The Role of the Universally Conserved ATPase YchF/Ola1 in Translation Regulation during Cellular Stress

The ability to respond to metabolic or environmental changes is an essential feature in all cells and involves both transcriptional and translational regulators that adjust the metabolic activity to fluctuating conditions. While transcriptional regulation has been studied in detail, the important role of the ribosome as an additional player in regulating gene expression is only beginning to emerge. Ribosome-interacting proteins are central to this translational regulation and include universally conserved ribosome interacting proteins, such as the ATPase YchF (Ola1 in eukaryotes). In both eukaryotes and bacteria, the cellular concentrations of YchF/Ola1 determine the ability to cope with different stress conditions and are linked to several pathologies in humans. The available data indicate that YchF/Ola1 regulates the stress response via controlling non-canonical translation initiation and via protein degradation. Although the molecular mechanisms appear to be different between bacteria and eukaryotes, increased non-canonical translation initiation is a common consequence of YchF/Ola1 regulated translational control in E. coli and H. sapiens. In this review, we summarize recent insights into the role of the universally conserved ATPase YchF/Ola1 in adapting translation to unfavourable conditions.

Programmable mammalian translational modulators by CRISPR-associated proteins

The complexity of synthetic genetic circuits relies on repertories of biological circuitry with high orthogonality. Although post-transcriptional circuitry relying on RNA-binding proteins (RBPs) qualifies as a repertory, the limited pool of regulatory devices hinders network modularity and scalability. Here we propose CaRTRIDGE (Cas-Responsive Translational Regulation Integratable into Diverse Genomic Engineering) to repurpose CRISPR-associated (Cas) proteins as translational modulators. We demonstrate that a set of Cas proteins are able to repress (OFF) or activate (ON) the translation of mRNAs that contain a Cas-binding RNA motif in the 5'-UTR. We designed 81 different types of translation OFF and ON switches and verified their functional characteristics. Many of them functioned as efficient translational regulators and showed orthogonality in mammalian cells. By interconnecting these switches, we designed and built artificial circuits, including 60 translational AND gates. Moreover, we show that various CRISPR-related technologies, including anti-CRISPR and split-Cas9 platforms, can be repurposed to control translation. Our Cas-mediated translational regulation is compatible with transcriptional regulation by Cas proteins and increases the complexity of synthetic circuits with fewer elements. CaRTRIDGE builds protein-responsive mRNA switches more than ever and leads to the development of both Cas-mediated genome editing and translational regulation technologies.

utr.annotation: a tool for annotating genomic variants that could influence post-transcriptional regulation

Summary Whole genome sequencing of patient populations is identifying thousands of new variants in UnTranslated Regions(UTRs). While the consequences of UTR mutations are not as easily predicted from primary sequence as coding mutations are, there are some known features of UTRs that modulate their function. utr.annotation is an R package that can be used to annotate potential deleterious variants in the UTR regions for both human and mouse species. Given a CSV or VCF format variant file, utr.annotation provides information of each variant on whether and how it alters known translational regulators including: upstream Open Reading Frames (uORFs), upstream Kozak sequences, polyA signals, Kozak sequences at the annotated translation start site, start codons, and stop codons, conservation scores in the variant position, and whether and how it changes ribosome loading based on a model derived from empirical data. Availability utr.annotation is freely available on Bitbucket (https://bitbucket.org/jdlabteam/utr.annotation/src/master/) and CRAN (https://cran.r-project.org/web/packages/utr.annotation/index.html) Supplementary information Supplementary data are available at https://wustl.box.com/s/yye99bryfin89nav45gv91l5k35fxo7z.

Contribution of Energy Dysfunction to Impaired Protein Translation in Neurodegenerative Diseases

Impaired energy homeostasis and aberrant translational control have independently been implicated in the pathogenesis of neurodegenerative diseases. AMP kinase (AMPK), regulated by the ratio of cellular AMP and ATP, is a major gatekeeper for cellular energy homeostasis. Abnormal regulation of AMPK has been reported in several neurodegenerative diseases, including Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS). Most importantly, AMPK activation is known to suppress the translational machinery by inhibiting the mechanistic target of rapamycin complex 1 (mTORC1), activating translational regulators, and phosphorylating nuclear transporter factors. In this review, we describe recent findings on the emerging role of protein translation impairment caused by energy dysregulation in neurodegenerative diseases.

utR.annotation: a tool for annotating genomic variants that could influence post-transcriptional regulation

Whole genome sequencing of patient populations is identifying thousands of new variants in UnTranslated Regions(UTRs). While the consequences of UTR mutations are not as easily predicted from primary sequence as coding mutations are, there are some known features of UTRs modulate their function. utR.annotation is an R package that can be used to annotate potential deleterious variants in the UTR regions for both human and mouse species. Given a CSV or VCF format variant file, utR.annotation provides information of each variant on whether and how it alters known translational regulators including:upstream Open Reading Frames (uORFs), upstream Kozak sequences, polyA signals, the Kozak sequence at the annotated translation initiation site, start codon, and stop codon, conservation scores in the variant position, and whether and how it changes ribosome loading based on a model from empirical data.

Pumilio proteins utilize distinct regulatory mechanisms to achieve complementary functions required for pluripotency and embryogenesis

Gene regulation in embryonic stem cells (ESCs) has been extensively studied at the epigenetic-transcriptional level, but not at the posttranscriptional level. Pumilio (Pum) proteins are among the few known translational regulators required for stem-cell maintenance in invertebrates and plants. Here we report the essential function of two murine Pum proteins, Pum1 and Pum2, in ESCs and early embryogenesis. Pum1/2 double-mutant ESCs display severely reduced self-renewal and differentiation, and Pum1/2 double-mutant mice are developmentally delayed at the morula stage and lethal by embryonic day 8.5. Remarkably, Pum1-deficient ESCs show increased expression of pluripotency genes but not differentiation genes, whereas Pum2-deficient ESCs show decreased pluripotency markers and accelerated differentiation. Thus, despite their high homology and overlapping target messenger RNAs (mRNAs), Pum1 promotes differentiation while Pum2 promotes self-renewal in ESCs. Pum1 and Pum2 achieve these two complementary aspects of pluripotency by forming a negative interregulatory feedback loop that directly regulates at least 1,486 mRNAs. Pum1 and Pum2 regulate target mRNAs not only by repressing translation, but also by promoting translation and enhancing or reducing mRNA stability of different target mRNAs. Together, these findings reveal distinct roles of individual mammalian Pum proteins in ESCs and their essential functions in ESC pluripotency and embryogenesis.

Shot-gun proteomics: why thousands of unidentified signals matter

ABSTRACT Mass spectrometry-based proteomics has become a constitutional part of the multi-omics toolbox in yeast research, advancing fundamental knowledge of molecular processes and guiding decisions in strain and product developmental pipelines. Nevertheless, post-translational protein modifications (PTMs) continue to challenge the field of proteomics. PTMs are not directly encoded in the genome; therefore, they require a sensitive analysis of the proteome itself. In yeast, the relevance of post-translational regulators has already been established, such as for phosphorylation, which can directly affect the reaction rates of metabolic enzymes. Whereas, the selective analysis of single modifications has become a broadly employed technique, the sensitive analysis of a comprehensive set of modifications still remains a challenge. At the same time, a large number of fragmentation spectra in a typical shot-gun proteomics experiment remain unidentified. It has been estimated that a good proportion of those unidentified spectra originates from unexpected modifications or natural peptide variants. In this review, recent advancements in microbial proteomics for unrestricted protein modification discovery are reviewed, and recent research integrating this additional layer of information to elucidate protein interaction and regulation in yeast is briefly discussed.

Phospho-dependent phase separation of FMRP and CAPRIN1 recapitulates regulation of translation and deadenylation

Membraneless organelles involved in RNA processing are biomolecular condensates assembled by phase separation. Despite the important role of intrinsically disordered protein regions (IDRs), the specific interactions underlying IDR phase separation and its functional consequences remain elusive. To address these questions, we used minimal condensates formed from the C-terminal disordered regions of two interacting translational regulators, FMRP and CAPRIN1. Nuclear magnetic resonance spectroscopy of FMRP-CAPRIN1 condensates revealed interactions involving arginine-rich and aromatic-rich regions. We found that different FMRP serine/threonine and CAPRIN1 tyrosine phosphorylation patterns control phase separation propensity with RNA, including subcompartmentalization, and tune deadenylation and translation rates in vitro. The resulting evidence for residue-specific interactions underlying co–phase separation, phosphorylation-modulated condensate architecture, and enzymatic activity within condensates has implications for how the integration of signaling pathways controls RNA processing and translation.

The target specificity of the RNA binding protein Pumilio is determined by distinct co-factors

Puf family proteins are translational regulators essential to a wide range of biological processes, including cell fate specification, stem cell self-renewal, and neural function. Yet, despite being associated with hundreds of RNAs, the underlying mechanisms of Puf target specification remain to be fully elucidated. In Drosophila, Pumilio – a sole Puf family protein – is known to collaborate with cofactors Nanos (Nos) and Brain Tumor (Brat); however, their roles in target specification are not clearly defined. Here, we identify Bag-of-marbles (Bam) as a new Pum cofactor in repression of Mothers against dpp (mad) mRNAs, for which Nos is known to be dispensable. Notably, our data show that Nos (but not Bam) was required for Pum association with hunchback (hb) mRNAs, a well-known target of Pum and Nos. In contrast, Bam (but not Nos) was required for Pum association with mad mRNAs. These findings show for the first time that Pum target specificity is determined not independently but in collaboration with cofactors.

The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A) translation

Cells have evolved quality control mechanisms to ensure protein homeostasis by detecting and degrading aberrant mRNAs and proteins. A common source of aberrant mRNAs is premature polyadenylation, which can result in non-functional protein products. Translating ribosomes that encounter poly(A) sequences are terminally stalled, followed by ribosome recycling and decay of the truncated nascent polypeptide via the ribosome-associated quality control (RQC). Here, we demonstrate that the conserved RNA-binding E3 ubiquitin ligase Makorin Ring Finger Protein 1 (MKRN1) promotes ribosome stalling at poly(A) sequences during RQC. We show that MKRN1 interacts with the cytoplasmic poly(A)-binding protein (PABP) and is positioned upstream of poly(A) tails in mRNAs. Ubiquitin remnant profiling uncovers PABP and ribosomal protein RPS10, as well as additional translational regulators as main ubiquitylation substrates of MKRN1. We propose that MKRN1 serves as a first line of poly(A) recognition at the mRNA level to prevent production of erroneous proteins, thus maintaining proteome integrity.