First person – Akruti Shah

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Akruti Shah is first author on ‘ IP6K1 upregulates the formation of processing bodies by influencing protein-protein interactions on the mRNA cap’, published in JCS. Akruti is a PhD student in the lab of Dr Rashna Bhandari at the Centre for DNA Fingerprinting and Diagnostics, Hyderabad, Telangana, India, investigating the effect of post-translational modifications and protein–protein interactions on mRNA metabolism in health and disease.

Nuclear mRNA metabolism drives selective basket assembly on a subset of nuclear pores in budding yeast

To determine which transcripts should reach the cytoplasm for translation, eukaryotic cells have established mechanisms to regulate selective mRNA export through the nuclear pore complex (NPC). The nuclear basket, a substructure of the NPC protruding into the nucleoplasm, is thought to function as a stable platform where mRNA-protein complexes (mRNPs) are rearranged and undergo quality control (QC) prior to export, ensuring that only mature mRNAs reach the cytoplasm. Here, we use proteomic, genetic, live-cell, and single-molecule resolution microscopy approaches in budding yeast to demonstrate that baskets assemble only on a subset of NPCs and that basket formation is dependent on RNA polymerase II (Pol II) transcription and subsequent mRNP processing. Specifically, we observe that the cleavage and polyadenylation machinery, the poly(A)-binding protein Pab1, and pre-mRNA-leakage factor Pml39 are required for basket assembly. We further show that while all nuclear pores can bind Mlp1, baskets assemble only on a subset of nucleoplasmic NPCs and these basket-containing pores associate a distinct protein and RNA interactome. Taken together, our data points towards nuclear pore heterogeneity and an RNA-dependent mechanism for functionalization of nuclear pores in budding yeast through nuclear basket assembly.

RNA Targeting in Inherited Neuromuscular Disorders: Novel Therapeutic Strategies to Counteract Mis-Splicing

Neuromuscular disorders represent multifaceted abnormal conditions, with little or no cure, leading to patient deaths from complete muscle wasting and atrophy. Despite strong efforts in the past decades, development of effective treatments is still urgently needed. Advent of next-generation sequencing technologies has allowed identification of novel genes and mutations associated with neuromuscular pathologies, highlighting splicing defects as essential players. Deciphering the significance and relative contributions of defective RNA metabolism will be instrumental to address and counteract these malignancies. We review here recent progress on the role played by alternative splicing in ensuring functional neuromuscular junctions (NMJs), and its involvement in the pathogenesis of NMJ-related neuromuscular disorders, with particular emphasis on congenital myasthenic syndromes and muscular dystrophies. We will also discuss novel strategies based on oligonucleotides designed to bind their cognate sequences in the RNA or targeting intermediary of mRNA metabolism. These efforts resulted in several chemical classes of RNA molecules that have recently proven to be clinically effective, more potent and better tolerated than previous strategies.

A system biology approach identifies candidate drugs to reduce mortality in severely ill COVID-19 patients

Despite the availability of highly efficacious vaccines, Coronavirus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) lacks effective drug treatment which results in a high rate of mortality. To address this therapeutic shortcoming, we applied a system biology approach to the study of patients hospitalized with severe COVID. We show that, at the time of hospital admission, patients who were equivalent on the clinical ordinal scale displayed significant differential monocyte epigenetic and transcriptomic attributes between those who would survive and those who would succumb to COVID-19. We identified mRNA metabolism, RNA splicing, and interferon signaling pathways as key host responses overactivated by patients who would not survive. Those pathways are prime drug targets to reduce mortality of critically ill COVID-19 patients leading us to identify Tacrolimus, Zotatifin, and Nintedanib as three strong candidates for treatment of severely ill patients at the time of hospital admission.

The Role of m6A Ribonucleic Acid Modification in the Occurrence of Atherosclerosis

The N6-methyladenosine (m6A) modification is the most abundant epitranscriptomic modification in eukaryotic messenger RNA (mRNA). The m6A modification process is jointly regulated by various enzymes and proteins, such as methyltransferases, demethylases and related m6A-binding proteins. The process is dynamic and reversible, and it plays an essential role in mRNA metabolism and various biological activities. Recently, an increasing number of researchers have confirmed that the onset and development of many diseases are closely associated with the molecular biological mechanism of m6A RNA methylation. This study focuses on the relationship between m6A RNA modification and atherosclerosis (AS). It thoroughly summarizes the mechanisms and processes of m6A RNA modification in AS-related cells and the relationships between m6A RNA modification and AS risk factors, and it provides a reference for exploring new targets for the early diagnosis and treatment of AS.

Star-PAP RNA Binding Landscape Reveals Novel Role of Star-PAP in mRNA Metabolism That Requires RBM10-RNA Association

Star-PAP is a non-canonical poly(A) polymerase that selects mRNA targets for polyadenylation. Yet, genome-wide direct Star-PAP targets or the mechanism of specific mRNA recognition is still vague. Here, we employ HITS-CLIP to map the cellular Star-PAP binding landscape and the mechanism of global Star-PAP mRNA association. We show a transcriptome-wide association of Star-PAP that is diminished on Star-PAP depletion. Consistent with its role in the 3′-UTR processing, we observed a high association of Star-PAP at the 3′-UTR region. Strikingly, there is an enrichment of Star-PAP at the coding region exons (CDS) in 42% of target mRNAs. We demonstrate that Star-PAP binding de-stabilises these mRNAs indicating a new role of Star-PAP in mRNA metabolism. Comparison with earlier microarray data reveals that while UTR-associated transcripts are down-regulated, CDS-associated mRNAs are largely up-regulated on Star-PAP depletion. Strikingly, the knockdown of a Star-PAP coregulator RBM10 resulted in a global loss of Star-PAP association on target mRNAs. Consistently, RBM10 depletion compromises 3′-end processing of a set of Star-PAP target mRNAs, while regulating stability/turnover of a different set of mRNAs. Our results establish a global profile of Star-PAP mRNA association and a novel role of Star-PAP in the mRNA metabolism that requires RBM10-mRNA association in the cell.

Transcriptome-wide m6A profiling reveals mRNA post-transcriptional modification of boar sperm during cryopreservation

Background Cryopreservation induces transcriptomic and epigenetic modifications that strongly impairs sperm quality and function, and thus decrease reproductive performance. N6-methyladenosine (m6A) RNA methylation varies in response to stress and has been implicated in multiple important biological processes, including post-transcriptional fate of mRNA, metabolism, and apoptosis. This study aimed to explore whether cryopreservation induces m6A modification of mRNAs associated with sperm energy metabolism, cryoinjuries, and freezability. Results The mRNA and protein expression of m6A modification enzymes were significantly dysregulated in sperm after cryopreservation. Furthermore, m6A peaks were mainly enriched in coding regions and near stop codons with classical RRACH motifs. The mRNAs containing highly methylated m6A peaks (fts vs. fs) were significantly associated with metabolism and gene expression, while the genes with less methylated m6A peaks were primarily involved in processes regulating RNA metabolism and transcription. Furthermore, the joint analysis of DMMGs and differentially expressed genes indicated that both of these play a vital role in sperm energy metabolism and apoptosis. Conclusions Our study is the first to reveal the dynamic m6A modification of mRNAs in boar sperm during cryopreservation. These epigenetic modifications may affect mRNA expression and are closely related to sperm motility, apoptosis, and metabolism, which will provide novel insights into understanding of the cryoinjuries or freezability of boar sperm during cryopreservation.

Splicing Factor SRSF1 Deficiency in the Liver Triggers NASH-like Pathology via R-Loop Induced DNA Damage and Cell Death

Regulation of RNA processing contributes profoundly to tissue development and physiology. Here, we report that serine–arginine–rich splicing factor 1 (SRSF1) is essential for hepatocyte function and survival. Although SRSF1 is mainly known for its many roles in mRNA metabolism, it is also crucial for maintaining genome stability. We show that acute liver damage in the setting of targeted SRSF1 deletion in mice is primarily mediated by the excessive formation of deleterious RNA–DNA hybrids (R–loops), which induce DNA damage. Combining hepatocyte–specific transcriptome, proteome, and RNA binding analyses, we demonstrate that widespread genotoxic stress following SRSF1 depletion results in global inhibition of mRNA transcription and protein synthesis, leading to impaired metabolism and trafficking of lipids. Lipid accumulation in SRSF1–deficient hepatocytes is followed by necroptotic cell death, inflammation, and fibrosis, resulting in NASH–like liver pathology. Importantly, SRSF1–depleted human liver cancer cells recapitulate this pathogenesis illustrating a conserved and fundamental role for SRSF1 in preserving genome integrity and tissue homeostasis. Thus, our study uncovers how accumulation of detrimental R–loops impedes hepatocellular gene expression, triggering metabolic derangement and liver failure.

Multivariate transcriptome analysis identifies networks and key drivers of chronic lymphocytic leukemia relapse risk and patient survival

Background Chronic lymphocytic leukemia (CLL) is an indolent heme malignancy characterized by the accumulation of CD5+ CD19+ B cells and episodes of relapse. The biological signaling that influence episodes of relapse in CLL are not fully described. Here, we identify gene networks associated with CLL relapse and survival risk. Methods Networks were investigated by using a novel weighted gene network co-expression analysis method and examining overrepresentation of upstream regulators and signaling pathways within co-expressed transcriptome modules across clinically annotated transcriptomes from CLL patients (N = 203). Gene Ontology analysis was used to identify biological functions overrepresented in each module. Differential Expression of modules and individual genes was assessed using an ANOVA (Binet Stage A and B relapsed patients) or T-test (SF3B1 mutations). The clinical relevance of biomarker candidates was evaluated using log-rank Kaplan Meier (survival and relapse interval) and ROC tests. Results Eight distinct modules (M2, M3, M4, M7, M9, M10, M11, M13) were significantly correlated with relapse and differentially expressed between relapsed and non-relapsed Binet Stage A CLL patients. The biological functions of modules positively correlated with relapse were carbohydrate and mRNA metabolism, whereas negatively correlated modules to relapse were protein translation associated. Additionally, M1, M3, M7, and M13 modules negatively correlated with overall survival. CLL biomarkers BTK, BCL2, and TP53 were co-expressed, while unmutated IGHV biomarker ZAP70 and cell survival-associated NOTCH1 were co-expressed in modules positively correlated with relapse and negatively correlated with survival days. Conclusions This study provides novel insights into CLL relapse biology and pathways associated with known and novel biomarkers for relapse and overall survival. The modules associated with relapse and overall survival represented both known and novel pathways associated with CLL pathogenesis and can be a resource for the CLL research community. The hub genes of these modules, e.g., ARHGAP27P2, C1S, CASC2, CLEC3B, CRY1, CXCR5, FUT5, MID1IP1, and URAHP, can be studied further as new therapeutic targets or clinical markers to predict CLL patient outcomes.

Hakai is required for stabilization of core components of the m6A mRNA methylation machinery

N6-methyladenosine (m6A) is the most abundant internal modification on mRNA which influences most steps of mRNA metabolism and is involved in several biological functions. The E3 ubiquitin ligase Hakai was previously found in complex with components of the m6A methylation machinery in plants and mammalian cells but its precise function remained to be investigated. Here we show that Hakai is a conserved component of the methyltransferase complex in Drosophila and human cells. In Drosophila, its depletion results in reduced m6A levels and altered m6A-dependent functions including sex determination. We show that its ubiquitination domain is required for dimerization and interaction with other members of the m6A machinery, while its catalytic activity is dispensable. Finally, we demonstrate that the loss of Hakai destabilizes several subunits of the methyltransferase complex, resulting in impaired m6A deposition. Our work adds functional and molecular insights into the mechanism of the m6A mRNA writer complex.