Adaptive translational pausing is a hallmark of the cellular response to severe environmental stress.

To survive, mammalian cells must adapt to environmental challenges. While the cellular response to mild stress has been widely studied, how cells respond to severe stress remains unclear. We show here that under severe hyperosmotic stress, cells enter a transient hibernation-like state in anticipation of recovery. We demonstrate this adaptive pausing response (APR) is a coordinated cellular response that limits ATP supply and consumption through mitochondrial fragmentation and widespread pausing of mRNA translation. This pausing is accomplished by ribosome stalling at translation initiation codons, which keeps mRNAs poised to resume translation upon recovery. We further show that recovery from severe stress involves ISR (integrated stress response) signaling that permits cell cycle progression, resumption of growth, and reversal of mitochondria fragmentation. Our findings indicate that cells can respond to severe stress via a hibernation-like mechanism that preserves vital elements of cellular function under harsh environmental conditions.

The kinetic landscape of an RNA-binding protein in cells.

Gene expression in higher eukaryotic cells orchestrates interactions between thousands of RNA-binding proteins (RBPs) and tens of thousands of RNAs1. The kinetics by which RBPs bind to and dissociate from their RNA sites are critical for the coordination of cellular RNA-protein interactions2. However, these kinetic parameters have not been experimentally measured in cells. Here we show that time-resolved RNA-protein cross-linking with a pulsed femtosecond ultraviolet laser, followed by immunoprecipitation and high-throughput sequencing, allows the determination of binding and dissociation kinetics of the RBP DAZL for thousands of individual RNA-binding sites in cells. This kinetic cross-linking and immunoprecipitation (KIN-CLIP) approach reveals that DAZL resides at individual binding sites for time periods of only seconds or shorter, whereas the binding sites remain DAZL-free for markedly longer. The data also indicate that DAZL binds to many RNAs in clusters of multiple proximal sites. The effect of DAZL on mRNA levels and ribosome association correlates with the cumulative probability of DAZL binding in these clusters. Integrating kinetic data with mRNA features quantitatively connects DAZL-RNA binding to DAZL function. Our results show how kinetic parameters for RNA-protein interactions can be measured in cells, and how these data link RBP-RNA binding to the cellular function of RBPs.

The helicase Ded1p controls use of near-cognate translation initiation codons in 5' UTRs.

The conserved and essential DEAD-box RNA helicase Ded1p from yeast and its mammalian orthologue DDX3 are critical for the initiation of translation1. Mutations in DDX3 are linked to tumorigenesis2-4 and intellectual disability5, and the enzyme is targeted by a range of viruses6. How Ded1p and its orthologues engage RNAs during the initiation of translation is unknown. Here we show, by integrating transcriptome-wide analyses of translation, RNA structure and Ded1p-RNA binding, that the effects of Ded1p on the initiation of translation are connected to near-cognate initiation codons in 5' untranslated regions. Ded1p associates with the translation pre-initiation complex at the mRNA entry channel and repressing the activity of Ded1p leads to the accumulation of RNA structure in 5' untranslated regions, the initiation of translation from near-cognate start codons immediately upstream of these structures and decreased protein synthesis from the corresponding main open reading frames. The data reveal a program for the regulation of translation that links Ded1p, the activation of near-cognate start codons and mRNA structure. This program has a role in meiosis, in which a marked decrease in the levels of Ded1p is accompanied by the activation of the alternative translation initiation sites that are seen when the activity of Ded1p is repressed. Our observations indicate that Ded1p affects translation initiation by controlling the use of near-cognate initiation codons that are proximal to mRNA structure in 5' untranslated regions.

Efficient GFP-labeling and analysis of spermatogenic cells using the IRG transgene and flow cytometry.

Spermatogenesis is a highly ordered developmental program that produces haploid male germ cells. The study of male germ cell development in the mouse has provided unique perspectives into the molecular mechanisms that control cell development and differentiation in mammals, including tissue-specific gene regulatory programs. An intrinsic challenge in spermatogenesis research is the heterogeneity of germ and somatic cell types present in the testis. Techniques to separate and isolate distinct mouse spermatogenic cell types have great potential to shed light on molecular mechanisms controlling mammalian cell development, while also providing new insights into cellular events important for human reproductive health. Here, we detail a versatile strategy that combines Cre-lox technology to fluorescently label germ cells, with flow cytometry to discriminate and isolate germ cells in different stages of development for cellular and molecular analyses.

Act1 drives chemoresistance via regulation of antioxidant RNA metabolism and redox homeostasis.

The IL-17 receptor adaptor molecule Act1, an RNA-binding protein, plays a critical role in IL-17-mediated cancer progression. Here, we report a novel mechanism of how IL-17/Act1 induces chemoresistance by modulating redox homeostasis through epitranscriptomic regulation of antioxidant RNA metabolism. Transcriptome-wide mapping of direct Act1-RNA interactions revealed that Act1 binds to the 5'UTR of antioxidant mRNAs and Wilms' tumor 1-associating protein (WTAP), a key regulator in m6A methyltransferase complex. Strikingly, Act1's binding sites are located in proximity to m6A modification sites, which allows Act1 to promote the recruitment of elF3G for cap-independent translation. Loss of Act1's RNA binding activity or Wtap knockdown abolished IL-17-induced m6A modification and translation of Wtap and antioxidant mRNAs, indicating a feedforward mechanism of the Act1-WTAP loop. We then developed antisense oligonucleotides (Wtap ASO) that specifically disrupt Act1's binding to Wtap mRNA, abolishing IL-17/Act1-WTAP-mediated antioxidant protein production during chemotherapy. Wtap ASO substantially increased the antitumor efficacy of cisplatin, demonstrating a potential therapeutic strategy for chemoresistance.

Pre-vaccination transcriptomic profiles of immune responders to the MUC1 peptide vaccine for colon cancer prevention.

Self-antigens abnormally expressed on tumors, such as MUC1, have been targeted by therapeutic cancer vaccines. We recently assessed in two clinical trials in a preventative setting whether immunity induced with a MUC1 peptide vaccine could reduce high colon cancer risk in individuals with a history of premalignant colon adenomas. In both trials, there were immune responders and non-responders to the vaccine. Here we used PBMC pre-vaccination and 2 weeks after the first vaccine of responders and non-responders selected from both trials to identify early biomarkers of immune response involved in long-term memory generation and prevention of adenoma recurrence. We performed flow cytometry, phosflow, and differential gene expression analyses on PBMCs collected from MUC1 vaccine responders and non-responders pre-vaccination and two weeks after the first of three vaccine doses. MUC1 vaccine responders had higher frequencies of CD4 cells pre-vaccination, increased expression of CD40L on CD8 and CD4 T-cells, and a greater increase in ICOS expression on CD8 T-cells. Differential gene expression analysis revealed that iCOSL, PI3K AKT MTOR, and B-cell signaling pathways are activated early in response to the MUC1 vaccine. We identified six specific transcripts involved in elevated antigen presentation, B-cell activation, and NF-kB1 activation that were directly linked to finding antibody response at week 12. Finally, a model using these transcripts was able to predict non-responders with accuracy. These findings suggest that individuals who can be predicted to respond to the MUC1 vaccine, and potentially other vaccines, have greater readiness in all immune compartments to present and respond to antigens. Predictive biomarkers of MUC1 vaccine response may lead to more effective vaccines tailored to individuals with high risk for cancer but with varying immune fitness.

Pre-vaccination transcriptomic profiles of immune responders to the MUC1 peptide vaccine for colon cancer prevention.

Introduction: Self-antigens abnormally expressed on tumors, such as MUC1, have been targeted by therapeutic cancer vaccines. We recently assessed in two clinical trials in a preventative setting whether immunity induced with a MUC1 peptide vaccine could reduce high colon cancer risk in individuals with a history of premalignant colon adenomas. In both trials, there were immune responders and non-responders to the vaccine. Methods: Here we used PBMC pre-vaccination and 2 weeks after the first vaccine of responders and non-responders selected from both trials to identify early biomarkers of immune response involved in long-term memory generation and prevention of adenoma recurrence. We performed flow cytometry, phosflow, and differential gene expression analyses on PBMCs collected from MUC1 vaccine responders and non-responders pre-vaccination and two weeks after the first of three vaccine doses. Results: MUC1 vaccine responders had higher frequencies of CD4 cells pre-vaccination, increased expression of CD40L on CD8 and CD4 T-cells, and a greater increase in ICOS expression on CD8 T-cells. Differential gene expression analysis revealed that iCOSL, PI3K AKT MTOR, and B-cell signaling pathways are activated early in response to the MUC1 vaccine. We identified six specific transcripts involved in elevated antigen presentation, B-cell activation, and NF-κB1 activation that were directly linked to finding antibody response at week 12. Finally, a model using these transcripts was able to predict non-responders with accuracy. Discussion: These findings suggest that individuals who can be predicted to respond to the MUC1 vaccine, and potentially other vaccines, have greater readiness in all immune compartments to present and respond to antigens. Predictive biomarkers of MUC1 vaccine response may lead to more effective vaccines tailored to individuals with high risk for cancer but with varying immune fitness.

A transcriptional evaluation of the melanoma and squamous cell carcinoma TIL compartment reveals an unexpected spectrum of exhausted and functional T cells.

Introduction: Significant heterogeneity exists within the tumor-infiltrating CD8 T cell population, and exhausted T cells harbor a subpopulation that may be replicating and may retain signatures of activation, with potential functional consequences in tumor progression. Dysfunctional immunity in the tumor microenvironment is associated with poor cancer outcomes, making exploration of these exhausted T cell subpopulations critical to the improvement of therapeutic approaches. Methods: To investigate mechanisms associated with terminally exhausted T cells, we sorted and performed transcriptional profiling of CD8+ tumor-infiltrating lymphocytes (TILs) co-expressing the exhaustion markers PD-1 and TIM-3 from large-volume melanoma tumors. We additionally performed immunologic phenotyping and functional validation, including at the single-cell level, to identify potential mechanisms that underlie their dysfunctional phenotype. Results: We identified novel dysregulated pathways in CD8+PD-1+TIM-3+ cells that have not been well studied in TILs; these include bile acid and peroxisome pathway-related metabolism and mammalian target of rapamycin (mTOR) signaling pathways, which are highly correlated with immune checkpoint receptor expression. Discussion: Based on bioinformatic integration of immunophenotypic data and network analysis, we propose unexpected targets for therapies to rescue the immune response to tumors in melanoma.

Newly synthesized mRNA escapes translational repression during the acute phase of the mammalian unfolded protein response.

Endoplasmic Reticulum (ER) stress, caused by the accumulation of misfolded proteins in the ER, elicits a homeostatic mechanism known as the Unfolded Protein Response (UPR). The UPR reprograms gene expression to promote adaptation to chronic ER stress. The UPR comprises an acute phase involving inhibition of bulk protein synthesis and a chronic phase of transcriptional induction coupled with the partial recovery of protein synthesis. However, the role of transcriptional regulation in the acute phase of the UPR is not well understood. Here we analyzed the fate of newly synthesized mRNA encoding the protective and homeostatic transcription factor X-box binding protein 1 (XBP1) during this acute phase. We have previously shown that global translational repression induced by the acute UPR was characterized by decreased translation and increased stability of XBP1 mRNA. We demonstrate here that this stabilization is independent of new transcription. In contrast, we show XBP1 mRNA newly synthesized during the acute phase accumulates with long poly(A) tails and escapes translational repression. Inhibition of newly synthesized RNA polyadenylation during the acute phase decreased cell survival with no effect in unstressed cells. Furthermore, during the chronic phase of the UPR, levels of XBP1 mRNA with long poly(A) tails decreased in a manner consistent with co-translational deadenylation. Finally, additional pro-survival, transcriptionally-induced mRNAs show similar regulation, supporting the broad significance of the pre-steady state UPR in translational control during ER stress. We conclude that the biphasic regulation of poly(A) tail length during the UPR represents a previously unrecognized pro-survival mechanism of mammalian gene regulation.

Mapping transcriptome-wide protein-RNA interactions to elucidate RNA regulatory programs.

BACKGROUND: Our understanding of post-transcriptional gene regulation has increased exponentially with the development of robust methods to define protein-RNA interactions across the transcriptome. In this review, we highlight the evolution and successful applications of crosslinking and immunoprecipitation (CLIP) methods to interrogate protein-RNA interactions in a transcriptome-wide manner. RESULTS: Here, we survey the vast array of in vitro and in vivo approaches used to identify protein-RNA interactions, including but not limited to electrophoretic mobility shift assays, systematic evolution of ligands by exponential enrichment (SELEX), and RIP-seq. We particularly emphasize the advancement of CLIP technologies, and detail protocol improvements and computational tools used to analyze the output data. Importantly, we discuss how profiling protein-RNA interactions can delineate biological functions including splicing regulation, alternative polyadenylation, cytoplasmic decay substrates, and miRNA targets. CONCLUSIONS: In summary, this review summarizes the benefits of characterizing RNA-protein networks to further understand the regulation of gene expression and disease pathogenesis. Our review comments on how future CLIP technologies can be adapted to address outstanding questions related to many aspects of RNA metabolism and further advance our understanding of RNA biology.

DAZL Regulates Germ Cell Survival through a Network of PolyA-Proximal mRNA Interactions.

The RNA binding protein DAZL is essential for gametogenesis, but its direct in vivo functions, RNA targets, and the molecular basis for germ cell loss in Dazl-null mice are unknown. Here, we mapped transcriptome-wide DAZL-RNA interactions in vivo, revealing DAZL binding to thousands of mRNAs via polyA-proximal 3' UTR interactions. In parallel, fluorescence-activated cell sorting and RNA-seq identified mRNAs sensitive to DAZL deletion in male germ cells. Despite binding a broad set of mRNAs, integrative analyses indicate that DAZL post-transcriptionally controls only a subset of its mRNA targets, namely those corresponding to a network of genes that are critical for germ cell proliferation and survival. In addition, we provide evidence that polyA sequences have key roles in specifying DAZL-RNA interactions across the transcriptome. Our results reveal a mechanism for DAZL-RNA binding and illustrate that DAZL functions as a master regulator of a post-transcriptional mRNA program essential for germ cell survival.

Ptbp2 Controls an Alternative Splicing Network Required for Cell Communication during Spermatogenesis.

Alternative splicing has essential roles in development. Remarkably, spermatogenic cells express more alternatively spliced RNAs compared to most whole tissues; however, regulation of these RNAs remains unclear. Here, we characterize the alternative splicing landscape during spermatogenesis and reveal an essential function for the RNA-binding protein Ptbp2 in this highly regulated developmental program. We found that Ptbp2 controls a network of genes involved in cell adhesion, migration, and polarity, suggesting that splicing regulation by Ptbp2 is critical for germ cell communication with Sertoli cells (multifunctional somatic cells necessary for spermatogenesis). Indeed, Ptbp2 ablation in germ cells resulted in disorganization of the filamentous actin (F-actin) cytoskeleton in Sertoli cells, indicating that alternative splicing regulation is necessary for cellular crosstalk during germ cell development. Collectively, the data delineate an alternative splicing regulatory network essential for spermatogenesis, the splicing factor that controls it, and its biological importance in germ-Sertoli communication.

RNA Binding Protein Ptbp2 Is Essential for Male Germ Cell Development.

RNA binding proteins (RBPs) are increasingly recognized as essential factors in tissue development and homeostasis. The polypyrimidine tract binding (PTB) protein family of RBPs are important posttranscriptional regulators of gene expression. In the nervous system, the function and importance of PTB protein 2 (Ptbp2) as a key alternative splicing regulator is well established. Ptbp2 is also abundantly expressed during spermatogenesis, but its role in this developmental program has not been explored. Additionally, the importance of alternative splicing regulation in spermatogenesis is unclear. Here, we demonstrate that Ptbp2 is essential for spermatogenesis. We also describe an improved dual fluorescence flow cytometry strategy to discriminate, quantify, and collect germ cells in different stages of development. Using this approach, in combination with traditional histological methods, we show that Ptbp2 ablation results in germ cell loss due to increased apoptosis of meiotic spermatocytes and postmeiotic arrest of spermatid differentiation. Furthermore, we show that Ptbp2 is required for alternative splicing regulation in the testis, as in brain. Strikingly, not all of the alternatively spliced RNAs examined were sensitive to Ptbp2 loss in both tissues. Collectively, the data provide evidence for an important role for alternative splicing regulation in germ cell development and a central role for Ptbp2 in this process.