Nudt21 Controls Cell Fate by Connecting Alternative Polyadenylation to Chromatin Signaling.

Cell fate transitions involve rapid gene expression changes and global chromatin remodeling, yet the underlying regulatory pathways remain incompletely understood. Here, we identified the RNA-processing factor Nudt21 as a novel regulator of cell fate change using transcription-factor-induced reprogramming as a screening assay. Suppression of Nudt21 enhanced the generation of induced pluripotent stem cells, facilitated transdifferentiation into trophoblast stem cells, and impaired differentiation of myeloid precursors and embryonic stem cells, suggesting a broader role for Nudt21 in cell fate change. We show that Nudt21 directs differential polyadenylation of over 1,500 transcripts in cells acquiring pluripotency, although only a fraction changed protein levels. Remarkably, these proteins were strongly enriched for chromatin regulators, and their suppression neutralized the effect of Nudt21 during reprogramming. Collectively, our data uncover Nudt21 as a novel post-transcriptional regulator of cell fate and establish a direct, previously unappreciated link between alternative polyadenylation and chromatin signaling.

Molecular Mechanisms for CFIm-Mediated Regulation of mRNA Alternative Polyadenylation.

Alternative mRNA processing is a critical mechanism for proteome expansion and gene regulation in higher eukaryotes. The SR family proteins play important roles in splicing regulation. Intriguingly, mammalian genomes encode many poorly characterized SR-like proteins, including subunits of the mRNA 3'-processing factor CFIm, CFIm68 and CFIm59. Here we demonstrate that CFIm functions as an enhancer-dependent activator of mRNA 3' processing. CFIm regulates global alternative polyadenylation (APA) by specifically binding and activating enhancer-containing poly(A) sites (PASs). Importantly, the CFIm activator functions are mediated by the arginine-serine repeat (RS) domains of CFIm68/59, which bind specifically to an RS-like region in the CPSF subunit Fip1, and this interaction is inhibited by CFIm68/59 hyper-phosphorylation. The remarkable functional similarities between CFIm and SR proteins suggest that interactions between RS-like domains in regulatory and core factors may provide a common activation mechanism for mRNA 3' processing, splicing, and potentially other steps in RNA metabolism.

Mechanism and consequences of herpes simplex virus 1-mediated regulation of host mRNA alternative polyadenylation.

Eukaryotic gene expression is extensively regulated by cellular stress and pathogen infections. We have previously shown that herpes simplex virus 1 (HSV-1) and several cellular stresses cause widespread disruption of transcription termination (DoTT) of RNA polymerase II (RNAPII) in host genes and that the viral immediate early factor ICP27 plays an important role in HSV-1-induced DoTT. Here, we show that HSV-1 infection also leads to widespread changes in alternative polyadenylation (APA) of host mRNAs. In the majority of cases, polyadenylation shifts to upstream poly(A) sites (PAS), including many intronic PAS. Mechanistically, ICP27 contributes to HSV-1-mediated APA regulation. HSV-1- and ICP27-induced activation of intronic PAS is sequence-dependent and does not involve general inhibition of U1 snRNP. HSV1-induced intronic polyadenylation is accompanied by early termination of RNAPII. HSV-1-induced mRNAs polyadenylated at intronic PAS (IPA) are exported into the cytoplasm while APA isoforms with extended 3' UTRs are sequestered in the nuclei, both preventing the expression of the full-length gene products. Finally we provide evidence that HSV-induced IPA isoforms are translated. Together with other recent studies, our results suggest that viral infection and cellular stresses induce a multi-faceted host response that includes DoTT and changes in APA profiles.

Diverse functional elements in RNA predicted transcriptome-wide by orthogonal RNA structure probing.

RNA molecules can fold into complex structures and interact with trans-acting factors to control their biology. Recent methods have been focused on developing novel tools to measure RNA structure transcriptome-wide, but their utility to study and predict RNA-protein interactions or RNA processing has been limited thus far. Here, we extend these studies with the first transcriptome-wide mapping method for cataloging RNA solvent accessibility, icLASER. By combining solvent accessibility (icLASER) with RNA flexibility (icSHAPE) data, we efficiently predict RNA-protein interactions transcriptome-wide and catalog RNA polyadenylation sites by RNA structure alone. These studies showcase the power of designing novel chemical approaches to studying RNA biology. Further, our study exemplifies merging complementary methods to measure RNA structure inside cells and its utility for predicting transcriptome-wide interactions that are critical for control of and regulation by RNA structure. We envision such approaches can be applied to studying different cell types or cells under varying conditions, using RNA structure and footprinting to characterize cellular interactions and processing involving RNA.

A conserved intronic U1 snRNP-binding sequence promotes trans-splicing in Drosophila.

Unlike typical cis-splicing, trans-splicing joins exons from two separate transcripts to produce chimeric mRNA and has been detected in most eukaryotes. Trans-splicing in trypanosomes and nematodes has been characterized as a spliced leader RNA-facilitated reaction; in contrast, its mechanism in higher eukaryotes remains unclear. Here we investigate mod(mdg4), a classic trans-spliced gene in Drosophila, and report that two critical RNA sequences in the middle of the last 5' intron, TSA and TSB, promote trans-splicing of mod(mdg4). In TSA, a 13-nucleotide (nt) core motif is conserved across Drosophila species and is essential and sufficient for trans-splicing, which binds U1 small nuclear RNP (snRNP) through strong base-pairing with U1 snRNA. In TSB, a conserved secondary structure acts as an enhancer. Deletions of TSA and TSB using the CRISPR/Cas9 system result in developmental defects in flies. Although it is not clear how the 5' intron finds the 3' introns, compensatory changes in U1 snRNA rescue trans-splicing of TSA mutants, demonstrating that U1 recruitment is critical to promote trans-splicing in vivo. Furthermore, TSA core-like motifs are found in many other trans-spliced Drosophila genes, including lola. These findings represent a novel mechanism of trans-splicing, in which RNA motifs in the 5' intron are sufficient to bring separate transcripts into close proximity to promote trans-splicing.

Splicing proofreading at 5' splice sites by ATPase Prp28p.

Fidelity and efficiency of pre-mRNA splicing are critical for generating functional mRNAs, but how such accuracy in 5' splice site (SS) selection is attained is not fully clear. Through a series of yeast genetic screens, we isolated alleles of prp28 that improve splicing of suboptimal 5'SS substrates, demonstrating that WT-Prp28p proofreads, and consequently rejects, poor 5'SS. Prp28p is thought to facilitate the disruption of 5'SS-U1 snRNA pairing to allow for 5'SS-U6 snRNA pairing in the catalytic spliceosome; unexpectedly, 5'SS proofreading by Prp28p is dependent on competition with the stability of the 5'SS:U6 duplex, but not the 5'SS:U1 duplex. E404K, the strongest prp28 allele containing a mutation located in the linker region between adenosine triphosphatase (ATPase) subdomains, exhibited lower RNA-binding activity and enhanced splicing of suboptimal substrates before first-step catalysis, suggesting that decreased Prp28p activity allows longer time for suboptimal 5'SS substrates to pair with U6 snRNA and thereby reduces splicing fidelity. Residue E404 is critical for providing high splicing activity, demonstrated here in both yeast and Drosophila cells. Thus, the subdomain linker in Prp28p plays important roles both in splicing efficiency across species and in proofreading of 5'SS.

Alternative splicing and trans-splicing events revealed by analysis of the Bombyx mori transcriptome.

Alternative splicing and trans-splicing events have not been systematically studied in the silkworm Bombyx mori. Here, the silkworm transcriptome was analyzed by RNA-seq. We identified 320 novel genes, modified 1140 gene models, and found thousands of alternative splicing and 58 trans-splicing events. Studies of three SR proteins show that both their alternative splicing patterns and mRNA products are conserved from insect to human, and one isoform of Srsf6 with a retained intron is expressed sex-specifically in silkworm gonads. Trans-splicing of mod(mdg4) in silkworm was experimentally confirmed. We identified integrations from a common 5'-gene with 46 newly identified alternative 3'-exons that are located on both DNA strands over a 500-kb region. Other trans-splicing events in B. mori were predicted by bioinformatic analysis, in which 12 events were confirmed by RT-PCR, six events were further validated by chimeric SNPs, and two events were confirmed by allele-specific RT-PCR in F(1) hybrids from distinct silkworm lines of JS and L10, indicating that trans-splicing is more widespread in insects than previously thought. Analysis of the B. mori transcriptome by RNA-seq provides valuable information of regulatory alternative splicing events. The conservation of splicing events across species and newly identified trans-splicing events suggest that B. mori is a good model for future studies.

Air-ground coordinated unmanned swarm systems: A multitasking framework for control design.

An air-ground heterogeneous unmanned swarm system coordination is considered. The system consists of N unmanned aerial vehicles (UAVs) and one unmanned ground vehicle (UGV). This forms a complicated mission, which consists of the following four different tasks. First, the aerial vehicles are in a compact formation, while avoiding collision with each other. Second, the aerial vehicles should stay close to the ground, while avoiding collision with the ground. Third, the aerial vehicles should stay close to the ground vehicle. Fourth, the ground vehicle should follow a desired trajectory. These tasks reflect two seemingly contradictory nature: close to (due to tracking) and away from (due to avoidance). The effective control design should address all four tasks even in the presence of uncertainty. By two creative transformations, this multitude of tasks are consolidated in a χ-measure. An adaptive robust control, which includes a robust control scheme and an online adaptation law, is then proposed to render guarantee boundedness performance of this χ-measure. As a result, the control design is able to accomplish the combined tracking-avoidance mission for the uncertain swarm system. Despite the presence of conflicting aspects between these tasks, the designed controller exhibits outstanding performance.

Satellite Formation-Containment Control Emphasis on Collision Avoidance and Uncertainty Suppression.

This article explores an adaptive robust control scheme for satellite formation-containment flying in a way of constraint following. For safe flight and uncertainty suppression, both collision avoidance and uncertainty suppression are addressed. First, for uncertainty suppression, (possibly fast) time-varying but bounded uncertainty is considered, and then an adaptive law is proposed to estimate the comprehensive uncertainty bounds online. Second, the problem of formation-containment control with collision avoidance is converted into another problem of constraint-following control by taking the objectives of collision avoidance, formation, and containment, respectively, as the collision-avoidance constraint, formation constraint, and containment constraint. Third, an η -measure is introduced to gauge the constraint-following error, and then an adaptive robust control is proposed to render the error to be uniformly bounded and uniformly ultimately bounded, regardless of the uncertainty. By this, the satellites can follow the above collision-avoidance constraint, formation constraint, and containment constraint approximately. As a result, satellite formation-containment control emphasis on collision avoidance and uncertainty suppression is achieved.

Adaptive Robust Formation Control of Connected and Autonomous Vehicle Swarm System Based on Constraint Following.

This article proposes an adaptive robust formation control scheme for the connected and autonomous vehicle (CAV) swarm system by utilizing swarm property, diffeomorphism transformation, and constraint following. The control design is processed by starting from a 2-D dynamics model with (possibly fast) time varying but bounded uncertainty. The uncertainty bounds are unknown. For compact formation, the CAV system is treated as an artificial swarm system, for which the ideal swarm performance is taken as a desired constraint. By this, formation control is converted into a problem of constraint following and then a performance measure ß is defined as the control object to evaluate the constraint following error. For collision avoidance, a diffeomorphism transformation on space measure between two vehicles is creatively performed, by which the space measure is positive restricted. For uncertainty handling, an adaptive robust control scheme is proposed to render the ß -measure to be uniformly bounded and uniformly ultimately bounded, that is, drive the controlled (CAV) swarm system to follow the desired constraint approximatively. As a result, the system can achieve the ideal swarm performance; thereout, compact formation is realized, regardless of the uncertainty. The main contribution of this article is exploring a 2-D formation control scheme for (CAV) swarm system under the consideration of collision avoidance and time-varying uncertainty.

The anti-cancer compound JTE-607 reveals hidden sequence specificity of the mRNA 3' processing machinery.

JTE-607 is a small molecule compound with anti-inflammation and anti-cancer activities. Upon entering the cell, it is hydrolyzed to Compound 2, which directly binds to and inhibits CPSF73, the endonuclease for the cleavage step in pre-mRNA 3' processing. Although CPSF73 is universally required for mRNA 3' end formation, we have unexpectedly found that Compound 2- mediated inhibition of pre-mRNA 3' processing is sequence-specific and that the sequences flanking the cleavage site (CS) are a major determinant for drug sensitivity. By using massively parallel in vitro assays, we have measured the Compound 2 sensitivities of over 260,000 sequence variants and identified key sequence features that determine drug sensitivity. A machine learning model trained on these data can predict the impact of JTE-607 on poly(A) site (PAS) selection and transcription termination genome-wide. We propose a biochemical model in which CPSF73 and other mRNA 3' processing factors bind to RNA of the CS region in a sequence-specific manner and the affinity of such interaction determines the Compound 2 sensitivity of a PAS. As the Compound 2-resistant CS sequences, characterized by U/A-rich motifs, are prevalent in PASs from yeast to human, the CS region sequence may have more fundamental functions beyond determining drug resistance. Together, our study not only characterized the mechanism of action of a compound with clinical implications, but also revealed a previously unknown and evolutionarily conserved sequence-specificity of the mRNA 3' processing machinery.

The anticancer compound JTE-607 reveals hidden sequence specificity of the mRNA 3' processing machinery.

JTE-607 is an anticancer and anti-inflammatory compound and its active form, compound 2, directly binds to and inhibits CPSF73, the endonuclease for the cleavage step in pre-messenger RNA (pre-mRNA) 3' processing. Surprisingly, compound 2-mediated inhibition of pre-mRNA cleavage is sequence specific and the drug sensitivity is predominantly determined by sequences flanking the cleavage site (CS). Using massively parallel in vitro assays, we identified key sequence features that determine drug sensitivity. We trained a machine learning model that can predict poly(A) site (PAS) relative sensitivity to compound 2 and provide the molecular basis for understanding the impact of JTE-607 on PAS selection and transcription termination genome wide. We propose that CPSF73 and associated factors bind to the CS region in a sequence-dependent manner and the interaction affinity determines compound 2 sensitivity. These results have not only elucidated the mechanism of action of JTE-607, but also unveiled an evolutionarily conserved sequence specificity of the mRNA 3' processing machinery.

Regulating Constraint-Following Bound for Fuzzy Mechanical Systems: Indirect Robust Control and Fuzzy Optimal Design.

This article proposes an optimal indirect approach of constraint-following control for fuzzy mechanical systems. The system contains (possibly fast) time-varying uncertainty that lies in a fuzzy set. It aims at an optimal controller for the system to render bounded constraint-following error such that it can stay within a predetermined bound at all time and be sufficiently small eventually. First, for deterministic performance, the original system is transformed into a constructed system. A deterministic (not the usual IF-THEN rules-based) robust control is then designed for the constructed system to render it to be uniformly bounded and uniformly ultimately bounded, regardless of the uncertainty. Second, for optimal performance, a performance index, including the average fuzzy system performance and control effort, is proposed based on the fuzzy information. An optimal design problem associated with the control gain is then formulated and solved by minimizing the performance index. Finally, it is proved when the constructed system renders uniform boundedness and uniform ultimate boundedness, the original system achieves the desired performance of bounded constraint following.

Robust Pointing Control of Marching Tank Gun With Matched and Mismatched Uncertainty.

This article focuses on a robust control scheme for pointing control of the marching tank gun. Both matched and mismatched uncertainties, which may be nonlinear (possibly fast) time varying but bounded, are considered. First, the pointing control system is constructed as a coupled, nonlinear, and uncertain dynamical system with two interconnected (horizontal and vertical) subsystems. Second, for the horizontal pointing control, robust control is proposed to render the horizontal subsystem to be practically stable. Third, for the vertical pointing control, an uncertainty bound-based state transformation is constructed in a similar way of backstepping to convert the original mismatched system (i.e., the vertical subsystem) to be locally matched and then robust control is proposed to render the transformed system to be practically stable. Finally, it is proved that when the transformed system is rendered to be practically stable, the original system renders the same performance; therefore, vertical pointing control is achieved. This work should be among the first ever endeavor to cast all the coupling, nonlinearity, and (both matched and mismatched) uncertainty into the pointing control framework of the marching tank gun.

Gene expression profiling of resistant and susceptible Bombyx mori strains reveals nucleopolyhedrovirus-associated variations in host gene transcript levels.

We investigated variations in the gene expression of Bombyx mori following infection with a nucleopolyhedrovirus (BmNPV). Two B. mori strains, KN and 306, which are highly resistant and susceptible to BmNPV infection, respectively, were used in this study. The infection profiles of BmNPV in the B. mori KN and 306 larvae revealed that the virus invaded the midguts of both these strains. However, its proliferation was notably inhibited in the midgut of the resistant strain. By using the suppression subtractive hybridization method, two cDNA libraries were constructed in order to compare the BmNPV responsive gene expressions between the two silkworm lines. In total, 62 differentially expressed genes were obtained. Real-time qPCR analysis confirmed that eight genes were significantly up-regulated in the midgut of the KN strain following BmNPV infection. Our results imply that these up-regulated genes may be involved in the B. mori immune response against BmNPV infection.

[Mapping of non-lepis wing gene nlw in silkworm (Bombyx mori) using SSR and STS markers].

The non-lepis wing of silkworm (Bombyx mori) is controlled by the recessive gene, nlw. Owning to lack of crossing over in females, the reciprocal backcrossed F(1) (BC(1)) progenies were used for linkage analysis and mapping of nlw based on the SSR linkage map and STS markers using the wild type (+(nlw)/+(nlw)) silkworm strain P50 and U06 with scaleless wing (nlw/nlw). The nlw gene was linked to eight SSR markers and one STS marker. All the individuals with the wild type in the BC1F (Using F(1) as female to backcross to the recessive parent, that is (U06xP50)xU06) showed heterozygous profile of (U06xP50) F(1), and the ones with non-lepis wing in BC1F exhibited the homozygous profile of the strain U06. Using a reciprocal BC1M (Using F1 as male to backcross to the recessive parent, that is U06x(U06xP50))cross, we constructed a linkage map of 125.6 cM, and the distance between nlw and the nearest marker cash2p was 11.4 cM.

Herpes simplex virus blocks host transcription termination via the bimodal activities of ICP27.

Infection by viruses, including herpes simplex virus-1 (HSV-1), and cellular stresses cause widespread disruption of transcription termination (DoTT) of RNA polymerase II (RNAPII) in host genes. However, the underlying mechanisms remain unclear. Here, we demonstrate that the HSV-1 immediate early protein ICP27 induces DoTT by directly binding to the essential mRNA 3' processing factor CPSF. It thereby induces the assembly of a dead-end 3' processing complex, blocking mRNA 3' cleavage. Remarkably, ICP27 also acts as a sequence-dependent activator of mRNA 3' processing for viral and a subset of host transcripts. Our results unravel a bimodal activity of ICP27 that plays a key role in HSV-1-induced host shutoff and identify CPSF as an important factor that mediates regulation of transcription termination. These findings have broad implications for understanding the regulation of transcription termination by other viruses, cellular stress and cancer.

Purification of mRNA Processing Complexes Using an RNA Affinity Approach.

Multiple mRNA processing steps, including splicing and 3' processing, take place in macromolecular complexes that contain many proteins and sometimes RNA molecules. A key challenge in the mRNA processing field has been to define the structure-function relationship of these sophisticated molecular machines. A prerequisite for addressing this challenge is to develop tools for purifying mRNA processing complexes in their native and intact forms that are suitable for functional and structural studies. Among many methods that have been developed, RNA affinity-based methods are most widely applied. In these methods, RNA molecules that are substrates to mRNA processing machineries are fused with an affinity tag, incubated with cellular extracts/lysates to allow for the assembly of mRNA processing complexes, and finally the assembled complexes are purified using RNA affinity tag. In this chapter, we will overview RNA affinity-based purification methods and describe in detail one such method, MS2-tagging, and its application in the purification of mRNA 3' processing complexes. Although these methods were originally developed for purifying mRNA processing complexes, they should be applicable to purification of other RNA-protein complexes as well.