Calorie restriction and rapamycin distinctly mitigate aging-associated protein phosphorylation changes in mouse muscles.

Calorie restriction (CR) and treatment with rapamycin (RM), an inhibitor of the mTORC1 growth-promoting signaling pathway, are known to slow aging and promote health from worms to humans. At the transcriptome and proteome levels, long-term CR and RM treatments have partially overlapping effects, while their impact on protein phosphorylation within cellular signaling pathways have not been compared. Here we measured the phosphoproteomes of soleus, tibialis anterior, triceps brachii and gastrocnemius muscles from adult (10 months) and 30-month-old (aged) mice receiving either a control, a calorie restricted or an RM containing diet from 15 months of age. We reproducibly detected and extensively analyzed a total of 6960 phosphosites, 1415 of which are not represented in standard repositories. We reveal the effect of these interventions on known mTORC1 pathway substrates, with CR displaying greater between-muscle variation than RM. Overall, CR and RM have largely consistent, but quantitatively distinct long-term effects on the phosphoproteome, mitigating age-related changes to different degrees. Our data expands the catalog of protein phosphorylation sites in the mouse, providing important information regarding their tissue-specificity, and revealing the impact of long-term nutrient-sensing pathway inhibition on mouse skeletal muscle.

Current limitations in predicting mRNA translation with deep learning models.

BACKGROUND: The design of nucleotide sequences with defined properties is a long-standing problem in bioengineering. An important application is protein expression, be it in the context of research or the production of mRNA vaccines. The rate of protein synthesis depends on the 5' untranslated region (5'UTR) of the mRNAs, and recently, deep learning models were proposed to predict the translation output of mRNAs from the 5'UTR sequence. At the same time, large data sets of endogenous and reporter mRNA translation have become available. RESULTS: In this study, we use complementary data obtained in two different cell types to assess the accuracy and generality of currently available models for predicting translational output. We find that while performing well on the data sets on which they were trained, deep learning models do not generalize well to other data sets, in particular of endogenous mRNAs, which differ in many properties from reporter constructs. CONCLUSIONS: These differences limit the ability of deep learning models to uncover mechanisms of translation control and to predict the impact of genetic variation. We suggest directions that combine high-throughput measurements and machine learning to unravel mechanisms of translation control and improve construct design.

Ribosomal protein RPL39L is an efficiency factor in the cotranslational folding of a subset of proteins with alpha helical domains.

Increasingly many studies reveal how ribosome composition can be tuned to optimally translate the transcriptome of individual cell types. In this study, we investigated the expression pattern, structure within the ribosome and effect on protein synthesis of the ribosomal protein paralog 39L (RPL39L). With a novel mass spectrometric approach we revealed the expression of RPL39L protein beyond mouse germ cells, in human pluripotent cells, cancer cell lines and tissue samples. We generated RPL39L knock-out mouse embryonic stem cell (mESC) lines and demonstrated that RPL39L impacts the dynamics of translation, to support the pluripotency and differentiation, spontaneous and along the germ cell lineage. Most differences in protein abundance between WT and RPL39L KO lines were explained by widespread autophagy. By CryoEM analysis of purified RPL39 and RPL39L-containing ribosomes we found that, unlike RPL39, RPL39L has two distinct conformations in the exposed segment of the nascent peptide exit tunnel, creating a distinct hydrophobic patch that has been predicted to support the efficient co-translational folding of alpha helices. Our study shows that ribosomal protein paralogs provide switchable modular components that can tune translation to the protein production needs of individual cell types.

CCAR1 promotes DNA repair via alternative splicing.

DNA repair is directly performed by hundreds of core factors and indirectly regulated by thousands of others. We massively expanded a CRISPR inhibition and Cas9-editing screening system to discover factors indirectly modulating homology-directed repair (HDR) in the context of ∼18,000 individual gene knockdowns. We focused on CCAR1, a poorly understood gene that we found the depletion of reduced both HDR and interstrand crosslink repair, phenocopying the loss of the Fanconi anemia pathway. CCAR1 loss abrogated FANCA protein without substantial reduction in the level of its mRNA or that of other FA genes. We instead found that CCAR1 prevents inclusion of a poison exon in FANCA. Transcriptomic analysis revealed that the CCAR1 splicing modulatory activity is not limited to FANCA, and it instead regulates widespread changes in alternative splicing that would damage coding sequences in mouse and human cells. CCAR1 therefore has an unanticipated function as a splicing fidelity factor.

MAPP unravels frequent co-regulation of splicing and polyadenylation by RNA-binding proteins and their dysregulation in cancer.

Maturation of eukaryotic pre-mRNAs via splicing and polyadenylation is modulated across cell types and conditions by a variety of RNA-binding proteins (RBPs). Although there exist over 1,500 RBPs in human cells, their binding motifs and functions still remain to be elucidated, especially in the complex environment of tissues and in the context of diseases. To overcome the lack of methods for the systematic and automated detection of sequence motif-guided pre-mRNA processing regulation from RNA sequencing (RNA-Seq) data we have developed MAPP (Motif Activity on Pre-mRNA Processing). Applying MAPP to RBP knock-down experiments reveals that many RBPs regulate both splicing and polyadenylation of nascent transcripts by acting on similar sequence motifs. MAPP not only infers these sequence motifs, but also unravels the position-dependent impact of the RBPs on pre-mRNA processing. Interestingly, all investigated RBPs that act on both splicing and 3' end processing exhibit a consistently repressive or activating effect on both processes, providing a first glimpse on the underlying mechanism. Applying MAPP to normal and malignant brain tissue samples unveils that the motifs bound by the PTBP1 and RBFOX RBPs coordinately drive the oncogenic splicing program active in glioblastomas demonstrating that MAPP paves the way for characterizing pre-mRNA processing regulators under physiological and pathological conditions.

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.

On the limits of inferring biophysical parameters of RBP-RNA interactions from in vitro RNA Bind'n Seq data.

We develop a thermodynamic model describing the binding of RNA binding proteins (RBP) to oligomers in vitro. We apply expectation-maximization to infer the specificity of RBPs, represented as position-specific weight matrices (PWMs), by maximizing the likelihood of RNA Bind'n Seq data from the ENCODE project. Analyzing these public data we find sequence motifs that can partly explain the data for more than half of the studied 111 RBPs, and for 48 of the proteins these motifs are consistent with the known specificity. Our code is publicly available, facilitating analysis of RBP binding data.

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.