Macrophages respond to inflammatory stimuli by modulating the expression of hundreds of genes in a defined temporal cascade, with diverse transcriptional and posttranscriptional mechanisms ...contributing to the regulatory network. We examined proinflammatory gene regulation in activated macrophages by performing RNA-seq with fractionated chromatin-associated, nucleoplasmic, and cytoplasmic transcripts. This methodological approach allowed us to separate the synthesis of nascent transcripts from transcript processing and the accumulation of mature mRNAs. In addition to documenting the subcellular locations of coding and noncoding transcripts, the results provide a high-resolution view of the relationship between defined promoter and chromatin properties and the temporal regulation of diverse classes of coexpressed genes. The data also reveal a striking accumulation of full-length yet incompletely spliced transcripts in the chromatin fraction, suggesting that splicing often occurs after transcription has been completed, with transcripts retained on the chromatin until fully spliced.
Display omitted
► Coding and noncoding transcripts exhibit characteristic subcellular distributions ► The most potently induced genes favor promoters with low CpG content ► Full-length, incompletely spliced transcripts accumulate on the chromatin ► Delayed transcript release may reflect a requirement for the completion of splicing
Sequencing analysis of subcellular fractions allows the separation of synthesis of nascent transcripts from RNA processing and the accumulation of mature mRNAs, revealing a high-resolution view of transcript dynamics of proinflammatory genes. Surprisingly, full-length yet incompletely spliced transcripts accumulate on chromatin, suggesting that splicing often follows the completion of transcription.
The neurogenetics of alternative splicing Vuong, Celine K; Black, Douglas L; Zheng, Sika
Nature reviews. Neuroscience,
05/2016, Volume:
17, Issue:
5
Journal Article
Peer reviewed
Open access
Alternative precursor-mRNA splicing is a key mechanism for regulating gene expression in mammals and is controlled by specialized RNA-binding proteins. The misregulation of splicing is implicated in ...multiple neurological disorders. We describe recent mouse genetic studies of alternative splicing that reveal its critical role in both neuronal development and the function of mature neurons. We discuss the challenges in understanding the extensive genetic programmes controlled by proteins that regulate splicing, both during development and in the adult brain.
Nuclear compartments have diverse roles in regulating gene expression, yet the molecular forces and components that drive compartment formation remain largely unclear
. The long non-coding RNA Xist ...establishes an intra-chromosomal compartment by localizing at a high concentration in a territory spatially close to its transcription locus
and binding diverse proteins
to achieve X-chromosome inactivation (XCI)
. The XCI process therefore serves as a paradigm for understanding how RNA-mediated recruitment of various proteins induces a functional compartment. The properties of the inactive X (Xi)-compartment are known to change over time, because after initial Xist spreading and transcriptional shutoff a state is reached in which gene silencing remains stable even if Xist is turned off
. Here we show that the Xist RNA-binding proteins PTBP1
, MATR3
, TDP-43
and CELF1
assemble on the multivalent E-repeat element of Xist
and, via self-aggregation and heterotypic protein-protein interactions, form a condensate
in the Xi. This condensate is required for gene silencing and for the anchoring of Xist to the Xi territory, and can be sustained in the absence of Xist. Notably, these E-repeat-binding proteins become essential coincident with transition to the Xist-independent XCI phase
, indicating that the condensate seeded by the E-repeat underlies the developmental switch from Xist-dependence to Xist-independence. Taken together, our data show that Xist forms the Xi compartment by seeding a heteromeric condensate that consists of ubiquitous RNA-binding proteins, revealing an unanticipated mechanism for heritable gene silencing.
•We present an overview of an ongoing EV vehicle-to-grid demonstration project.•We discuss practical issues of bidding EV capacity in frequency regulation markets.•We formulate a MILP optimization ...model to plan EV charging and day-ahead bidding.•We analyze the model sensitivity and bidding strategy to variation of key inputs.•Model behavior is highly sensitive to predicted resource utilization and prices.
The Los Angeles Air Force Base Electric Vehicle Demonstration is a currently ongoing vehicle-to-grid demonstration project with the objective of minimizing the cost of operation of a fleet of approximately 30 electric vehicles (EVs) through participation in the California Independent System Operator (CAISO) frequency regulation market. To accomplish this, a hierarchical control system has been developed to optimize, plan, and control the charging, market bidding, and response to grid system operator control of the EVs. This paper presents an overview of the day-ahead optimization model component of the hierarchy. The model is a mixed integer linear program that optimizes daily EV charging and regulation capacity bids strategies in order to minimize operation costs and maximize ancillary service revenue. A deterministic approach is used due to several practical concerns of the demonstration project, including model complexity and the availability and uncertainty of input data in day-ahead decision making, and the limited size of the fleet. The model includes additional user-defined parameters to tune model behavior to better match real-world conditions and minimize the risks of uncertainty.
The paper conducts scenario analysis to explore the impact of these parameters on high level model behavior and resulting bid strategy. The parameters explored include hourly regulation prices, local load conditions leading to retail demand charges, forced symmetry constraints for regulation bids, SOC penalty values to reserve higher states-of-charge in vehicles, and expected regulation resource utilization while providing reserves. These analyses show significant sensitivity in the frequency regulation bidding strategy to the regulation utilization, as well as large differences in the regulation prices between regulation up (discharging capacity) and regulation down (charging capacity). Results also suggest enforcing symmetry in regulation appears to have significant impacts in regulation revenue when there is large relative disparities between prices in the up and down direction. Finally, imposing a small cost on low SOC values significantly impacts the fleet-wide average SOC, making the system more resilient to uncertainty in the mobility demands gathered at the time of making day ahead decisions.
Human genetic studies have identified the neuronal RNA binding protein, Rbfox1, as a candidate gene for autism spectrum disorders. While Rbfox1 functions as a splicing regulator in the nucleus, it is ...also alternatively spliced to produce cytoplasmic isoforms. To investigate the function of cytoplasmic Rbfox1, we knocked down Rbfox proteins in mouse neurons and rescued with cytoplasmic or nuclear Rbfox1. Transcriptome profiling showed that nuclear Rbfox1 rescued splicing changes, whereas cytoplasmic Rbfox1 rescued changes in mRNA levels. iCLIP-seq of subcellular fractions revealed that Rbfox1 bound predominantly to introns in nascent RNA, while cytoplasmic Rbox1 bound to 3ʹ UTRs. Cytoplasmic Rbfox1 binding increased target mRNA stability and translation, and Rbfox1 and miRNA binding sites overlapped significantly. Cytoplasmic Rbfox1 target mRNAs were enriched in genes involved in cortical development and autism. Our results uncover a new Rbfox1 regulatory network and highlight the importance of cytoplasmic RNA metabolism to cortical development and disease.
Display omitted
•Nuclear and cytoplasmic Rbfox1 isoforms regulate distinct neuronal mRNAs•Cytoplasmic Rbfox1 regulates the stability and translation of its target mRNAs•Rbfox1 and miRNA binding sites overlap significantly in target mRNA 3′ UTRs•Cytoplasmic Rbfox1 targets are enriched in cortical development and autism genes
Rbfox1 regulates the splicing of many exons in the nucleus of neurons. Lee at al. demonstrate that Rbfox1 also binds to the 3ʹ UTR of target mRNAs in the cytoplasm to upregulate the expression of synaptic and autism-related genes.
In metazoan organisms, pre-mRNA splicing is thought to occur during transcription, and it is postulated that these two processes are functionally coupled via still-unknown mechanisms. Current ...evidence supports co-transcriptional spliceosomal assembly, but there is little quantitative information on how much splicing is completed during RNA synthesis. Here we isolate nascent chromatin-associated RNA from free, nucleoplasmic RNA already released from the DNA template. Using a quantitative RT-PCR assay, we show that the majority of introns separating constitutive exons are already excised from the human c-Src and fibronectin pre-mRNAs that are still in the process of synthesis, and that these introns are removed in a general 5'-to-3' order. Introns flanking alternative exons in these transcripts are also removed during synthesis, but show differences in excision efficiency between cell lines with different regulatory conditions. Our data suggest that skipping of an exon can induce a lag in splicing compared to intron removal under conditions of exon inclusion. Nevertheless, excision of the long intron encompassing the skipped exon is still completed prior to transcript release into the nucleoplasm. Thus, we demonstrate that the decision to include or skip an alternative exon is made during transcription and not post-transcriptionally.
Highlights • Alternative splicing coupled to mRNA decay can control gene expression. • Choice of splicing pattern can also determine later mRNA utilization. • These aspects of splicing regulation ...profoundly affect neuronal cell biology.
Alternative pre-mRNA splicing is a central mode of genetic regulation in
higher eukaryotes. Variability in splicing patterns is a major source of
protein diversity from the genome. In this review, I ...describe what is currently
known of the molecular mechanisms that control changes in splice site choice. I
start with the best-characterized systems from the
Drosophila
sex
determination pathway, and then describe the regulators of other systems about
whose mechanisms there is some data. How these regulators are combined into
complex systems of tissue-specific splicing is discussed. In conclusion, very
recent studies are presented that point to new directions for understanding
alternative splicing and its mechanisms.
Rbfox proteins control alternative splicing and posttranscriptional regulation in mammalian brain and are implicated in neurological disease. These proteins recognize the RNA sequence (U)GCAUG, but ...their structures and diverse roles imply a variety of protein-protein interactions. We find that nuclear Rbfox proteins are bound within a large assembly of splicing regulators (LASR), a multimeric complex containing the proteins hnRNP M, hnRNP H, hnRNP C, Matrin3, NF110/NFAR-2, NF45, and DDX5, all approximately equimolar to Rbfox. We show that splicing repression mediated by hnRNP M is stimulated by Rbfox. Virtually all the intron-bound Rbfox is associated with LASR, and hnRNP M motifs are enriched adjacent to Rbfox crosslinking sites in vivo. These findings demonstrate that Rbfox proteins bind RNA with a defined set of cofactors and affect a broader set of exons than previously recognized. The function of this multimeric LASR complex has implications for deciphering the regulatory codes controlling splicing networks.
Display omitted
•Rbfox proteins fractionate with high-molecular-weight nuclear material•Nuclear Rbfox proteins bind RNA within a large assembly of splicing regulators (LASR)•Rbfox proteins can alter splicing via a binding site for LASR component hnRNP M•HnRNP M and other LASR-binding motifs are found adjacent to Rbfox binding sites
Rbfox family splicing factors function within a large complex of RNA-binding proteins that extends the influence of these regulators, capitalizing on the different binding specificities of the constituent proteins.
Alternative pre-mRNA splicing determines many changes in gene expression during development. Two regulators known to control splicing patterns during neuron and muscle differentiation are the ...polypyrimidine tract-binding protein (PTB) and its neuronal homolog nPTB. These proteins repress certain exons in early myoblasts, but upon differentiation of mature myotubes PTB/nPTB expression is reduced, leading to increased inclusion of their target exons. We show here that the repression of nPTB expression during myoblast differentiation results from its targeting by the muscle-restricted microRNA miR-133. During differentiation of C2C12 myoblasts, nPTB protein but not mRNA expression is strongly reduced, concurrent with the up-regulation of miR-133 and the induction of splicing for several PTB-repressed exons. Introduction of synthetic miR-133 into undifferentiated C2C12 cells leads to a decrease in endogenous nPTB expression. Both the miR-133 and the coexpressed miR-1/206 microRNAs are extremely conserved across animal species, and PTB proteins are predicted targets for these miRNAs in Drosophila, mice, and humans. There are two potential miR-133-responsive elements (MRE) within the nPTB 3' untranslated region (UTR), and a luciferase reporter carrying this 3' UTR is repressed by miR-133 in an MRE-dependent manner. Transfection of locked nucleic acid (LNA) oligonucleotides designed to block the function of miR-133 and miR-1/206 increases expression of nPTB and decreases the inclusion of PTB dependent exons. These results indicate that miR-133 directly down-regulates a key splicing factor during muscle development and establishes a role for microRNAs in the control of a developmentally dynamic splicing program.
PDF
