacemaking, through their shared control of sodium channel transcript splicing.

Integration of a splicing regulatory network within the meiotic gene expression program of Saccharomyces cerevisiae.

Splicing regulatory networks are essential components of eukaryotic gene expression programs, yet little is known about how they are integrated with transcriptional regulatory networks into coherent gene expression programs. Here we define the MER1 splicing regulatory network and examine its role in the gene expression program during meiosis in budding yeast. Mer1p splicing factor promotes splicing of just four pre-mRNAs. All four Mer1p-responsive genes also require Nam8p for splicing activation by Mer1p; however, other genes require Nam8p but not Mer1p, exposing an overlapping meiotic splicing network controlled by Nam8p. MER1 mRNA and three of the four Mer1p substrate pre-mRNAs are induced by the transcriptional regulator Ume6p. This unusual arrangement delays expression of Mer1p-responsive genes relative to other genes under Ume6p control. Products of Mer1p-responsive genes are required for initiating and completing recombination and for activation of Ndt80p, the activator of the transcriptional network required for subsequent steps in the program. Thus, the MER1 splicing regulatory network mediates the dependent relationship between the UME6 and NDT80 transcriptional regulatory networks in the meiotic gene expression program. This study reveals how splicing regulatory networks can be interlaced with transcriptional regulatory networks in eukaryotic gene expression programs.

Rbfox1 downregulation and altered calpain 3 splicing by FRG1 in a mouse model of Facioscapulohumeral muscular dystrophy (FSHD).

Facioscapulohumeral muscular dystrophy (FSHD) is a common muscle disease whose molecular pathogenesis remains largely unknown. Over-expression of FSHD region gene 1 (FRG1) in mice, frogs, and worms perturbs muscle development and causes FSHD-like phenotypes. FRG1 has been implicated in splicing, and we asked how splicing might be involved in FSHD by conducting a genome-wide analysis in FRG1 mice. We find that splicing perturbations parallel the responses of different muscles to FRG1 over-expression and disease progression. Interestingly, binding sites for the Rbfox family of splicing factors are over-represented in a subset of FRG1-affected splicing events. Rbfox1 knockdown, over-expression, and RNA-IP confirm that these are direct Rbfox1 targets. We find that FRG1 is associated to the Rbfox1 RNA and decreases its stability. Consistent with this, Rbfox1 expression is down-regulated in mice and cells over-expressing FRG1 as well as in FSHD patients. Among the genes affected is Calpain 3, which is mutated in limb girdle muscular dystrophy, a disease phenotypically similar to FSHD. In FRG1 mice and FSHD patients, the Calpain 3 isoform lacking exon 6 (Capn3 E6-) is increased. Finally, Rbfox1 knockdown and over-expression of Capn3 E6- inhibit muscle differentiation. Collectively, our results suggest that a component of FSHD pathogenesis may arise by over-expression of FRG1, reducing Rbfox1 levels and leading to aberrant expression of an altered Calpain 3 protein through dysregulated splicing.

Quaking and PTB control overlapping splicing regulatory networks during muscle cell differentiation.

Alternative splicing contributes to muscle development, but a complete set of muscle-splicing factors and their combinatorial interactions are unknown. Previous work identified ACUAA ("STAR" motif) as an enriched intron sequence near muscle-specific alternative exons such as Capzb exon 9. Mass spectrometry of myoblast proteins selected by the Capzb exon 9 intron via RNA affinity chromatography identifies Quaking (QK), a protein known to regulate mRNA function through ACUAA motifs in 3' UTRs. We find that QK promotes inclusion of Capzb exon 9 in opposition to repression by polypyrimidine tract-binding protein (PTB). QK depletion alters inclusion of 406 cassette exons whose adjacent intron sequences are also enriched in ACUAA motifs. During differentiation of myoblasts to myotubes, QK levels increase two- to threefold, suggesting a mechanism for QK-responsive exon regulation. Combined analysis of the PTB- and QK-splicing regulatory networks during myogenesis suggests that 39% of regulated exons are under the control of one or both of these splicing factors. This work provides the first evidence that QK is a global regulator of splicing during muscle development in vertebrates and shows how overlapping splicing regulatory networks contribute to gene expression programs during differentiation.

Exploring functional relationships between components of the gene expression machinery.

Eukaryotic gene expression requires the coordinated activity of many macromolecular machines including transcription factors and RNA polymerase, the spliceosome, mRNA export factors, the nuclear pore, the ribosome and decay machineries. Yeast carrying mutations in genes encoding components of these machineries were examined using microarrays to measure changes in both pre-mRNA and mRNA levels. We used these measurements as a quantitative phenotype to ask how steps in the gene expression pathway are functionally connected. A multiclass support vector machine was trained to recognize the gene expression phenotypes caused by these mutations. In several cases, unexpected phenotype assignments by the computer revealed functional roles for specific factors at multiple steps in the gene expression pathway. The ability to resolve gene expression pathway phenotypes provides insight into how the major machineries of gene expression communicate with each other.

Author Correction: Origin and evolution of the octoploid strawberry genome.

In the version of this article originally published, author Joshua R. Puzey was incorrectly listed as having affiliation 7 (School of Plant Sciences, University of Arizona, Tucson, AZ, USA); affiliation 6 (Department of Biology, College of William and Mary, Williamsburg, VA, USA) is the correct affiliation. The error has been corrected in the HTML and PDF versions of the article.

Origin and evolution of the octoploid strawberry genome.

Cultivated strawberry emerged from the hybridization of two wild octoploid species, both descendants from the merger of four diploid progenitor species into a single nucleus more than 1 million years ago. Here we report a near-complete chromosome-scale assembly for cultivated octoploid strawberry (Fragaria × ananassa) and uncovered the origin and evolutionary processes that shaped this complex allopolyploid. We identified the extant relatives of each diploid progenitor species and provide support for the North American origin of octoploid strawberry. We examined the dynamics among the four subgenomes in octoploid strawberry and uncovered the presence of a single dominant subgenome with significantly greater gene content, gene expression abundance, and biased exchanges between homoeologous chromosomes, as compared with the other subgenomes. Pathway analysis showed that certain metabolomic and disease-resistance traits are largely controlled by the dominant subgenome. These findings and the reference genome should serve as a powerful platform for future evolutionary studies and enable molecular breeding in strawberry.

Haplotype-phased genome and evolution of phytonutrient pathways of tetraploid blueberry.

BACKGROUND: Highbush blueberry (Vaccinium corymbosum) has long been consumed for its unique flavor and composition of health-promoting phytonutrients. However, breeding efforts to improve fruit quality in blueberry have been greatly hampered by the lack of adequate genomic resources and a limited understanding of the underlying genetics encoding key traits. The genome of highbush blueberry has been particularly challenging to assemble due, in large part, to its polyploid nature and genome size. FINDINGS: Here, we present a chromosome-scale and haplotype-phased genome assembly of the cultivar "Draper," which has the highest antioxidant levels among a diversity panel of 71 cultivars and 13 wild Vaccinium species. We leveraged this genome, combined with gene expression and metabolite data measured across fruit development, to identify candidate genes involved in the biosynthesis of important phytonutrients among other metabolites associated with superior fruit quality. Genome-wide analyses revealed that both polyploidy and tandem gene duplications modified various pathways involved in the biosynthesis of key phytonutrients. Furthermore, gene expression analyses hint at the presence of a spatial-temporal specific dominantly expressed subgenome including during fruit development. CONCLUSIONS: These findings and the reference genome will serve as a valuable resource to guide future genome-enabled breeding of important agronomic traits in highbush blueberry.

Developmental expression profile of quaking, a candidate gene for schizophrenia, and its target genes in human prefrontal cortex and hippocampus shows regional specificity.

Decreased expression of oligodendrocyte/myelin-related (OMR) genes, including quaking (QKI), is a consistent finding in gene expression studies of post-mortem brain from subjects with schizophrenia, and these changes are most prominent in the hippocampus vs. the prefrontal cortex (PFC). Although expression of QKI and other OMR genes has been examined in rodents, little is known about their developmental trajectory in the human brain. Therefore, we examined expression of QKI and several putative mRNA targets of QKI in human PFC and hippocampus at different ages. The pattern of QKI expression in the PFC resembled that reported in rodents, with high QKI-5 in the fetal brain and an increase in QKI-6 and QKI-7 during the period of active myelination, although QKI-5 expression did not decrease substantially during postnatal development in the PFC in humans as it does in rodent brain. Most of the putative QKI target genes also showed linear increases in expression with increasing age in the PFC. In contrast, expression of these genes showed little evidence of developmental regulation in the hippocampus. Correlations between expression levels of the nuclear vs. cytoplasmic QKI isoforms, and putative splicing targets of the former, also differed between tissues. Thus, we speculate that a robust increase in OMR gene expression normally occurs with age in the PFC, but not in the hippocampus, which may explain why decreases in OMR gene expression in schizophrenia are more pronounced in the latter tissue. We also suggest that OMR transcripts might be processed by different splicing proteins in different tissues.

Cell type and culture condition-dependent alternative splicing in human breast cancer cells revealed by splicing-sensitive microarrays.

Growing evidence indicates that alternative or aberrant pre-mRNA splicing takes place during the development, progression, and metastasis of breast cancer. However, which splicing changes that might contribute directly to tumorigenesis or cancer progression remain to be elucidated. We used splicing-sensitive microarrays to detect differences in alternative splicing between two breast cancer cell lines, MCF7 (estrogen receptor positive) and MDA-MB-231 (estrogen receptor negative), as well as cultured human mammary epithelial cells. Several splicing alterations in genes, including CD44, FAS, RBM9, hnRNPA/B, APLP2, and MYL6, were detected by the microarray and verified by reverse transcription-PCR. We also compared splicing in these breast cancer cells cultured in either two-dimensional flat dishes or in three-dimensional Matrigel conditions. Only a subset of the splicing differences that distinguish MCF7 cells from MDA-MB-231 cells under two-dimensional culture condition is retained under three-dimensional conditions, suggesting that alternative splicing events are influenced by the geometry of the culture conditions of these cells. Further characterization of splicing patterns of several genes in MCF7 cells grown in Matrigel and in xenograft in nude mice shows that splicing is similar under both conditions. Thus, our oligonucleotide microarray can effectively detect changes in alternative splicing in different cells or in the same cells grown in different environments. Our findings also illustrate the potential for understanding gene expression with resolution of alternative splicing in the study of breast cancer.

Analysis of a splice array experiment elucidates roles of chromatin elongation factor Spt4-5 in splicing.

Splicing is an important process for regulation of gene expression in eukaryotes, and it has important functional links to other steps of gene expression. Two examples of these linkages include Ceg1, a component of the mRNA capping enzyme, and the chromatin elongation factors Spt4-5, both of which have recently been shown to play a role in the normal splicing of several genes in the yeast Saccharomyces cerevisiae. Using a genomic approach to characterize the roles of Spt4-5 in splicing, we used splicing-sensitive DNA microarrays to identify specific sets of genes that are mis-spliced in ceg1, spt4, and spt5 mutants. In the context of a complex, nested, experimental design featuring 22 dye-swap array hybridizations, comprising both biological and technical replicates, we applied five appropriate statistical models for assessing differential expression between wild-type and the mutants. To refine selection of differential expression genes, we then used a robust model-synthesizing approach, Differential Expression via Distance Synthesis, to integrate all five models. The resultant list of differentially expressed genes was then further analyzed with regard to select attributes: we found that highly transcribed genes with long introns were most sensitive to spt mutations. QPCR confirmation of differential expression was established for the limited number of genes evaluated. In this paper, we showcase splicing array technology, as well as powerful, yet general, statistical methodology for assessing differential expression, in the context of a real, complex experimental design. Our results suggest that the Spt4-Spt5 complex may help coordinate splicing with transcription under conditions that present kinetic challenges to spliceosome assembly or function.

Distinct and shared functions of ALS-associated proteins TDP-43, FUS and TAF15 revealed by multisystem analyses.

The RNA-binding protein (RBP) TAF15 is implicated in amyotrophic lateral sclerosis (ALS). To compare TAF15 function to that of two ALS-associated RBPs, FUS and TDP-43, we integrate CLIP-seq and RNA Bind-N-Seq technologies, and show that TAF15 binds to ∼4,900 RNAs enriched for GGUA motifs in adult mouse brains. TAF15 and FUS exhibit similar binding patterns in introns, are enriched in 3' untranslated regions and alter genes distinct from TDP-43. However, unlike FUS and TDP-43, TAF15 has a minimal role in alternative splicing. In human neural progenitors, TAF15 and FUS affect turnover of their RNA targets. In human stem cell-derived motor neurons, the RNA profile associated with concomitant loss of both TAF15 and FUS resembles that observed in the presence of the ALS-associated mutation FUS R521G, but contrasts with late-stage sporadic ALS patients. Taken together, our findings reveal convergent and divergent roles for FUS, TAF15 and TDP-43 in RNA metabolism.

Quaking promotes monocyte differentiation into pro-atherogenic macrophages by controlling pre-mRNA splicing and gene expression.

A hallmark of inflammatory diseases is the excessive recruitment and influx of monocytes to sites of tissue damage and their ensuing differentiation into macrophages. Numerous stimuli are known to induce transcriptional changes associated with macrophage phenotype, but posttranscriptional control of human macrophage differentiation is less well understood. Here we show that expression levels of the RNA-binding protein Quaking (QKI) are low in monocytes and early human atherosclerotic lesions, but are abundant in macrophages of advanced plaques. Depletion of QKI protein impairs monocyte adhesion, migration, differentiation into macrophages and foam cell formation in vitro and in vivo. RNA-seq and microarray analysis of human monocyte and macrophage transcriptomes, including those of a unique QKI haploinsufficient patient, reveal striking changes in QKI-dependent messenger RNA levels and splicing of RNA transcripts. The biological importance of these transcripts and requirement for QKI during differentiation illustrates a central role for QKI in posttranscriptionally guiding macrophage identity and function.