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Massively parallel reporter assays (MPRAs) are powerful tools for quantifying the impacts of sequence variation on gene expression. Reading out molecular phenotypes with sequencing enables interrogating the impact of sequence variation beyond genome scale. Machine learning models integrate and codify information learned from MPRAs and enable generalization by predicting sequences outside the training data set. Models can provide a quantitative understanding of cis-regulatory codes controlling gene expression, enable variant stratification, and guide the design of synthetic regulatory elements for applications from synthetic biology to mRNA and gene therapy. This review focuses on cis-regulatory MPRAs, particularly those that interrogate cotranscriptional and post-transcriptional processes: alternative splicing, cleavage and polyadenylation, translation, and mRNA decay.

Endogenous retroviruses (ERVs) are remnants of ancient parasitic infections and comprise sizable portions of most genomes. Although epigenetic mechanisms silence most ERVs by generating a repressive environment that prevents their expression (heterochromatin), little is known about mechanisms silencing ERVs residing in open regions of the genome (euchromatin). This is particularly important during embryonic development, where induction and repression of distinct classes of ERVs occur in short temporal windows. Here, we demonstrate that transcription-associated RNA degradation by the nuclear RNA exosome and Integrator is a regulatory mechanism that controls the productive transcription of most genes and many ERVs involved in preimplantation development. Disrupting nuclear RNA catabolism promotes dedifferentiation to a totipotent-like state characterized by defects in RNAPII elongation and decreased expression of long genes (gene-length asymmetry). Our results indicate that RNA catabolism is a core regulatory module of gene networks that safeguards RNAPII activity, ERV expression, cell identity, and developmental potency.

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.

RNA-binding motif 8A (RBM8A) is a core component of the exon junction complex (EJC) that binds pre-mRNAs and regulates their splicing, transport, translation, and nonsense-mediated decay (NMD). Dysfunction in the core proteins has been linked to several detriments in brain development and neuropsychiatric diseases. To understand the functional role of Rbm8a in brain development, we have generated brain-specific Rbm8a knockout mice and used next-generation RNA-sequencing to identify differentially expressed genes (DEGs) in mice with heterozygous, conditional knockout (cKO) of Rbm8a in the brain at postnatal day 17 (P17) and at embryonic day 12. Additionally, we analyzed enriched gene clusters and signaling pathways within the DEGs. At the P17 time point, between the control and cKO mice, about 251 significant DEGs were identified. At E12, only 25 DEGs were identified in the hindbrain samples. Bioinformatics analyses have revealed many signaling pathways related to the central nervous system (CNS). When E12 and P17 results were compared, three DEGs, Spp1, Gpnmb, and Top2a, appeared to peak at different developmental time points in the Rbm8a cKO mice. Enrichment analyses suggested altered activity in pathways affecting cellular proliferation, differentiation, and survival. The results support the hypothesis that loss of Rbm8a causes decreased cellular proliferation, increased apoptosis, and early differentiation of neuronal subtypes, which may lead ultimately to an altered neuronal subtype composition in the brain.

It is every biochemist's dream to reconstitute a biological process in vitro using defined components, because doing so not only reduces a biological phenomenon to one or a series of biochemical reactions, but also defines the minimal list of essential components. In this issue of Genes & Development, Boreikaite and colleagues (pp. 210-224) and Schmidt and colleagues (pp. 195-209) report their independent reconstitution of human pre-mRNA 3' end processing.

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.

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.

Alternative polyadenylation (APA) is a widespread phenomenon in eukaryotes that contributes to regulating gene expression and generating proteomic diversity. APA plays critical roles in development and its mis-regulation has been implicated in a wide variety of human diseases, including cancer. To study APA on the transcriptome-wide level, numerous deep sequencing methods that capture 3' end of mRNAs have been developed in the past decade, but they generally require a large amount of hands-on time and/or high RNA input. Here, we introduce PAS-seq 2, a fast and sensitive method for global and quantitative profiling of polyadenylated RNAs. Compared to our original PAS-seq, this method takes less time and requires much lower total RNA input due to improvement in the reverse transcription process. PAS-seq 2 can be applied to both APA and differential gene expression analyses.

The majority of eukaryotic genes produce multiple mRNA isoforms by using alternative poly(A) sites in a process called alternative polyadenylation (APA). APA is a dynamic process that is highly regulated in development and in response to extrinsic or intrinsic stimuli. Mis-regulation of APA has been linked to a wide variety of diseases, including cancer, neurological and immunological disorders. Since the first example of APA was described 40 years ago, the regulatory mechanisms of APA have been actively investigated. Conventionally, research in this area has focused primarily on the roles of regulatory cis-elements and trans-acting RNA-binding proteins. Recent studies, however, have revealed important functions for epigenetic mechanisms, including DNA and histone modifications and higher-order chromatin structures, in APA regulation. Here we will discuss these recent findings and their implications for our understanding of the crosstalk between epigenetics and mRNA 3'-end processing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Intestinal stem cells are non-quiescent, dividing epithelial cells that rapidly differentiate into progenitor cells of the absorptive and secretory cell lineages. The kinetics of this process is rapid such that the epithelium is replaced weekly. To determine how the transcriptome and proteome keep pace with rapid differentiation, we developed a new cell sorting method to purify mouse colon epithelial cells. Here we show that alternative mRNA splicing and polyadenylation dominate changes in the transcriptome as stem cells differentiate into progenitors. In contrast, as progenitors differentiate into mature cell types, changes in mRNA levels dominate the transcriptome. RNA processing targets regulators of cell cycle, RNA, cell adhesion, SUMOylation, and Wnt and Notch signaling. Additionally, global proteome profiling detected >2,800 proteins and revealed RNA:protein patterns of abundance and correlation. Paired together, these data highlight new potentials for autocrine and feedback regulation and provide new insights into cell state transitions in the crypt.

The mammalian pre-mRNA 3'-end-processing machinery consists of cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), and other proteins, but the overall architecture of this machinery remains unclear. CPSF contains two functionally distinct modules: a cleavage factor (mCF) and a polyadenylation specificity factor (mPSF). Here, we have produced recombinant human CPSF and CstF and examined these factors by electron microscopy (EM). We find that mPSF is the organizational core of the machinery, while the conformations of mCF and CstF and the position of mCF relative to mPSF are highly variable. We have identified by cryo-EM a segment in CPSF100 that tethers mCF to mPSF, and we have named it the PSF interaction motif (PIM). Mutations in the PIM can abolish CPSF formation, indicating that it is a crucial contact in CPSF. We have also obtained reconstructions of mCF and CstF77 by cryo-EM, assembled around the mPSF core.

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.

3'-untranslated regions (UTRs) can vary through the use of alternative polyadenylation sites during pre-mRNA processing. Multiple publically available pipelines combining high profiling technologies and bioinformatics tools have been developed to catalog changes in 3'-UTR lengths. In our recent RNA-seq experiments using cells with hyper-activated mammalian target of rapamycin (mTOR), we found that cellular mTOR activation leads to transcriptome-wide alternative polyadenylation (APA), resulting in the activation of multiple cellular pathways. Here, we developed a novel bioinformatics algorithm, IntMAP, which integrates RNA-Seq and PolyA Site (PAS)-Seq data for a comprehensive characterization of APA events. By applying IntMAP to the datasets from cells with hyper-activated mTOR, we identified novel APA events that could otherwise not be identified by either profiling method alone. Several transcription factors including Cebpg (CCAAT/enhancer binding protein gamma) were among the newly discovered APA transcripts, indicating that diverse transcriptional networks may be regulated by mTOR-coordinated APA. The prevention of APA in Cebpg using the CRISPR/cas9-mediated genome editing tool showed that mTOR-driven 3'-UTR shortening in Cebpg is critical in protecting cells from endoplasmic reticulum (ER) stress. Taken together, we present IntMAP as a new bioinformatics algorithm for APA analysis by which we expand our understanding of the physiological role of mTOR-coordinated APA events to ER stress response. IntMAP toolbox is available at http://compbio.cs.umn.edu/IntMAP/.

Nearly all eukaryotic messenger RNA precursors must undergo cleavage and polyadenylation at their 3-end for maturation. A crucial step in this process is the recognition of the AAUAAA polyadenylation signal (PAS), and the molecular mechanism of this recognition has been a long-standing problem. Here, we report the cryo-electron microscopy structure of a quaternary complex of human CPSF-160, WDR33, CPSF-30, and an AAUAAA RNA at 3.4-Å resolution. Strikingly, the AAUAAA PAS assumes an unusual conformation that allows this short motif to be bound directly by both CPSF-30 and WDR33. The A1 and A2 bases are recognized specifically by zinc finger 2 (ZF2) of CPSF-30 and the A4 and A5 bases by ZF3. Interestingly, the U3 and A6 bases form an intramolecular Hoogsteen base pair and directly contact WDR33. CPSF-160 functions as an essential scaffold and preorganizes CPSF-30 and WDR33 for high-affinity binding to AAUAAA. Our findings provide an elegant molecular explanation for how PAS sequences are recognized for mRNA 3-end formation.

(Cell 172, 106–120; January 11, 2018) Due to a production error, the original publication of this article included typographic errors in Figure 1. In that figure, four instances of “THY-1” appeared as “HY-1”. These errors have been corrected in print and online, and we apologize for any inconvenience. [Figure presented] A Serial siRNA Screen Identifies Nudt21 as a Potent Barrier to Reprogramming (corrected) [Figure presented] A Serial siRNA Screen Identifies Nudt21 as a Potent Barrier to Reprogramming (original)

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.

mRNA 3' end processing is an essential step in gene expression. It is well established that canonical eukaryotic pre-mRNA 3' processing is carried out within a macromolecular machinery consisting of dozens of trans-acting proteins. However, it is unknown whether RNAs play any role in this process. Unexpectedly, we found that a subset of small nucleolar RNAs (snoRNAs) are associated with the mammalian mRNA 3' processing complex. These snoRNAs primarily interact with Fip1, a component of cleavage and polyadenylation specificity factor (CPSF). We have functionally characterized one of these snoRNAs and our results demonstrated that the U/A-rich SNORD50A inhibits mRNA 3' processing by blocking the Fip1-poly(A) site (PAS) interaction. Consistently, SNORD50A depletion altered the Fip1-RNA interaction landscape and changed the alternative polyadenylation (APA) profiles and/or transcript levels of a subset of genes. Taken together, our data revealed a novel function for snoRNAs and provided the first evidence that non-coding RNAs may play an important role in regulating mRNA 3' processing.

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.

Alternative polyadenylation has been implicated as an important regulator of gene expression. In some cases, alternative polyadenylation is known to couple with alternative splicing to influence last intron removal. However, it is unknown whether alternative polyadenylation events influence alternative splicing decisions at upstream exons. Knockdown of the polyadenylation factors CFIm25 or CstF64 in HeLa cells was used as an approach in identifying alternative polyadenylation and alternative splicing events on a genome-wide scale. Although hundreds of alternative splicing events were found to be differentially spliced in the knockdown of CstF64, genes associated with alternative polyadenylation did not exhibit an increased incidence of alternative splicing. These results demonstrate that the coupling between alternative polyadenylation and alternative splicing is usually limited to defining the last exon. The striking influence of CstF64 knockdown on alternative splicing can be explained through its effects on UTR selection of known splicing regulators such as hnRNP A2/B1, thereby indirectly influencing splice site selection. We conclude that changes in the expression of the polyadenylation factor CstF64 influences alternative splicing through indirect effects.

mRNA alternative polyadenylation (APA) is a critical mechanism for post-transcriptional gene regulation and is often regulated in a tissue- and/or developmental stage-specific manner. An ultimate goal for the APA field has been to be able to computationally predict APA profiles under different physiological or pathological conditions. As a first step toward this goal, we have assembled a poly(A) code for predicting tissue-specific poly(A) sites (PASs). Based on a compendium of over 600 features that have known or potential roles in PAS selection, we have generated and refined a machine-learning algorithm using multiple high-throughput sequencing-based data sets of tissue-specific and constitutive PASs. This code can predict tissue-specific PASs with >85% accuracy. Importantly, by analyzing the prediction performance based on different RNA features, we found that PAS context, including the distance between alternative PASs and the relative position of a PAS within the gene, is a key feature for determining the susceptibility of a PAS to tissue-specific regulation. Our poly(A) code provides a useful tool for not only predicting tissue-specific APA regulation, but also for studying its underlying molecular mechanisms.

Aiming at the problem of dynamic pressure induced by overburden fracture in the square area of the first face in western mining area, based on theoretical analysis, we studied the risk of dynamic pressure in the square area of faces of different widths by analyzing the probability of overburden fracture and the dynamic pressure. The results show that, the number of key strata differs in different buried mines, breaking interval of each key strata distributes randomly and uniformly. Each key layer has the possibility of breaking in the square area. When the width the first mining face are 100 m, 200 m and 300 m, the probabilities of strata breaking in the square area are 18.2%, 9.1% and 6.1%. With the expectation loss method, it is found that the risk of dynamic pressure in the square increases with face width increases. To strengthen the protections of dynamic pressure in the square area is needed, and the measures of adjusting face, strengthening support, and directional hydraulic crack in high strata are given.

Temperature difference and its duration are two main factors that affect thermoelectric performance. One can obtain the desired temperature distributions by manipulating heat flow directions; however, it is generally neglected when designing thermoelectric generators (TEGs). In this study, thermal rectifiers work in forward directions to produce in-plane temperature differences (ΔTh), where hot and cold zones are, respectively, provided by the small terminals of rectifiers and gaps between these areas. Thermoelectric legs placed above are arranged in an "X"-shape, keep TEGs' internal resistances, and have a stable range from 0.7 to 2 Ω; even heating temperatures Th have a significant range from 30 to 80 °C. When the rectification coefficient of thermal rectifiers was 1.63 and the thickness of thermoelectric legs decreased from 1 mm to 10 μm, simulated-ΔTh in the steady state rises from 2.62 to 27.10 °C rather than falling. An experimental thermal rectifier with a PI film thickness of 25 μm demonstrates that ΔTh can reach up to 14.7 °C, and the time duration is more than 60 s, where Th and ambient are 50 and 20 °C, respectively. The maximum output power can reach up to 92.48 μW when the temperature bias between Th and ambient increases to 65.33 °C. These novel thin-TEGs with designed in-plane temperature gradient zones by asymmetric thermal rectifiers are expected to be applied in distributed sensors, wearable devices, etc. [ABSTRACT FROM AUTHOR]