Family Of RNA-binding Proteins Research Articles

#4976 DIFFERENTIAL ROLES OF REGNASE3 IN RESIDENT MACROPHAGE VERSUS TUBULAR EPITHELIAL CELLS IN THE HEALTHY OR POSTISCHEMIC KIDNEY

Abstract Background and Aims Acute kidney injury (AKI) is a prevalent yet severe condition that occurs in clinical settings. The prognosis for AKI is poor, with many patients progressing to AKD, CKD, and even ESKD especially in those who were admitted to the ICU. RNA-binding proteins (RBPs) are a class of proteins that play a vital role in regulating gene expression and have been implicated in a wide range of diseases. Regnase3 is a member of the Regnase RBPs family and has been shown to promote inflammation by increasing TNF in macrophages. Despite this knowledge, the full extent of the role played by Regnase3 in AKI remains unknown. Therefore, we hypothesized that Regnase3 plays a role in both macrophages and tubular epithelial cells, influencing inflammation and tubular repair in AKI. Method In order to investigate the role of Regnase3 in kidney injury, a series of genetically-modified mice were developed on the C57BL/6J genetic background. These mice included the paired box gene 8 (Pax8)-reverse tetracycline transactivator (rTtA, Pax8rTtA), tetracycline resistance protein (TetO)-Cre (TetOCre), transgenic receptor activator of nuclear factor kappa-Β (Rank)-Cre (RankCre), and Regnase3 floxed (Regnase-3fl/fl) mice. Using these genetically modified mice, we applied a range of experimental animal models to study the effects of Regnase3 on kidney injury, including unilateral kidney ischemia-reperfusion surgery with or without nephrectomy and nephrocalcinosis-related kidney injury. Additionally, we employed in vitro models utilizing primary cells to study Regnase3 on the cellular level. Furthermore, we utilized scRNA-seq and RNA-seq to investigate the underlying mechanisms of Regnase3’s function. Results We conducted a thorough investigation and discovered that the Regnase3 is highly expressed in macrophages located within the kidney after AKI. The scRNA-seq showed the expression Regnase3 positively correlated with the phagocytosis, chemokines, and monocyte maturation. Furthermore, Rank-Regnase3 mice suffered from more inflammation and kidney injury after AKI, characterized by an increase in CCR2 positive leukocyte infiltration. Additionally, in vitro experiments revealed that Regnase3 is involved in modulating macrophage behavior, acting as a regulator of macrophage polarization towards an M1 and M2 phenotype and influencing the capacity of cell migration. These findings indicate that Regnase3 is essential in the migration of macrophages after AKI, which contribute to both early phase inflammation and progression towards AKI- CKD. Next, we aimed to examine the role of Regnase3 in the context of renal tubule injury. We observed that Regnase3 is highly expressed in healthy renal tubules, but its expression is reduced following injury. Through in silico, in vivo and in vitro experiments, we found that Regnase3 controls early apoptosis, cell death, proliferation, and wound healing capability of tubular epithelial cells. These effects contribute to kidney injury in postischemic kidneys. Additionally, we discovered that the deletion of Regnase3 provides protection against ischemic AKI (Figure 1). Mechanistically, Regnase3 targets pre-RNA and modulates alternative splicing by increasing the proportion of targeted skipped exon events and decreasing the probability of targeted retained intron events. Overall, our findings indicate that Regnase3 plays a vital role in tubular epithelial cells in the response to kidney injury by regulating alternative splicing. Conclusion In summary, our findings indicate that Regnase3 contributes to kidney injury in postischemic kidneys. However, the impact of Regnase3 is contingent upon the specific cell lineage in question. The Rank-Regnase3 leads to an exacerbation of kidney injury by increasing macrophage recruitment, whereas the Pax8-Regnase3 leads to an improvement in kidney injury through its effects on cell death and wound healing capability of tubular epithelial cells. Therefore, the role of Regnase3 in kidney injury is dependent on the cell lineage, which ultimately determines the detrimental or beneficial impact on kidney injury.

A combined computational and functional approach identifies IGF2BP2 as a driver of chemoresistance in a wide array of pre-clinical models of colorectal cancer

AimChemoresistance is a major cause of treatment failure in colorectal cancer (CRC) therapy. In this study, the impact of the IGF2BP family of RNA-binding proteins on CRC chemoresistance was investigated using in silico, in vitro, and in vivo approaches.MethodsGene expression data from a well-characterized cohort and publicly available cross-linking immunoprecipitation sequencing (CLIP-Seq) data were collected. Resistance to chemotherapeutics was assessed in patient-derived xenografts (PDXs) and patient-derived organoids (PDOs). Functional studies were performed in 2D and 3D cell culture models, including proliferation, spheroid growth, and mitochondrial respiration analyses.ResultsWe identified IGF2BP2 as the most abundant IGF2BP in primary and metastastatic CRC, correlating with tumor stage in patient samples and tumor growth in PDXs. IGF2BP2 expression in primary tumor tissue was significantly associated with resistance to selumetinib, gefitinib, and regorafenib in PDOs and to 5-fluorouracil and oxaliplatin in PDX in vivo. IGF2BP2 knockout (KO) HCT116 cells were more susceptible to regorafenib in 2D and to oxaliplatin, selumitinib, and nintedanib in 3D cell culture. Further, a bioinformatic analysis using CLIP data suggested stabilization of target transcripts in primary and metastatic tumors. Measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) revealed a decreased basal OCR and an increase in glycolytic ATP production rate in IGF2BP2 KO. In addition, real-time reverse transcriptase polymerase chain reaction (qPCR) analysis confirmed decreased expression of genes of the respiratory chain complex I, complex IV, and the outer mitochondrial membrane in IGF2BP2 KO cells.ConclusionsIGF2BP2 correlates with CRC tumor growth in vivo and promotes chemoresistance by altering mitochondrial respiratory chain metabolism. As a druggable target, IGF2BP2 could be used in future CRC therapy to overcome CRC chemoresistance.

Alternative Splicing Outcomes Across an RNA-Binding Protein Concentration Gradient

Alternative splicing (AS) is a dynamic RNA processing step that produces multiple RNA isoforms from a single pre-mRNA transcript and contributes to the complexity of the cellular transcriptome and proteome. This process is regulated through a network of cis-regulatory sequence elements and trans-acting factors, most-notably RNA binding proteins (RBPs). The muscleblind-like (MBNL) and RNA binding fox-1 homolog (RBFOX) are two well characterized families of RBPs that regulate fetal to adult AS transitions critical for proper muscle, heart, and central nervous system development. To better understand how the concentration of these RBPs influences AS transcriptome wide, we engineered a MBNL1 and RBFOX1 inducible HEK-293 cell line. Modest induction of exogenous RBFOX1 in this cell line modulated MBNL1-dependent AS outcomes in 3 skipped exon events, despite significant levels of endogenous RBFOX1 and RBFOX2. Due to background RBFOX levels, we conducted a focused analysis of dose-dependent MBNL1 skipped exon AS outcomes and generated transcriptome wide dose–response curves. Analysis of this data demonstrates that MBNL1-regulated exclusion events may require higher concentrations of MBNL1 protein to properly regulate AS outcomes compared to inclusion events and that multiple arrangements of YGCY motifs can produce similar splicing outcomes. These results suggest that rather than a simple relationship between the organization of RBP binding sites and a specific splicing outcome, that complex interaction networks govern both AS inclusion and exclusion events across a RBP gradient.

Pbp1 associates with Puf3 and promotes translation of its target mRNAs involved in mitochondrial biogenesis.

Pbp1 (poly(A)-binding protein-binding protein 1) is a cytoplasmic stress granule marker that is capable of forming condensates that function in the negative regulation of TORC1 signaling under respiratory conditions. Polyglutamine expansions in its mammalian ortholog ataxin-2 lead to spinocerebellar dysfunction due to toxic protein aggregation. Here, we show that loss of Pbp1 in S. cerevisiae leads to decreased amounts of mRNAs and mitochondrial proteins which are targets of Puf3, a member of the PUF (Pumilio and FBF) family of RNA-binding proteins. We found that Pbp1 supports the translation of Puf3-target mRNAs in respiratory conditions, such as those involved in the assembly of cytochrome c oxidase and subunits of mitochondrial ribosomes. We further show that Pbp1 and Puf3 interact through their respective low complexity domains, which is required for Puf3-target mRNA translation. Our findings reveal a key role for Pbp1-containing assemblies in enabling the translation of mRNAs critical for mitochondrial biogenesis and respiration. They may further explain prior associations of Pbp1/ataxin-2 with RNA, stress granule biology, mitochondrial function, and neuronal health.

RBFOX2 confers tumor growth by PI3K/AKT and MAPK signaling in gastric cancer.

RNA-binding Fox (RBFOX)2, a member of a family of RNA-binding proteins, is well known as a regulator of alternative pre-mRNA splicing. However, its possible role in gastric cancer is unknown. In this study, we investigated the biologic role and clinical significance of RBFOX2 in gastric cancer growth and elucidated its underlying molecular mechanisms. We found that RBFOX2 was highly expressed in gastric cancer cell lines and tumor tissue compared with the adjacent nontumor tissue. We also found that RBFOX2 overexpression was correlated with poor overall survival in patients with gastric cancers. Multivariate survival analyses revealed that higher RBFOX2 expression was an independent prognostic factor for the overall survival of patients with gastric cancers. Suppression of RBFOX2 by shRNA inhibited gastric cancer cell proliferation, colony formation and induced apoptosis. Mechanism studies revealed that these effects were achieved through the simultaneous modulation of multiple signaling pathways. Knockdown of RBFOX2 expression by shRNA markedly inhibited the phosphorylation of phosphatidylinositol 3-hydroxy kinase, threonine kinase and extracellular signal-regulated kinase and Jun N-terminal kinases proteins. In contrast, the ectopic expression of RBFOX2 had the opposite effects. Moreover, RBFOX2 knockdown also induced the cleavage of caspase-3 and caspase-9 proteins. Collectively, these results demonstrate that RBFOX2 plays a critical role in regulating gastric cancer cell proliferation and survival and may be a potential prognostic biomarker and therapeutic target for gastric cancer.

PO-01-145 DYNAMIC UTR COMPOSITION IMPACTS RNA BINDING PROTEIN COMPLEXING TO CONTROL TRANSLATION AND LIMIT CARDIAC GAP JUNCTION FORMATION DURING STRESS

Translational control has emerged as a critical yet dynamic process, representing one of the most rapid means by which the cardiomyocyte can alter its proteome. Several key cardiac mRNAs, including connexin43 (Cx43, gene name GJA1) are understood to undergo alternative translation initiation with resulting products (ie, GJA1-20k) impacting cardiac function. We previously found alterations to GJA1 mRNA untranslated region (UTR) length occur in aged and stressed heart tissue, suppressing internal translation of GJA1-20k, and limiting Cx43 gap junction formation. Having therefore identified dynamic mRNA cis element-based regulation of Cx43/GJA1-20k translation, we next asked what trans-acting factors are at play to alter GJA1 mRNA translation during stress. The IMP family of RNA binding proteins (RBPs) regulate alternative and canonical translation initiation of several genes, which led us to investigate how they may impact GJA1 mRNA. Using GJA1 as a model RNA, we test the hypothesis that alternative transcription start site usage controls inclusion of cis-acting elements in the mRNA 5’UTR affecting RBP binding and regulation of translation. Pathological hypertrophy was induced by isoproterenol deliverey to adult mice via implanted minipumps. Mouse models are complemented with matured human IPSC-derived cardiomyocytes, primary mouse ventricular cardiomyocytes, exposed to chronic hypoxia or TGF-β in vitro. Single molecule fluorescence in situ hybridization (smFISH) coupled with super-resolution microscopy localize GJA1 mRNA and RBPs. Hypertrophic hearts show reduced GJA1-20k and IMP1 expression. Using smFISH to label GJA1 mRNA, we find IMP1 complexes with GJA1 mRNA predominantly in comparison to IMP2 and 3, and that this complexing is reduced during stress, concomitant with loss of GJA1-20k internal translation. Biochemical assays confirm GJA1 mRNA/IMP1 binding by RNA-IP and synthetic reduction of IMP1 expression by RNAi or CRISPR/Cas9 alters GJA1 mRNA localization, reduces internal translation of GJA1-20k, and limits gap junction formation. We find comparable results during hypoxia in primary mouse cardiomyocytes and human induced pluripotent stem cell derived cardiomyocytes. Dynamic GJA1-5’UTR composition therefore represents a powerful mechanism connecting alterations in transcription to gene-specific translational regulation by RBPs such as IMP1, disruption of intercellular communication during stress, and arrhythmogenesis.

Splicing of ultraconserved poison exon elements in RNA binding proteins reprograms the T effector immune response

Abstract Our current understanding of pathways governing the strength and longevity of T cell mediated immune responses primarily focuses on transcriptional and epigenetic changes. However, our prior work demonstrated that optimal T cell effector function is also dependent on RNA binding proteins (RBPs) that carry out post-transcriptional RNA splicing and polyadenylation. Here, we find that a specific family of RBPs, serine- and arginine-rich (SR) RBPs, are significantly upregulated by T cell activation and required for CD8 +T cell mediated responses. SR transcripts contain ultraconserved poison exon (PE) elements that are alternatively spliced and introduce premature stop codons that trigger nonsense-mediated decay. We show that differential splicing of specific RBP PE regions is both induced by TCR stimulation and required for CD8 +T cell activation in both human and mouse. Further, we demonstrate that PEs are cell-state associated by using single-cell RNA sequencing analysis of splicing changes across mouse thymic T cell subsets, in CD8 +T cells over the course of an acute Listeria infection, as well as in human lymphoblastic leukemia, atherosclerosis, and melanoma patient T cells. CRISPR-Cas9 mediated deletion of the PE element of SR-like RBP Tra2β in mouse T effector cells during influenza infection resulted in a significant increase in proliferating and surviving T cells and altered the splicing pattern of critical cell survival, proliferation, and adhesion genes. In total, our work demonstrates a fundamental role for alternatively spliced ultraconserved elements in defining T cell states, controlling T effector-mediated immunity, and altering the T cell response in pathologic conditions. This work was supported in part by NIH grants AI136955 and R21AI139891 awarded to A.T.V, institutional support, and Boehringer Ingelheim endowed Chair in Immunology.

ZFP36-mediated mRNA decay regulates metabolism

Cellular metabolism is tightly regulated by growth factor signaling, which promotes metabolic rewiring to support growth and proliferation. While growth factor-induced transcriptional and post-translational modes of metabolic regulation have been well defined, whether post-transcriptional mechanisms impacting mRNA stability regulate this process is less clear. Here, we present the ZFP36/L1/L2 family of RNA-binding proteins and mRNA decay factors as key drivers of metabolic regulation downstream of acute growth factor signaling. We quantitatively catalog metabolic enzyme and nutrient transporter mRNAs directly bound by ZFP36 following growth factor stimulation-many of which encode rate-limiting steps in metabolic pathways. Further, we show that ZFP36 directly promotes the mRNA decay of Enolase 2 (Eno2), altering Eno2 protein expression and enzymatic activity, and provide evidence of a ZFP36/Eno2 axis during VEGF-stimulated developmental retinal angiogenesis. Thus, ZFP36-mediated mRNA decay serves as an important mode of metabolic regulation downstream of growth factor signaling within dynamic cell and tissue states.

The HNRNPF/H RNA binding proteins and disease.

The members of the HNRNPF/H family of heterogeneous nuclear RNA proteins-HNRNPF, HNRNPH1, HNRNPH2, HNRNPH3, and GRSF1, are critical regulators of RNA maturation. Documented functions of these proteins include regulating splicing, particularly alternative splicing, 5' capping and 3' polyadenylation of RNAs, and RNA export. The assignment of these proteins to the HNRNPF/H protein family members relates to differences in the amino acid composition of their RNA recognition motifs, which differ from those of other RNA binding proteins (RBPs). HNRNPF/H proteins typically bind RNA sequences enriched with guanine (G) residues, including sequences that, in the presence of a cation, have the potential to form higher-order G-quadruplex structures. The need to further investigate members of the HNRNPF/H family of RBPs has intensified with the recent descriptions of their involvement in several disease states, including the pediatric tumor Ewing sarcoma and the hematological malignancy mantle cell lymphoma; newly described groups of developmental syndromes; and neuronal-related disorders, including addictive behavior. Here, to foster the study of the HNRNPF/H family of RBPs, we discuss features of the genes encoding these proteins, their structures and functions, and emerging contributions to disease. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Processing > Splicing Regulation/Alternative Splicing RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Arabidopsis SRPKII family proteins regulate flowering via phosphorylation of SR proteins and effects on gene expression and alternative splicing.

Alternative splicing of pre-mRNAs is crucial for plant growth and development. Serine/arginine-rich (SR) proteins are a conserved family of RNA binding proteins that are critical for both constitutive and alternative splicing. However, how phosphorylation of SR proteins regulates gene transcription and alternative splicing during plant development is poorly understood. We found that the Arabidopsis thaliana L. SR protein-specific kinase II family proteins (SRPKIIs) play an important role in plant development, including flowering. SRPKIIs regulate the phosphorylation status of a subset of specific SR proteins, including SR45 and SC35, which subsequently mediates their subcellular localization. A phospho-dead SR45 mutant inhibits the assembly of the ASAP (apoptosis-and splicing-associated protein) complex and thereby upregulates the expression of FLC (FLOWERING LOCUS C) via epigenetic modification. The splicing efficiency of FLC introns was significantly increased in the shoot apex of the srpkii mutant. Transcriptomic analysis revealed that SRPKIIs regulate the alternative splicing of approximately four hundred genes, which largely overlap with those regulated by SR45 and SC35-SCL family proteins. In summary, we found that Arabidopsis SRPKIIs specifically affect the phosphorylation status of a subset SR proteins and regulate the expression and alternative splicing of FLC to control flowering time.

The evolution and immunomodulatory role of Zc3h12 proteins in zebrafish (Danio rerio)

Zc3h12 family is an important RNA-binding protein family regulating mRNA of inflammatory cytokines in mammals. However, there are few studies on their post-transcriptional level regulation of inflammatory cytokines in fish. Here, we investigated the evolution of zebrafish Zc3h12 family and explored their immunomodulatory role. Phylogenetic and syntenic analysis indicated the number of zc3h12 family members had increased ranging from a single member in invertebrates to a single copy of four members in mammals. As the most evolutionarily diverse group of vertebrates, the number of zc3h12 family members was more complex and diverse in the teleost, each member experienced different fates and followed different rules in multiple rounds of whole-genome duplication events. Thereinto, zebrafish contained three zc3h12 genes, among which zc3h12aa and zc3h12ab were duplicated from the same gene. Zebrafish Zc3h12 family could recognize the 3′-UTR regions of inflammatory cytokines through binding to the specific RNA secondary structure and negatively regulate their expression. Deletion of either Zc3h12 domains or mutation of the key amino acid in RNAase domain attenuated their modulatory effect, suggesting both domain and RNAase activity are important to the immunomodulatory role. These results elucidated the evolution of Zc3h12 family and uncovered Zc3h12-mediated post-transcriptional regulation of cytokines in zebrafish.

Comprehensive Identification and Expression Analysis of the YTH Family of RNA-Binding Proteins in Strawberry.

Plant growth and development processes are tightly regulated at multiple levels, including transcriptional and post-transcriptional levels, and the RNA-binding protein YTH regulates gene expression during growth and development at the post-transcriptional level by regulating RNA splicing, processing, stability, and translation. We performed a systematic characterization of YTH genes in diploid forest strawberry and identified a total of nine YTH genes. With the help of phylogenetic analysis, these nine genes were found to belong to two different groups, YTHDC and YTHDF, with YTHDF being further subdivided into three subfamilies. Replication analysis showed that YTH3 and YTH4 are a gene pair generated by tandem repeat replication. These two genes have similarities in gene structure, number of motifs, and distribution patterns. Promoter analysis revealed the presence of multiple developmental, stress response, and hormone-response-related cis-elements. Analysis of available transcriptome data showed that the expression levels of most of the YTH genes were stable with no dramatic changes during development in different tissues. However, YTH3 maintained high expression levels in all tissues and during fruit development, and YTH4 was expressed at higher levels in tissues such as flowers, leaves, and seedlings, while it was significantly lower than YTH3 in white fruits and ripening fruits with little fluctuation. Taken together, our study provides insightful and comprehensive basic information for the study of YTH genes in strawberry.

Pathogenic Mis-splicing of CPEB4 in Schizophrenia

BackgroundSchizophrenia (SCZ) is caused by an interplay of polygenic risk and environmental factors, which may alter regulators of gene expression leading to pathogenic misexpression of SCZ risk genes. The CPEB family of RNA-binding proteins (CPEB1–4) regulates translation of target RNAs (approximately 40% of overall genes). We previously identified CPEB4 as a key dysregulated translational regulator in autism spectrum disorder (ASD) because its neuronal-specific microexon (exon 4) is mis-spliced in ASD brains, causing underexpression of numerous ASD risk genes. The genetic factors and pathogenic mechanisms shared between SCZ and ASD led us to hypothesize CPEB4 mis-splicing in SCZ leading to underexpression of multiple SCZ-related genes. MethodsWe performed MAGMA-enrichment analysis on Psychiatric Genomics Consortium genome-wide association study data and analyzed RNA sequencing data from the PsychENCODE Consortium. Reverse transcriptase polymerase chain reaction and Western blot were performed on postmortem brain tissue, and the presence/absence of antipsychotics was assessed through toxicological analysis. Finally, mice with mild overexpression of exon 4–lacking CPEB4 (CPEB4Δ4) were generated and analyzed biochemically and behaviorally. ResultsFirst, we found enrichment of SCZ-associated genes for CPEB4-binder transcripts. We also found decreased usage of CPEB4 microexon in SCZ probands, which was correlated with decreased protein levels of CPEB4-target SCZ-associated genes only in antipsychotic-free individuals. Interestingly, differentially expressed genes fit those reported for SCZ, specifically in the SCZ probands with decreased CPEB4-microexon inclusion. Finally, we demonstrated that mice with mild overexpression of CPEB4Δ4 showed decreased protein levels of CPEB4-target SCZ genes and SCZ-linked behaviors. ConclusionsWe identified aberrant CPEB4 splicing and downstream misexpression of SCZ risk genes as a novel etiological mechanism in SCZ.

Role of Heterogeneous Nuclear Ribonucleoproteins in the Cancer-Immune Landscape

Cancer remains the second leading cause of death, accounting for approximately 20% of all fatalities. Evolving cancer cells and a dysregulated immune system create complex tumor environments that fuel tumor growth, metastasis, and resistance. Over the past decades, significant progress in deciphering cancer cell behavior and recognizing the immune system as a hallmark of tumorigenesis has been achieved. However, the underlying mechanisms controlling the evolving cancer-immune landscape remain mostly unexplored. Heterogeneous nuclear ribonuclear proteins (hnRNP), a highly conserved family of RNA-binding proteins, have vital roles in critical cellular processes, including transcription, post-transcriptional modifications, and translation. Dysregulation of hnRNP is a critical contributor to cancer development and resistance. HnRNP contribute to the diversity of tumor and immune-associated aberrant proteomes by controlling alternative splicing and translation. They can also promote cancer-associated gene expression by regulating transcription factors, binding to DNA directly, or promoting chromatin remodeling. HnRNP are emerging as newly recognized mRNA readers. Here, we review the roles of hnRNP as regulators of the cancer-immune landscape. Dissecting the molecular functions of hnRNP will provide a better understanding of cancer-immune biology and will impact the development of new approaches to control and treat cancer.

Genome-Wide Identification and Evolutionary Analysis of Gossypium YTH Domain-Containing RNA-Binding Protein Family and the Role of GhYTH8 in Response to Drought Stress.

YTH domain-containing proteins are one kind of RNA-binding protein involved in post-transcriptional regulation and play multiple roles in regulating the growth, development, and abiotic stress responses of plants. However, the YTH domain-containing RNA-binding protein family has not been previously studied in cotton. In this study, a total of 10, 11, 22, and 21 YTH genes were identified in Gossypium arboreum, Gossypium raimondii, Gossypium barbadense, and Gossypium hirsutum, respectively. These Gossypium YTH genes were categorized into three subgroups by phylogenetic analysis. The chromosomal distribution, synteny analysis, structures of Gossypium YTH genes, and the motifs of YTH proteins were analyzed. Furthermore, the cis-element of GhYTH genes promoter, miRNA targets of GhYTH genes, and subcellular localization of GhYTH8 and GhYTH16 were characterized. Expression patterns of GhYTH genes in different tissues, organs, and in response to different stresses were also analyzed. Moreover, functional verifications revealed that silencing GhYTH8 attenuated the drought tolerance in the upland cotton TM-1 line. These findings provide useful clues for the functional and evolutionary analysis of YTH genes in cotton.

Up-regulated Circular RNAs in Colorectal Cancer: New Entities for Therapy and Tools for Identification of Therapeutic Targets.

Patients with disseminated colorectal cancer have a dismal prognosis with a 5-year survival rate of only 13%. In order to identify new treatment modalities and new targets, we searched the literature for up-regulated circular RNAs in colorectal cancer which induce tumor growth in corresponding preclinical in vivo models. We identified nine circular RNAs that mediate resistance against chemotherapeutic agents, seven that up-regulate transmembrane receptors, five that induce secreted factors, nine that activate signaling components, five which up-regulate enzymes, six which activate actin-related proteins, six which induce transcription factors and two which up-regulate the MUSASHI family of RNA binding proteins. All of the circular RNAs discussed in this paper induce the corresponding targets by sponging microRNAs (miRs) and can be inhibited by RNAi or shRNA in vitro and in xenograft models. We have focused on circular RNAs with demonstrated activity in preclinical in vivo models because the latter is an important milestone in drug development. All circular RNAs with in vitro activity only data are not referenced in this review. The translational impact of inhibition of these circular RNAs and of the identified targets for treatment of colorectal cancer (CRC) are discussed.

RNA Helicase DDX6 Regulates A-to-I Editing and Neuronal Differentiation in Human Cells

The DEAD-box proteins, one family of RNA-binding proteins (RBPs), participate in post-transcriptional regulation of gene expression with multiple aspects. Among them, DDX6 is an essential component of the cytoplasmic RNA processing body (P-body) and is involved in translational repression, miRNA-meditated gene silencing, and RNA decay. In addition to the cytoplasmic function, DDX6 is also present in the nucleus, but the nuclear function remains unknown. To decipher the potential role of DDX6 in the nucleus, we performed mass spectrometry analysis of immunoprecipitated DDX6 from a HeLa nuclear extract. We found that adenosine deaminases that act on RNA 1 (ADAR1) interact with DDX6 in the nucleus. Utilizing our newly developed dual-fluorescence reporter assay, we elucidated the DDX6 function as negative regulators in cellular ADAR1p110 and ADAR2. In addition, depletion of DDX6 and ADARs results in the opposite effect on facilitation of RA-induced differentiation of neuronal lineage cells. Our data suggest the impact of DDX6 in regulation of the cellular RNA editing level, thus contributing to differentiation in the neuronal cell model.

Fission Yeast PUF Proteins Puf3 and Puf4 Are Novel Regulators of PI4P5K Signaling.

Phosphatidylinositol-4-phosphate 5-kinase (PI4P5K) is a highly conserved enzyme that generates phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) by phosphorylating phosphatidylinositol 4-phosphate (PI(4)P). Schizosaccharomyces pombe (S. pombe) its3-1 is a loss-of-function mutation in the essential its3+ gene that encodes a PI4P5K. Its3 regulates cell proliferation, cytokinesis, cell integrity, and membrane trafficking, but little is known about the regulatory mechanisms of Its3. To identify regulators of Its3, we performed a genetic screening utilizing the high-temperature sensitivity (TS) of its3-1 and identified puf3+ and puf4+, encoding Pumilio/PUF family RNA-binding proteins as multicopy suppressors of its3-1 cells. The deletions of the PUF domains in the puf3+ and puf4+ genes resulted in the reduced ability to suppress its3-1, suggesting that the suppression by Puf3 and Puf4 may involve their RNA-binding activities. The gene knockout of Puf4, but not that of Puf3, exacerbated the TS of its3-1. Interestingly, mutant Its3 expression levels both at mRNA and protein levels were lower than those of the wild-type (WT) Its3. Consistently, the overexpression of the mutant its3-1 gene suppressed the its3-1 phenotypes. Notably, Puf3 and Puf4 overexpression increased the mRNA and protein expression levels of both Its3 and Its3-1. Collectively, our genetic screening revealed a functional relationship between the Pumilio/PUF family RNA-binding proteins and PI4P5K.

Multivalent interactions between RNA and RNA binding proteins drive differential intra-speckle positioning of RNA transcripts.

Nuclear speckles represent a type of membraneless organelles in higher eukaryotic cells which are rich in snRNP components, certain splicing factors, including SR proteins (a family of RNA binding proteins (RBPs) named for containing regions with repetitive serine and arginine residues), polyA+ RNAs, and certain long noncoding RNAs (lncRNA). Multivalent interactions between RNAs and residing protein components drive the formation, as well as layered organization of components in nuclear speckles. Furthermore, RBPs have differential localization with respect to nuclear speckles; while SRSF1 proteins is enriched in nuclear speckles, heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) is either uniformly distributed in the nucleoplasm or slightly depleted from nuclear speckles. Using super-resolution imaging we show that the pre-mRNA transcripts containing SRSF1 motifs in the exon and hnRNPA1 motifs in the intron, are orientated in a way such that the regions containing the SRSF1 binding motifs are positioned relatively towards the interior of nuclear speckles and hnRNPA1 binding motifs are placed relatively towards the periphery of speckles. Knocking down of SRSF1 protein leads to the migration of RNA transcripts further towards the periphery of nuclear speckles, whereas knocking down of hnRNPA1 protein leads to the inward migration of RNA transcripts with respect to the speckle center. A similar effect of protein knockdown is seen on the endogenous nuclear speckle associated lncRNA, MALAT1. Our results collectively demonstrate that RNAs can adopt non-random orientation and positioning within the membraneless organelles. These observations might help explain the need for nuclear RNAs for structural integrity of nuclear speckles and the context dependent effect of SR and hnRNP proteins on splicing outcomes.

Alternative splicing mediates the compensatory upregulation of MBNL2 upon MBNL1 loss-of-function.

Loss of gene function can be compensated by paralogs with redundant functions. An example of such compensation are the paralogs of the Muscleblind-Like (MBNL) family of RNA-binding proteins that are sequestered and lose their function in Myotonic Dystrophy Type 1 (DM1). Loss of MBNL1 increases the levels of its paralog MBNL2 in tissues where Mbnl2 expression is low, allowing MBNL2 to functionally compensate for MBNL1 loss. Here, we show that loss of MBNL1 increases the inclusion of Mbnl2 exon 6 and exon 9. We find that inclusion of Mbnl2 exon 6 increases the translocation of MBNL2 to the nucleus, while the inclusion of Mbnl2 exon 9 shifts the reading frame to an alternative C-terminus. We show that the C-terminus lacking exon 9 contains a PEST domain which causes proteasomal degradation. Loss of MBNL1 increases the inclusion of exon 9, resulting in an alternative C-terminus lacking the PEST domain and the increase of MBNL2. We further find that the compensatory mechanism is active in a mouse DM1 model. Together, this study uncovers the compensatory mechanism by which loss of MBNL1 upregulates its paralog MBNL2 and highlights a potential role of the compensatory mechanism in DM1.