T helper cells exhibit a dynamic and reversible 3'-UTR landscape.

3' untranslated regions (3' UTRs) are critical elements of messenger RNAs, as they contain binding sites for RNA-binding proteins (RBPs) and microRNAs that affect various aspects of the RNA life cycle including transcript stability and cellular localization. In response to T cell receptor activation, T cells undergo massive expansion during the effector phase of the immune response and dynamically modify their 3' UTRs. Whether this serves to directly regulate the abundance of specific mRNAs or is a secondary effect of proliferation remains unclear. To study 3'-UTR dynamics in T helper cells, we investigated division-dependent alternative polyadenylation (APA). In addition, we generated 3' end UTR sequencing data from naive, activated, memory, and regulatory CD4+ T cells. 3'-UTR length changes were estimated using a nonnegative matrix factorization approach and were compared with those inferred from long-read PacBio sequencing. We found that APA events were transient and reverted after effector phase expansion. Using an orthogonal bulk RNA-seq data set, we did not find evidence of APA association with differential gene expression or transcript usage, indicating that APA has only a marginal effect on transcript abundance. 3'-UTR sequence analysis revealed conserved binding sites for T cell-relevant microRNAs and RBPs in the alternative 3' UTRs. These results indicate that poly(A) site usage could play an important role in the control of cell fate decisions and homeostasis.

Extensible benchmarking of methods that identify and quantify polyadenylation sites from RNA-seq data.

The tremendous rate with which data is generated and analysis methods emerge makes it increasingly difficult to keep track of their domain of applicability, assumptions, limitations, and consequently, of the efficacy and precision with which they solve specific tasks. Therefore, there is an increasing need for benchmarks, and for the provision of infrastructure for continuous method evaluation. APAeval is an international community effort, organized by the RNA Society in 2021, to benchmark tools for the identification and quantification of the usage of alternative polyadenylation (APA) sites from short-read, bulk RNA-sequencing (RNA-seq) data. Here, we reviewed 17 tools and benchmarked eight on their ability to perform APA identification and quantification, using a comprehensive set of RNA-seq experiments comprising real, synthetic, and matched 3'-end sequencing data. To support continuous benchmarking, we have incorporated the results into the OpenEBench online platform, which allows for continuous extension of the set of methods, metrics, and challenges. We envisage that our analyses will assist researchers in selecting the appropriate tools for their studies, while the containers and reproducible workflows could easily be deployed and extended to evaluate new methods or data sets.

Identification of experimentally-supported poly(A) sites in single-cell RNA-seq data with SCINPAS.

Alternative polyadenylation is a main driver of transcriptome diversity in mammals, generating transcript isoforms with different 3' ends via cleavage and polyadenylation at distinct polyadenylation (poly(A)) sites. The regulation of cell type-specific poly(A) site choice is not completely resolved, and requires quantitative poly(A) site usage data across cell types. 3' end-based single-cell RNA-seq can now be broadly used to obtain such data, enabling the identification and quantification of poly(A) sites with direct experimental support. We propose SCINPAS, a computational method to identify poly(A) sites from scRNA-seq datasets. SCINPAS modifies the read deduplication step to favor the selection of distal reads and extract those with non-templated poly(A) tails. This approach improves the resolution of poly(A) site recovery relative to standard software. SCINPAS identifies poly(A) sites in genic and non-genic regions, providing complementary information relative to other tools. The workflow is modular, and the key read deduplication step is general, enabling the use of SCINPAS in other typical analyses of single cell gene expression. Taken together, we show that SCINPAS is able to identify experimentally-supported, known and novel poly(A) sites from 3' end-based single-cell RNA sequencing data.

Impaired age-associated mitochondrial translation is mitigated by exercise and PGC-1α.

Sarcopenia, the age-related loss of skeletal muscle mass and function, can dramatically impinge on quality of life and mortality. While mitochondrial dysfunction and imbalanced proteostasis are recognized as hallmarks of sarcopenia, the regulatory and functional link between these processes is underappreciated and unresolved. We therefore investigated how mitochondrial proteostasis, a crucial process that coordinates the expression of nuclear- and mitochondrial-encoded mitochondrial proteins with supercomplex formation and respiratory activity, is affected in skeletal muscle aging. Intriguingly, a robust mitochondrial translation impairment was observed in sarcopenic muscle, which is regulated by the peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α) with the estrogen-related receptor α (ERRα). Exercise, a potent inducer of PGC-1α activity, rectifies age-related reduction in mitochondrial translation, in conjunction with quality control pathways. These results highlight the importance of mitochondrial proteostasis in muscle aging, and elucidate regulatory interactions that underlie the powerful benefits of physical activity in this context.

Extensible benchmarking of methods that identify and quantify polyadenylation sites from RNA-seq data.

The tremendous rate with which data is generated and analysis methods emerge makes it increasingly difficult to keep track of their domain of applicability, assumptions, and limitations and consequently, of the efficacy and precision with which they solve specific tasks. Therefore, there is an increasing need for benchmarks, and for the provision of infrastructure for continuous method evaluation. APAeval is an international community effort, organized by the RNA Society in 2021, to benchmark tools for the identification and quantification of the usage of alternative polyadenylation (APA) sites from short-read, bulk RNA-sequencing (RNA-seq) data. Here, we reviewed 17 tools and benchmarked eight on their ability to perform APA identification and quantification, using a comprehensive set of RNA-seq experiments comprising real, synthetic, and matched 3'-end sequencing data. To support continuous benchmarking, we have incorporated the results into the OpenEBench online platform, which allows for seamless extension of the set of methods, metrics, and challenges. We envisage that our analyses will assist researchers in selecting the appropriate tools for their studies. Furthermore, the containers and reproducible workflows generated in the course of this project can be seamlessly deployed and extended in the future to evaluate new methods or datasets.

Improved analysis of (e)CLIP data with RCRUNCH yields a compendium of RNA-binding protein binding sites and motifs.

We present RCRUNCH, an end-to-end solution to CLIP data analysis for identification of binding sites and sequence specificity of RNA-binding proteins. RCRUNCH can analyze not only reads that map uniquely to the genome but also those that map to multiple genome locations or across splice boundaries and can consider various types of background in the estimation of read enrichment. By applying RCRUNCH to the eCLIP data from the ENCODE project, we have constructed a comprehensive and homogeneous resource of in-vivo-bound RBP sequence motifs. RCRUNCH automates the reproducible analysis of CLIP data, enabling studies of post-transcriptional control of gene expression.

Dual roles of mTORC1-dependent activation of the ubiquitin-proteasome system in muscle proteostasis.

Muscle size is controlled by the PI3K-PKB/Akt-mTORC1-FoxO pathway, which integrates signals from growth factors, energy and amino acids to activate protein synthesis and inhibit protein breakdown. While mTORC1 activity is necessary for PKB/Akt-induced muscle hypertrophy, its constant activation alone induces muscle atrophy. Here we show that this paradox is based on mTORC1 activity promoting protein breakdown through the ubiquitin-proteasome system (UPS) by simultaneously inducing ubiquitin E3 ligase expression via feedback inhibition of PKB/Akt and proteasome biogenesis via Nuclear Factor Erythroid 2-Like 1 (Nrf1). Muscle growth was restored by reactivation of PKB/Akt, but not by Nrf1 knockdown, implicating ubiquitination as the limiting step. However, both PKB/Akt activation and proteasome depletion by Nrf1 knockdown led to an immediate disruption of proteome integrity with rapid accumulation of damaged material. These data highlight the physiological importance of mTORC1-mediated PKB/Akt inhibition and point to juxtaposed roles of the UPS in atrophy and proteome integrity.

A novel approach to single-cell analysis reveals intrinsic differences in immune marker expression in unstimulated BALB/c and C57BL/6 macrophages.

The origin of functional heterogeneity among macrophages, key innate immune system components, is still debated. While mouse strains differ in their immune responses, the range of gene expression variation among their pre-stimulation macrophages is unknown. With a novel approach to scRNA-seq analysis, we reveal the gene expression variation in unstimulated macrophage populations from BALB/c and C57BL/6 mice. We show that intrinsic strain-to-strain differences are detectable before stimulation and we place the unstimulated single cells within the gene expression landscape of stimulated macrophages. C57BL/6 mice show stronger evidence of macrophage polarization than BALB/c mice, which may contribute to their relative resistance to pathogens. Our computational methods can be generally adopted to uncover biological variation between cell populations.

Cholesterol Deprivation Drives DHEA Biosynthesis in Human Adrenals.

Adrenarche is an early event in sexual maturation in prepubertal children and corresponds to the postnatal development of the adrenocortical zona reticularis (zR). However, the molecular mechanisms that govern the onset and maturation of zR remain unknown. Using tissue laser microdissection combined with transcript quantification and immunodetection, we showed that the human zR receives low levels of cholesterol in comparison with other adrenal layers. To model this metabolic condition, we challenged adrenal cells in vitro using cholesterol deprivation. This resulted in reprogramming the steroidogenic pathway toward inactivation of 3-beta-hydroxysteroid dehydrogenase type 2 (HSD3B2), increased CYB5A expression, and increased biosynthesis of dehydroepiandrosterone (DHEA), 3 key features of zR maturation during adrenarche. Finally, we found that cholesterol deprivation leads to decreased transcriptional activity of POU3F2, which normally stimulates the expression of HSD3B2 by directly binding to its promoter. These findings demonstrate that cholesterol deprivation can account, at least in part, for the acquisition of a zR-like androgenic program in humans.

Distinct and additive effects of calorie restriction and rapamycin in aging skeletal muscle.

Preserving skeletal muscle function is essential to maintain life quality at high age. Calorie restriction (CR) potently extends health and lifespan, but is largely unachievable in humans, making "CR mimetics" of great interest. CR targets nutrient-sensing pathways centering on mTORC1. The mTORC1 inhibitor, rapamycin, is considered a potential CR mimetic and is proven to counteract age-related muscle loss. Therefore, we tested whether rapamycin acts via similar mechanisms as CR to slow muscle aging. Here we show that long-term CR and rapamycin unexpectedly display distinct gene expression profiles in geriatric mouse skeletal muscle, despite both benefiting aging muscles. Furthermore, CR improves muscle integrity in mice with nutrient-insensitive, sustained muscle mTORC1 activity and rapamycin provides additive benefits to CR in naturally aging mouse muscles. We conclude that rapamycin and CR exert distinct, compounding effects in aging skeletal muscle, thus opening the possibility of parallel interventions to counteract muscle aging.

CFIm-mediated alternative polyadenylation remodels cellular signaling and miRNA biogenesis.

The mammalian cleavage factor I (CFIm) has been implicated in alternative polyadenylation (APA) in a broad range of contexts, from cancers to learning deficits and parasite infections. To determine how the CFIm expression levels are translated into these diverse phenotypes, we carried out a multi-omics analysis of cell lines in which the CFIm25 (NUDT21) or CFIm68 (CPSF6) subunits were either repressed by siRNA-mediated knockdown or over-expressed from stably integrated constructs. We established that >800 genes undergo coherent APA in response to changes in CFIm levels, and they cluster in distinct functional classes related to protein metabolism. The activity of the ERK pathway traces the CFIm concentration, and explains some of the fluctuations in cell growth and metabolism that are observed upon CFIm perturbations. Furthermore, multiple transcripts encoding proteins from the miRNA pathway are targets of CFIm-dependent APA. This leads to an increased biogenesis and repressive activity of miRNAs at the same time as some 3' UTRs become shorter and presumably less sensitive to miRNA-mediated repression. Our study provides a first systematic assessment of a core set of APA targets that respond coherently to changes in CFIm protein subunit levels (CFIm25/CFIm68). We describe the elicited signaling pathways downstream of CFIm, which improve our understanding of the key role of CFIm in integrating RNA processing with other cellular activities.

Untargeted sequencing of circulating microRNAs in a healthy and diseased older population.

We performed untargeted profiling of circulating microRNAs (miRNAs) in a well characterized cohort of older adults to verify associations of health and disease-related biomarkers with systemic miRNA expression. Differential expression analysis revealed 30 miRNAs that significantly differed between healthy active, healthy sedentary and sedentary cardiovascular risk patients. Increased expression of miRNAs miR-193b-5p, miR-122-5p, miR-885-3p, miR-193a-5p, miR-34a-5p, miR-505-3p, miR-194-5p, miR-27b-3p, miR-885-5p, miR-23b-5b, miR-365a-3p, miR-365b-3p, miR-22-5p was associated with a higher metabolic risk profile, unfavourable macro- and microvascular health, lower physical activity (PA) as well as cardiorespiratory fitness (CRF) levels. Increased expression of miR-342-3p, miR-1-3p, miR-92b-5p, miR-454-3p, miR-190a-5p and miR-375-3p was associated with a lower metabolic risk profile, favourable macro- and microvascular health as well as higher PA and CRF. Of note, the first two principal components explained as much as 20% and 11% of the data variance. miRNAs and their potential target genes appear to mediate disease- and health-related physiological and pathophysiological adaptations that need to be validated and supported by further downstream analysis in future studies.Clinical Trial Registration: ClinicalTrials.gov: NCT02796976 ( https://clinicaltrials.gov/ct2/show/NCT02796976 ).

Shortening of 3' UTRs in most cell types composing tumor tissues implicates alternative polyadenylation in protein metabolism.

During pre-mRNA maturation 3' end processing can occur at different polyadenylation sites in the 3' untranslated region (3' UTR) to give rise to transcript isoforms that differ in the length of their 3' UTRs. Longer 3' UTRs contain additional cis-regulatory elements that impact the fate of the transcript and/or of the resulting protein. Extensive alternative polyadenylation (APA) has been observed in cancers, but the mechanisms and roles remain elusive. In particular, it is unclear whether the APA occurs in the malignant cells or in other cell types that infiltrate the tumor. To resolve this, we developed a computational method, called SCUREL, that quantifies changes in 3' UTR length between groups of cells, including cells of the same type originating from tumor and control tissue. We used this method to study APA in human lung adenocarcinoma (LUAD). SCUREL relies solely on annotated 3' UTRs and on control systems such as T cell activation, and spermatogenesis gives qualitatively similar results at much greater sensitivity compared to the previously published scAPA method. In the LUAD samples, we find a general trend toward 3' UTR shortening not only in cancer cells compared to the cell type of origin, but also when comparing other cell types from the tumor vs. the control tissue environment. However, we also find high variability in the individual targets between patients. The findings help in understanding the extent and impact of APA in LUAD, which may support improvements in diagnosis and treatment.

Nanopore sequencing reveals endogenous NMD-targeted isoforms in human cells.

BACKGROUND: Nonsense-mediated mRNA decay (NMD) is a eukaryotic, translation-dependent degradation pathway that targets mRNAs with premature termination codons and also regulates the expression of some mRNAs that encode full-length proteins. Although many genes express NMD-sensitive transcripts, identifying them based on short-read sequencing data remains a challenge. RESULTS: To identify and analyze endogenous targets of NMD, we apply cDNA Nanopore sequencing and short-read sequencing to human cells with varying expression levels of NMD factors. Our approach detects full-length NMD substrates that are highly unstable and increase in levels or even only appear when NMD is inhibited. Among the many new NMD-targeted isoforms that our analysis identifies, most derive from alternative exon usage. The isoform-aware analysis reveals many genes with significant changes in splicing but no significant changes in overall expression levels upon NMD knockdown. NMD-sensitive mRNAs have more exons in the 3΄UTR and, for those mRNAs with a termination codon in the last exon, the length of the 3΄UTR per se does not correlate with NMD sensitivity. Analysis of splicing signals reveals isoforms where NMD has been co-opted in the regulation of gene expression, though the main function of NMD seems to be ridding the transcriptome of isoforms resulting from spurious splicing events. CONCLUSIONS: Long-read sequencing enables the identification of many novel NMD-sensitive mRNAs and reveals both known and unexpected features concerning their biogenesis and their biological role. Our data provide a highly valuable resource of human NMD transcript targets for future genomic and transcriptomic applications.

Transcriptional Enhancer Factor Domain Family member 4 Exerts an Oncogenic Role in Hepatocellular Carcinoma by Hippo-Independent Regulation of Heat Shock Protein 70 Family Members.

Transcriptional enhancer factor domain family member 4 (TEAD4) is a downstream effector of the conserved Hippo signaling pathway, regulating the expression of genes involved in cell proliferation and differentiation. It is up-regulated in several cancer types and is associated with metastasis and poor prognosis. However, its role in hepatocellular carcinoma (HCC) remains largely unexplored. Using data from The Cancer Genome Atlas, we found that TEAD4 was overexpressed in HCC and was associated with aggressive HCC features and worse outcome. Overexpression of TEAD4 significantly increased proliferation and migration rates in HCC cells in vitro as well as tumor growth in vivo. Additionally, RNA sequencing analysis of TEAD4-overexpressing HCC cells demonstrated that TEAD4 overexpression was associated with the up-regulation of genes involved in epithelial-to-mesenchymal transition, proliferation, and protein-folding pathways. Among the most up-regulated genes following TEAD4 overexpression were the 70-kDa heat shock protein (HSP70) family members HSPA6 and HSPA1A. Chromatin immunoprecipitation-quantitative real-time polymerase chain reaction experiments demonstrated that TEAD4 regulates HSPA6 and HSPA1A expression by directly binding to their promoter and enhancer regions. The pharmacologic inhibition of HSP70 expression in TEAD4-overexpressing cells reduced the effect of TEAD4 on cell proliferation. Finally, by overexpressing TEAD4 in yes-associated protein (YAP)/transcriptional coactivator with PDZ binding motif (TAZ)-knockdown HCC cells, we showed that the effect of TEAD4 on cell proliferation and its regulation of HSP70 expression does not require YAP and TAZ, the main effectors of the Hippo signaling pathway. Conclusion: A novel Hippo-independent mechanism for TEAD4 promotes cell proliferation and tumor growth in HCC by directly regulating HSP70 family members.

Bayesian inference of gene expression states from single-cell RNA-seq data.

Despite substantial progress in single-cell RNA-seq (scRNA-seq) data analysis methods, there is still little agreement on how to best normalize such data. Starting from the basic requirements that inferred expression states should correct for both biological and measurement sampling noise and that changes in expression should be measured in terms of fold changes, we here derive a Bayesian normalization procedure called Sanity (SAmpling-Noise-corrected Inference of Transcription activitY) from first principles. Sanity estimates expression values and associated error bars directly from raw unique molecular identifier (UMI) counts without any tunable parameters. Using simulated and real scRNA-seq datasets, we show that Sanity outperforms other normalization methods on downstream tasks, such as finding nearest-neighbor cells and clustering cells into subtypes. Moreover, we show that by systematically overestimating the expression variability of genes with low expression and by introducing spurious correlations through mapping the data to a lower-dimensional representation, other methods yield severely distorted pictures of the data.

The transcriptional landscape of a hepatoma cell line grown on scaffolds of extracellular matrix proteins.

BACKGROUND: The behavior of cells in vivo is complex and highly dynamic, as it results from an interplay between intercellular matrix proteins with surface receptors and other microenvironmental cues. Although the effects of the cellular niche have been investigated for a number of cell types using different molecular approaches, comprehensive assessments of how the global transcriptome responds to 3D scaffolds composed of various extracellular matrix (ECM) constituents at different concentrations are still lacking. RESULTS: In this study, we explored the effects of two diverse extracellular matrix (ECM) components, Collagen I and Matrigel, on the transcriptional profile of cells in a cell culture system. Culturing Huh-7 cells on traditional cell culture plates (Control) or on the ECM components at different concentrations to modulate microenvironment properties, we have generated transcriptomics data that may be further explored to understand the differentiation and growth potential of this cell type for the development of 3D cultures. Our analysis infers transcription factors that are most responsible for the transcriptome response to the extracellular cues. CONCLUSION: Our data indicates that the Collagen I substrate induces a robust transcriptional response in the Huh-7 cells, distinct from that induced by Matrigel. Enhanced hepatocyte markers (ALB and miR-122) reveal a potentially robust remodelling towards primary hepatocytes. Our results aid in defining the appropriate culture and transcription pathways while using hepatoma cell lines. As systems mimicking the in vivo structure and function of liver cells are still being developed, our study could potentially circumvent bottlenecks of limited availability of primary hepatocytes for preclinical studies of drug targets.