Specific RNA-binding Proteins Research Articles

The RNA binding protein HNRNPA2B1 regulates RNA abundance and motor protein activity in neurites.

RNA molecules are localized to subcellular regions through interactions between localization-regulatory cis-elements and trans-acting RNA binding proteins (RBPs). However, the identities of RNAs whose localization is regulated by a specific RBP as well as the impacts of that RNA localization on cell function have generally remained unknown. Here, we demonstrate that the RBP HNRNPA2B1 acts to keep specific RNAs out of neuronal projections. Using subcellular fractionation, high-throughput sequencing, and single molecule RNA FISH, we find that hundreds of RNAs demonstrate markedly increased abundance in neurites in HNRNPA2B1 knockout cells. These RNAs often encode motor proteins and are enriched for known HNRNPA2B1 binding sites and motifs in their 3' UTRs. The speed and processivity of microtubule-based transport is impaired in these cells, specifically in their neurites. HNRNPA2B1 point mutations that increase its cytoplasmic abundance relative to wildtype lead to stronger suppression of RNA mislocalization defects than seen with wildtype HNRNPA2B1. We further find that the subcellular localizations of HNRNPA2B1 target RNAs are sensitive to perturbations of RNA decay machinery, suggesting that it is HNRNPA2B1's known role in regulating RNA stability that may explain these observations. These findings establish HNRNPA2B1 as a negative regulator of neurite RNA abundance and link the subcellular activities of motor proteins with the subcellular abundance of the RNAs that encode them.

CircHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA

Circular RNAs represent a class of endogenous RNAs that regulate gene expression and influence cell biological decisions with implications for the pathogenesis of several diseases. Here, we disclose a novel gene-regulatory role of circHIPK3 by combining analyses of large genomics datasets and mechanistic cell biological follow-up experiments. Using time-course depletion of circHIPK3 and specific candidate RNA-binding proteins, we identify several perturbed genes by RNA sequencing analyses. Expression-coupled motif analyses identify an 11-mer motif within circHIPK3, which also becomes enriched in genes that are downregulated upon circHIPK3 depletion. By mining eCLIP datasets and combined with RNA immunoprecipitation assays, we demonstrate that the 11-mer motif constitutes a strong binding site for IGF2BP2 in bladder cancer cell lines. Our results suggest that circHIPK3 can sequester IGF2BP2 as a competing endogenous RNA (ceRNA), leading to target mRNA stabilization. As an example of a circHIPK3-regulated gene, we focus on the STAT3 mRNA as a specific substrate of IGF2BP2 and validate that manipulation of circHIPK3 regulates IGF2BP2-STAT3 mRNA binding and, thereby, STAT3 mRNA levels. Surprisingly, absolute copy number quantifications demonstrate that IGF2BP2 outnumbers circHIPK3 by orders of magnitude, which is inconsistent with a simple 1:1 ceRNA hypothesis. Instead, we show that circHIPK3 can nucleate multiple copies of IGF2BP2, potentially via phase separation, to produce IGF2BP2 condensates. Our results support a model where a few cellular circHIPK3 molecules can induce IGF2BP2 condensation, thereby regulating key factors for cell proliferation.

CircHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA.

Circular RNAs represent a class of endogenous RNAs that regulate gene expression and influence cell biological decisions with implications for the pathogenesis of several diseases. Here, we disclose a novel gene-regulatory role of circHIPK3 by combining analyses of large genomics datasets and mechanistic cell biological follow-up experiments. Using time-course depletion of circHIPK3 and specific candidate RNA-binding proteins, we identify several perturbed genes by RNA sequencing analyses. Expression-coupled motif analyses identify an 11-mer motif within circHIPK3, which also becomes enriched in genes that are downregulated upon circHIPK3 depletion. By mining eCLIP datasets and combined with RNA immunoprecipitation assays, we demonstrate that the 11-mer motif constitutes a strong binding site for IGF2BP2 in bladder cancer cell lines. Our results suggest that circHIPK3 can sequester IGF2BP2 as a competing endogenous RNA (ceRNA), leading to target mRNA stabilization. As an example of a circHIPK3-regulated gene, we focus on the STAT3 mRNA as a specific substrate of IGF2BP2 and validate that manipulation of circHIPK3 regulates IGF2BP2-STAT3 mRNA binding and, thereby, STAT3 mRNA levels. Surprisingly, absolute copy number quantifications demonstrate that IGF2BP2 outnumbers circHIPK3 by orders of magnitude, which is inconsistent with a simple 1:1 ceRNA hypothesis. Instead, we show that circHIPK3 can nucleate multiple copies of IGF2BP2, potentially via phase separation, to produce IGF2BP2 condensates. Our results support a model where a few cellular circHIPK3 molecules can induce IGF2BP2 condensation, thereby regulating key factors for cell proliferation.

Crosstalk between Regnase-1 and -3 shapes mast cell survival and cytokine expression.

Post-transcriptional regulation of immune-related transcripts by RNA-binding proteins (RBPs) impacts immune cell responses, including mast cell functionality. Despite their importance in immune regulation, the functional role of most RBPs remains to be understood. By manipulating the expression of specific RBPs in murine mast cells, coupled with mass spectrometry and transcriptomic analyses, we found that the Regnase family of proteins acts as a potent regulator of mast cell physiology. Specifically, Regnase-1 is required to maintain basic cell proliferation and survival, whereas both Regnase-1 and -3 cooperatively regulate the expression of inflammatory transcripts upon activation, with Tnf being a primary target in both human and mouse cells. Furthermore, Regnase-3 directly interacts with Regnase-1 in mast cells and is necessary to restrain Regnase-1 expression through the destabilization of its transcript. Overall, our study identifies protein interactors of endogenously expressed Regnase factors, characterizes the regulatory interplay between Regnase family members in mast cells, and establishes their role in the control of mast cell homeostasis and inflammatory responses.

Sex-dependent interaction of PTGS2 with miR-146a as risk factor for melanoma and the impact of sex hormones in gene expression in skin cells.

Gender disparity in melanoma is a complex issue where sex hormones could be engaged. Differences in genetic variations are important in understanding the mechanisms of sex disparity in melanoma. Post-transcriptional regulation of prostaglandin-endoperoxide synthase (PTGS2) mRNA occurs through a complex interplay of specific trans-acting RNA-binding proteins and microRNAs. MiR-146a is a key player in melanoma, modulating immune responses and tumor microenvironment (TME). Polymorphisms in PTGS2 gene rs20415G<C and miR-146a gene rs2910164G>C have been associated with an increased risk of melanoma. Epistasis between polymorphisms rs20415G<C and rs2910164G>C was investigated by genotyping 453 melanoma patients and 382 control individuals. The effects of testosterone and 17β-estradiol were analyzed in keratinocytes and two melanoma cell lines. The rs2910164GG showed a higher risk in the presence of the genotype rs20417CC in the male population. Testosterone and 17β-estradiol act differently on PTGS2 and miR-146a expression, depending on the cell type. Testosterone augments PTGS2 gene expression in keratinocytes and miR-146a in melanoma cells. While 17β-estradiol only increases miR-146a expression in HaCaT cells. The present study indicates a sex-specific relation between miR-146a and PTGS2 polymorphisms with melanoma cancer risk. Testosterone and 17β-estradiol act differently on the expression of PTGS2 and miR-146a depending on the skin cell type.

Identification and validation of RNA-binding protein SLC3A2 regulates melanocyte ferroptosis in vitiligo by integrated analysis of single-cell and bulk RNA-sequencing

BackgroundThe pathogenesis of vitiligo remains unclear. The genes encoding vitiligo-related RNA-binding proteins (RBPs) and their underlying pathogenic mechanism have not been determined.ResultsSingle-cell transcriptome sequencing (scRNA-seq) data from the CNCB database was obtained to identify distinct cell types and subpopulations and the relative proportion changes in vitiligo and healthy samples. We identified 14 different cell types and 28 cell subpopulations. The proportion of each cell subpopulation significantly differed between the patients with vitiligo and healthy groups. Using RBP genes for unsupervised clustering, we obtained the specific RBP genes of different cell types in vitiligo and healthy groups. The RBP gene expression was highly heterogeneous; there were significant differences in some cell types, such as keratinocytes, Langerhans, and melanocytes, while there were no significant differences in other cells, such as T cells and fibroblasts, in the two groups. The melanocyte-specific RBP genes were enriched in the apoptosis and immune-related pathways in the patients with vitiligo. Combined with the bulk RNA-seq data of melanocytes, key RBP genes related to melanocytes were identified, including eight upregulated RBP genes (CDKN2A, HLA-A, RPL12, RPL29, RPL31, RPS19, RPS21, and RPS28) and one downregulated RBP gene (SLC3A2). Cell experiments were conducted to explore the role of the key RBP gene SLC3A2 in vitiligo. Cell experiments confirmed that melanocyte proliferation decreased, whereas apoptosis increased, after SLC3A2 knockdown. SLC3A2 knockdown in melanocytes also decreased the SOD activity and melanin content; increased the Fe2+, ROS, and MDA content; significantly increased the expression levels of TYR and COX2; and decreased the expression levels of glutathione and GPX4.ConclusionWe identified the RBP genes of different cell subsets in patients with vitiligo and confirmed that downregulating SLC3A2 can promote ferroptosis in melanocytes. These findings provide new insights into the pathogenesis of vitiligo.

Predicting circRNA-RBP Binding Sites Using a Hybrid Deep Neural Network.

Circular RNAs (circRNAs) are non-coding RNAs generated by reverse splicing. They are involved in biological process and human diseases by interacting with specific RNA-binding proteins (RBPs). Due to traditional biological experiments being costly, computational methods have been proposed to predict the circRNA-RBP interaction. However, these methods have problems of single feature extraction. Therefore, we propose a novel model called circ-FHN, which utilizes only circRNA sequences to predict circRNA-RBP interactions. The circ-FHN approach involves feature coding and a hybrid deep learning model. Feature coding takes into account the physicochemical properties of circRNA sequences and employs four coding methods to extract sequence features. The hybrid deep structure comprises a convolutional neural network (CNN) and a bidirectional gated recurrent unit (BiGRU). The CNN learns high-level abstract features, while the BiGRU captures long-term dependencies in the sequence. To assess the effectiveness of circ-FHN, we compared it to other computational methods on 16 datasets and conducted ablation experiments. Additionally, we conducted motif analysis. The results demonstrate that circ-FHN exhibits exceptional performance and surpasses other methods. circ-FHN is freely available at https://github.com/zhaoqi106/circ-FHN .

TcZC3HTTP, a regulatory element that contributes to Trypanosoma cruzi cell proliferation.

Post-transcriptional regulation of gene expression is a critical process for adapting to and surviving Trypanosoma cruzi, a parasite with a complex life cycle. RNA-binding proteins (RBPs) are key players in this regulation, forming ribonucleoprotein complexes (messenger ribonucleoproteins) and RNA granules that control transcript stability, localization, degradation, and translation modulation. Understanding the specific roles of individual RBPs is crucial for unraveling the details of this regulatory network. In this study, we generated null mutants of the TcZC3HTTP gene, a specific RBP in the Trypanosoma family characterized by a C3H zinc finger and a DNAJ domain associated with RNA and protein binding, respectively. Through cell growth assays, we demonstrated that the absence of TcZC3HTTP or the expression of an additional tagged version impacted epimastigote growth, indicating its contribution to cell proliferation. TcZC3HTTP was found to associate with mRNAs involved in cell cycle and division in epimastigotes, while in nutritionally stressed parasites it exhibited associations with mRNAs coding for other RBPs and rRNA. Furthermore, our analysis identified that TcZC3HTTP protein partners were different during normal growth conditions compared to starvation conditions, with the latter showing enrichment of ribosomal proteins and other RBPs. Therefore, this study provides insights into TcZC3HTTP's role in the post-transcriptional regulation of gene expression during normal growth and nutritional stress in T. cruzi, uncovering its versatile functions in different cellular contexts.IMPORTANCEUnderstanding how Trypanosoma cruzi, the causative agent of Chagas disease, regulates gene expression is crucial for developing targeted interventions. In this study, we investigated the role of TcZC3HTTP, an RNA-binding protein, in post-transcriptional regulation. Our findings demonstrate that TcZC3HTTP is relevant for the growth and proliferation of epimastigotes, a stage of the parasite's life cycle. We identified its associations with specific mRNAs involved in cell cycle and division and its interactions with enzymes and other RNA-binding proteins (RBPs) under normal and starvation conditions. These insights shed light on the regulatory network underlying gene expression in T. cruzi and reveal the multifaceted functions of RBPs in this parasite. Such knowledge enhances our understanding of the parasite's biology and opens avenues for developing novel therapeutic strategies targeting post-transcriptional gene regulation in T. cruzi.

Neofunctionalization driven by positive selection led to the retention of the loqs2 gene encoding an Aedes specific dsRNA binding protein

BackgroundMosquito borne viruses, such as dengue, Zika, yellow fever and Chikungunya, cause millions of infections every year. These viruses are mostly transmitted by two urban-adapted mosquito species, Aedes aegypti and Aedes albopictus. Although mechanistic understanding remains largely unknown, Aedes mosquitoes may have unique adaptations that lower the impact of viral infection. Recently, we reported the identification of an Aedes specific double-stranded RNA binding protein (dsRBP), named Loqs2, that is involved in the control of infection by dengue and Zika viruses in mosquitoes. Preliminary analyses suggested that the loqs2 gene is a paralog of loquacious (loqs) and r2d2, two co-factors of the RNA interference (RNAi) pathway, a major antiviral mechanism in insects.ResultsHere we analyzed the origin and evolution of loqs2. Our data suggest that loqs2 originated from two independent duplications of the first double-stranded RNA binding domain of loqs that occurred before the origin of the Aedes Stegomyia subgenus, around 31 million years ago. We show that the loqs2 gene is evolving under relaxed purifying selection at a faster pace than loqs, with evidence of neofunctionalization driven by positive selection. Accordingly, we observed that Loqs2 is localized mainly in the nucleus, different from R2D2 and both isoforms of Loqs that are cytoplasmic. In contrast to r2d2 and loqs, loqs2 expression is stage- and tissue-specific, restricted mostly to reproductive tissues in adult Ae. aegypti and Ae. albopictus. Transgenic mosquitoes engineered to express loqs2 ubiquitously undergo developmental arrest at larval stages that correlates with massive dysregulation of gene expression without major effects on microRNAs or other endogenous small RNAs, classically associated with RNA interference.ConclusionsOur results uncover the peculiar origin and neofunctionalization of loqs2 driven by positive selection. This study shows an example of unique adaptations in Aedes mosquitoes that could ultimately help explain their effectiveness as virus vectors.

Role of C9orf72 hexanucleotide repeat expansions in ALS/FTD pathogenesis.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are progressive neurological disorders that share neurodegenerative pathways and features. The most prevalent genetic causes of ALS/FTD is the GGGGCC hexanucleotide repeat expansions in the first intron region of the chromosome 9 open reading frame 72 (C9orf72) gene. In this review, we comprehensively summarize the accumulating evidences elucidating the pathogenic mechanism associated with hexanucleotide repeat expansions in ALS/FTD. These mechanisms encompass the structural polymorphism of DNA and transcribed RNA, the formation of RNA foci via phase separation, and the cytoplasmic accumulation and toxicities of dipeptide-repeat proteins. Additionally, the formation of G-quadruplex structures significantly impairs the expression and normal function of the C9orf72 protein. We also discuss the sequestration of specific RNA binding proteins by GGGGCC RNA, which further contributes to the toxicity of C9orf72 hexanucleotide repeat expansions. The deeper understanding of the pathogenic mechanism of hexanucleotide repeat expansions in ALS/FTD provides multiple potential drug targets for these devastating diseases.

Abstract C082: Decoding the KRAS-dependent proteome through the ribosomal lens

Abstract Our study addresses the gap in understanding the KRAS-specific translation landscape and functional proteome in cancer. We demonstrate that mutant KRAS drives distinct mechanisms of translational control to fuel cancer growth. By leveraging ribosome footprinting and advanced sequencing techniques, we investigate the impact of sotorasib, a targeted inhibitor of G12C mutant KRAS activity, on translation. Notably, KRAS (G12C) inhibition profoundly alters the translation of ribosomal proteins and translation elongation factors. Additionally, we reveal that KRAS inhibition resulted in ribosome stalling on a specific subset of mRNAs. Acute treatment with sotorasib hampers the translation of crucial oncogenic proteins, including MYC, as well as proteins involved in metabolism. These translational changes are facilitated by specific RNA motifs and RNA binding proteins that respond to KRAS activity. In essence, our study delineates the KRAS (G12C)-specific regulation of translation and elucidates the mechanism underlying KRAS (G12C)-mediated control over the cancer proteome. Citation Format: Kamini Singh, Trang Uyen Nguyen, Eric Nels Pederson, Vinay Kumar, Xiaoling Yun, Zhengqing Ouyang. Decoding the KRAS-dependent proteome through the ribosomal lens [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C082.

RNA structure profiling at single-cell resolution reveals new determinants of cell identity.

RNA structure is critical for multiple steps in gene regulation. However, how the structures of transcripts differ both within and between individual cells is unknown. Here we develop a SHAPE-inspired method called single-cell structure probing of RNA transcripts that enables simultaneous determination of transcript secondary structure and abundance at single-cell resolution. We apply single-cell structure probing of RNA transcripts to human embryonic stem cells and differentiating neurons. Remarkably, RNA structure is more homogeneous in human embryonic stem cells compared with neurons, with the greatest homogeneity found in coding regions. More extensive heterogeneity is found within 3' untranslated regions and is determined by specific RNA-binding proteins. Overall RNA structure profiles better discriminate cell type identity and differentiation stage than gene expression profiles alone. We further discover a cell-type variable region of 18S ribosomal RNA that is associated with cell cycle and translation control. Our method opens the door to the systematic characterization of RNA structure-function relationships at single-cell resolution.

RNA binding proteins in cardiovascular development and disease.

Congenital heart disease (CHD) is the most common birth defect affecting>1.35 million newborn babies worldwide. CHD can lead to prenatal, neonatal, postnatal lethality or life-long cardiac complications. RNA binding protein (RBP) mutations or variants are emerging as contributors to CHDs. RBPs are wizards of gene regulation and are major contributors to mRNA and protein landscape. However, not much is known about RBPs in the developing heart and their contributions to CHD. In this chapter, we will discuss our current knowledge about specific RBPs implicated in CHDs. We are in an exciting era to study RBPs using the currently available and highly successful RNA-based therapies and methodologies. Understanding how RBPs shape the developing heart will unveil their contributions to CHD. Identifying their target RNAs in the embryonic heart will ultimately lead to RNA-based treatments for congenital heart disease.

Identification of bona fide RNA G-quadruplex binding proteins.

RNAs often accomplish their diverse functions through direct interactions with RNA-binding proteins (RBPs) in a sequence- and/or structure-dependent manner. RNA G-quadruplexes (rG4s) are unique secondary structures formed by guanine-rich RNA sequences which impact RNA function independently and in combination with RBPs. Efforts from several labs have identified dozens of rG4 specific RBPs (rG4BPs), although the research is still in the growing phase. Here we present methods for the systematic identification of rG4BPs using a pull-down approach that takes advantage of the chemical modification of guanine bases. This allows abolishing the rG4 structures while still maintaining the base composition intact, and hence helps in recognizing true rG4BPS (in contrast to G-rich motif binders). In combination with other biochemical assays, such an approach can be efficiently used for the identification and characterization of bona fide rG4BPs.

Post-transcriptional mechanisms controlling neurogenesis and direct neuronal reprogramming.

Neurogenesis is a tightly regulated process in time and space both in the developing embryo and in adult neurogenic niches. A drastic change in the transcriptome and proteome of radial glial cells or neural stem cells towards the neuronal state is achieved due to sophisticated mechanisms of epigenetic, transcriptional, and post-transcriptional regulation. Understanding these neurogenic mechanisms is of major importance, not only for shedding light on very complex and crucial developmental processes, but also for the identification of putative reprogramming factors, that harbor hierarchically central regulatory roles in the course of neurogenesis and bare thus the capacity to drive direct reprogramming towards the neuronal fate. The major transcriptional programs that orchestrate the neurogenic process have been the focus of research for many years and key neurogenic transcription factors, as well as repressor complexes, have been identified and employed in direct reprogramming protocols to convert non-neuronal cells, into functional neurons. The post-transcriptional regulation of gene expression during nervous system development has emerged as another important and intricate regulatory layer, strongly contributing to the complexity of the mechanisms controlling neurogenesis and neuronal function. In particular, recent advances are highlighting the importance of specific RNA binding proteins that control major steps of mRNA life cycle during neurogenesis, such as alternative splicing, polyadenylation, stability, and translation. Apart from the RNA binding proteins, microRNAs, a class of small non-coding RNAs that block the translation of their target mRNAs, have also been shown to play crucial roles in all the stages of the neurogenic process, from neural stem/progenitor cell proliferation, neuronal differentiation and migration, to functional maturation. Here, we provide an overview of the most prominent post-transcriptional mechanisms mediated by RNA binding proteins and microRNAs during the neurogenic process, giving particular emphasis on the interplay of specific RNA binding proteins with neurogenic microRNAs. Taking under consideration that the molecular mechanisms of neurogenesis exert high similarity to the ones driving direct neuronal reprogramming, we also discuss the current advances in in vitro and in vivo direct neuronal reprogramming approaches that have employed microRNAs or RNA binding proteins as reprogramming factors, highlighting the so far known mechanisms of their reprogramming action.

LhCLIP reveals the in vivo RNA-RNA interactions recognized by hnRNPK.

RNA-RNA interactions play a crucial role in regulating gene expression and various biological processes, but identifying these interactions on a transcriptomic scale remains a challenge. To address this, we have developed a new biochemical technique called pCp-biotin labelled RNA hybrid and ultraviolet crosslinking and immunoprecipitation (lhCLIP) that enables the transcriptome-wide identification of intra- and intermolecular RNA-RNA interactions mediated by a specific RNA-binding protein (RBP). Using lhCLIP, we have uncovered a diverse landscape of intermolecular RNA interactions recognized by hnRNPK in human cells, involving all major classes of noncoding RNAs (ncRNAs) and mRNA. Notably, hnRNPK selectively binds with snRNA U4, U11, and U12, and shapes the secondary structure of these snRNAs, which may impact RNA splicing. Our study demonstrates the potential of lhCLIP as a user-friendly and widely applicable method for discovering RNA-RNA interactions mediated by a particular protein of interest and provides a valuable tool for further investigating the role of RBPs in gene expression and biological processes.

Crosstalk between m6A modification and alternative splicing during cancer progression.

Background N6-methyladenosine (m6A), the most prevalent internal mRNA modification in eukaryotes, isadded bym6Amethyltransferases, removed by m6A demethylases and recognised by m6A-binding proteins. This modification significantly influences carious facets of RNA metabolism and plays a pivotal role in cellular and physiological processes. Main body Pre-mRNA alternative splicing, a process that generates multiple splice isoforms from multi-exon genes, contributes significantly to the protein diversity in mammals. Moreover, the presence of crosstalk between m6A modification and alternative splicing, with m6A modifications on pre-mRNAs exerting regulatory control, has been established. The m6A modification modulates alternative splicing patterns by recruiting specific RNA-binding proteins (RBPs) that regulate alternative splicing or by directly influencing the interaction between RBPs and their target RNAs. Conversely, alternative splicing can impact the deposition or recognition of m6A modification on mRNAs. The integration of m6A modifications has expanded the scope of therapeutic strategies for cancer treatment, while alternative splicing offers novel insights into the mechanistic role of m6A methylation in cancer initiation and progression. Conclusion This review aims to highlight the biological functions of alternative splicing of m6A modification machinery and its implications in tumourigenesis. Furthermore, we discuss the clinical relevance of understanding m6A-dependent alternative splicing in tumour therapies.

Stress granules: stress-induced cytoplasmic mRNPs compartments linked to mRNA translational regulatory pathways

Stress granules (SG) are macro-complexes that assemble as phase-separated and dynamic RNA biocondensates in the cytoplasm of the eukaryotic cell when the initiation step of the general translation of mRNAs is stalled. This occurs mainly as an adaptive cell response to either environmental (i.e., radiation, exposure to chemical drugs), pathological (i.e., viral treatment), physiological (i.e., oxygen-, amino acids-, and glucose-deprivation), or therapeutic (i.e., treatment with anti-cancer drugs) translational stress. SG also formed when translation initiation is blocked through stress-independent events including alteration of the activities of specific translation initiation factors and RNA-binding proteins. Both stress-dependent and–independent inhibition of translation initiation results in the accumulation of untranslated mRNAs, considered as integral components of SG. Consistently, in vivo assays of SG assembly combined with in vitro-based assembly of SG-like biocondensates studies support a fundamental role of the accumulation of untranslated mRNA in initiating the formation of SG, which then further promote their repression, potentially in a feed-back regulatory mechanism. The potential role of SG in actively repressing translation of associated mRNAs has been supported by a number of functional studies, establishing SG as critical regulatory sites of RNA homeostasis, in particular during stress. The view that the SG environment restricts translation of associated mRNAs was however challenged in studies showing that stress-induced translation repression can occur similarly in absence and presence of SG, leading to the emerging concept that formation of SG and translation repression are uncoupled processes. While it still a debate if mRNA recruitment to SG contributes to their translation repression, recent finding reported translation of reporter mRNAs in SG, suggesting rather an active translational role of SG. In this review, we describe the main translational signaling pathways that regulate the biology of SG, summarize current data supporting RNA as an integral functional component of SG, and then discuss evidence supporting or not the role of SG in regulating translation either negatively or positively during stress.

Abstract P2103: Cardiac Fibroblast-specific Role Of PTBP1 In Maintaining Heart Function

Cardiac fibroblasts (CFs) are the primary nonmyocyte in the heart that maintain cardiac homeostasis by regulating myocardial function and cardiac remodeling. CFs secrete an extracellular matrix to stabilize the myocardial wall while maintaining the heart’s mechanical integrity during development and pathogenesis. Developmental and pathogenic transitions are directed through CFs by transcriptional and post-transcriptional modifications, such as alternative splicing. Alternative splicing is a central element in post-transcriptional gene regulation and proteomic diversity. Impaired and aberrant splicing is known to cause developmental defects and drive cardiac pathogenesis. RNA binding proteins (RBPs), which function as alternative splicing factors, are upregulated in cardiovascular disease states. While nearly a third of all CF gene regulation changes during cardiac fibrosis are subject to transcriptional regulation via RBPs, the role of CF specific splicing RBPs in the adult heart and during cardiac fibrosis is unknown. Our group previously identified high expression of well-known RBP and splicing factor polypyrimidine tract-binding protein (PTBP1) in CFs when compared to cardiomyocytes in the adult heart. With single cell transcriptomics we found PTBP1 regulates CF identity and plasticity, suggesting an emergent role of PTBP1 within CF biology. We hypothesize PTBP1 is essential for maintaining cardiac fibroblast function in the adult heart. Preliminary in vitro data suggests partial Ptbp1 knockdown impacts mechanical function by decreasing overall CF migration. To further characterize PTBP1 in vivo , we have validated our novel mouse model to specifically delete PTBP1 from CFs in adult mice. Following PTBP1 deletion, we will utilize an established model of fibrosis by Angiotensin II osmotic minipumps to characterize the CF specific PTBP1 loss during fibrosis. We will evaluate CF biology with our in vitro and in vivo models to examine PTBP1’s physiological and mechanistic role in maintaining CF biology during cardiac homeostasis and cardiac fibrosis pathogenesis.

Two RNA binding proteins, ADAD2 and RNF17, interact to form a heterogeneous population of novel meiotic germ cell granules with developmentally dependent organelle association.

Mammalian male germ cell differentiation relies on complex RNA biogenesis events, many of which occur in non-membrane bound organelles termed RNA germ cell granules that are rich in RNA binding proteins (RBPs). Though known to be required for male germ cell differentiation, we understand little of the relationships between the numerous granule subtypes. ADAD2, a testis specific RBP, is required for normal male fertility and forms a poorly characterized granule in meiotic germ cells. This work aimed to understand the role of ADAD2 granules in male germ cell differentiation by clearly defining their molecular composition and relationship to other granules. Biochemical analyses identified RNF17, a testis specific RBP that forms meiotic male germ cell granules, as an ADAD2-interacting protein. Phenotypic analysis of Adad2 and Rnf17 mutants identified a rare post-meiotic chromatin defect, suggesting shared biological roles. ADAD2 and RNF17 were found to be dependent on one another for granularization and together form a previously unstudied set of germ cell granules. Based on co-localization studies with well-characterized granule RBPs and organelle-specific markers, a subset of the ADAD2-RNF17 granules are found to be associated with the intermitochondrial cement and piRNA biogenesis. In contrast, a second, morphologically distinct population of ADAD2-RNF17 granules co-localized with the translation regulators NANOS1 and PUM1, along with the molecular chaperone PDI. These large granules form a unique funnel-shaped structure that displays distinct protein subdomains and is tightly associated with the endoplasmic reticulum. Developmental studies suggest the different granule populations represent different phases of a granule maturation process. Lastly, a double Adad2-Rnf17 mutant model suggests the interaction between ADAD2 and RNF17, as opposed to loss of either, is the likely driver of the Adad2 and Rnf17 mutant phenotypes. These findings shed light on the relationship between germ cell granule pools and define new genetic approaches to their study.