Family Of RNA-binding Proteins Research Articles

Effects of PUMILIO1 and PUMILIO2 knockdown on cardiomyogenic differentiation of human embryonic stem cells culture.

Posttranscriptional regulation plays a fundamental role in the biology of embryonic stem cells (ESCs). Many studies have demonstrated that multiple mRNAs are coregulated by one or more RNA-binding proteins (RBPs) that orchestrate mRNA expression. A family of RBPs, which is known as the Pumilio-FBF (PUF) family, is highly conserved among different species and has been associated with the undifferentiated and differentiated states of different cell lines. In humans, two homologs of the PUF family have been found: Pumilio 1 (PUM1) and Pumilio 2 (PUM2). To understand the role of these proteins in human ESCs (hESCs), we first assessed the influence of the silencing of PUM1 and PUM2 on pluripotency genes and found that the knockdown of Pumilio genes significantly decreased the OCT4 and NANOG mRNA levels and reduced the amount of nuclear OCT4, which suggests that Pumilio proteins play a role in the maintenance of pluripotency in hESCs. Furthermore, we observed that PUM1-and-PUM2-silenced hESCs exhibited improved efficiency of in vitro cardiomyogenic differentiation. Through an in silico analysis, we identified mRNA targets of PUM1 and PUM2 that are expressed at the early stages of cardiomyogenesis, and further investigation will determine whether these target mRNAs are active and involved in the progression of cardiomyogenesis. Our findings contribute to the understanding of the role of Pumilio proteins in hESC maintenance and differentiation.

Musashi 2 (MSI2) expression as an independent prognostic biomarker in non-small cell lung cancer (NSCLC).

e21583 Background: Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death in the world, and metastasis is the most common cause of death in lung cancer patients. Musashi-2 (MSI2) is a member of RNA-binding protein family and regulates mRNA translation of numerous intracellular targets, and influences multiple biological processes, including maintenance of stem cell identity. Previously we reported that MSI2 is upregulated in the metastasis-competent NSCLC murine and human cell lines, drives NSCLC metastasis, and is progressively elevated in lung cancer patient samples. This study assessed MSI2 as potential clinical biomarker in NSCLC. Methods: The current study included 40 patients with NSCLC, of whom 15 presented with stage II and III respectively (37,5% each), and 10 patients (25%) had stage IV. All patients received treatment in accordance with the NCCN guidelines, and did not participate in clinical trials. All patient samples were obtained before treatment started. We used immunohistochemical (IHC) approach to measure MSI2 expression on the protein level in matching specimens of normal versus tumor tissues, and primary tumor vs. metastasis, followed by correlative analysis in relation to clinical outcomes. In parallel, clinical correlative analysis of MSI2 mRNA expression was performed in silico using publicly available datasets (TCGA/ICGC) as of January 2020. Results: MSI2 protein expression in patient samples was significantly elevated in NSCLC primary tumors and metastatic sites versus normal tissue (p = 0.03). MSI2 elevated expression positively correlated with a decreased progression free survival (PFS) (p = 0.026) at all stages and overall survival at stage IV (p = 0.013). Elevated MSI2 expression on RNA level was observed in primary tumor versus normal tissue samples in TCGA (p < 0.0001), and positively correlated with decreased OS (p = 0.028). No correlation was observed between MSI2 expression and age, sex, smoking, and treatment type. Conclusions: Elevated MSI2 expression in primary NSCLC tumors is associated with poor prognosis and can be used as a novel potential prognostic biomarker in NSCLC patients. Future studies in an extended patient cohort are strongly warranted.

Conditionally Replicative Adenovirus Controlled by the Stabilization System of AU-Rich Elements Containing mRNA.

AU-rich elements (AREs) are RNA elements that enhance the rapid decay of mRNAs, including those of genes required for cell growth and proliferation. HuR, a member of the embryonic lethal abnormal vision (ELAV) family of RNA-binding proteins, is involved in the stabilization of ARE-mRNA. The level of HuR in the cytoplasm is up-regulated in most cancer cells, resulting in the stabilization of ARE-mRNA. We developed the adenoviruses AdARET and AdAREF, which include the ARE of TNF-α and c-fos genes in the 3′-untranslated regions of the E1A gene, respectively. The expression of the E1A protein was higher in cancer cells than in normal cells, and virus production and cytolytic activities were also higher in many types of cancer cells. The inhibition of ARE-mRNA stabilization resulted in a reduction in viral replication, demonstrating that the stabilization system was required for production of the virus. The growth of human tumors that formed in nude mice was inhibited by an intratumoral injection of AdARET and AdAREF. These results indicate that these viruses have potential as oncolytic adenoviruses in the vast majority of cancers in which ARE-mRNA is stabilized.

Nop9 recognizes structured and single-stranded RNA elements of preribosomal RNA

Nop9 is an essential factor in the processing of preribosomal RNA. Its absence in yeast is lethal, and defects in the human ortholog are associated with breast cancer, autoimmunity, and learning/language impairment. PUF family RNA-binding proteins are best known for sequence-specific RNA recognition, and most contain eight α-helical repeats that bind to the RNA bases of single-stranded RNA. Nop9 is an unusual member of this family in that it contains eleven repeats and recognizes both RNA structure and sequence. Here we report a crystal structure of Saccharomyces cerevisiae Nop9 in complex with its target RNA within the 20S preribosomal RNA. This structure reveals that Nop9 brings together a carboxy-terminal module recognizing the 5′ single-stranded region of the RNA and a bifunctional amino-terminal module recognizing the central double-stranded stem region. We further show that the 3′ single-stranded region of the 20S target RNA adds sequence-independent binding energy to the RNA–Nop9 interaction. Both the amino- and carboxy-terminal modules retain the characteristic sequence-specific recognition of PUF proteins, but the amino-terminal module has also evolved a distinct interface, which allows Nop9 to recognize either single-stranded RNA sequences or RNAs with a combination of single-stranded and structured elements.

Zfp36 family members redundantly protect against T cell-mediated autoinflammation and premature mortality

Abstract Cytokine production must be tightly regulated in order to prevent auto-inflammatory diseases. The zinc finger 36 (Zfp36) family of RNA-binding proteins, including Zfp36, Zfp36l1, and Zfp36l2, are known to negatively regulate mRNA stability or translation of many transcripts, including cytokines. Polymorphisms in ZFP36L1 and ZFP36L2 have been identified in GWAS studies of a variety of human autoimmune diseases, necessitating understanding the functions of these genes. While there are reports of all three family members controlling cytokine production from T cells, delineating the exact functions of these genes has been challenging due to spontaneous phenotypes or mortality upon global deletion of single genes and potential redundancy in their functions. To overcome this, we generated Cd4-Cre+ Zfp36fl/fl Zfp36l1fl/fl Zfp36l2fl/fl mice. Only upon triple deletion, but not individual or various paired deletions, do mice spontaneously develop an inflammatory disease characterized by early mortality and immune cell infiltration into various organs, including the central nervous system, kidneys, and liver. These mice have drastically elevated levels of many cytokines in their sera. Our findings demonstrate a novel redundancy of the Zfp36 family members in regulating T cell homeostasis and suppressing cytokine-driven inflammation. We are currently investigating the specific mechanisms and mRNA targets that contribute to this phenotype and whether disease is primarily driven by CD4+ or CD8+ T cells. Understanding the individual and redundant functions of the Zfp36 family members may lead to opportunities to target them for suppression of T cell-driven autoimmunity or for activating anti-tumor or anti-pathogen T cell responses.

Abstract A43: Investigating novel inhibitors of the IMP-1-KRAS mRNA interaction

Abstract K-Ras functionality is integral for cell growth and differentiation due its canonical role in signaling the PI3K and MAPK pathways. Genetic analysis on various cancers revealed that mutations in the K-Ras gene are responsible for 21% of all cases, which include lung, pancreatic and colorectal cancer. Prior efforts at directly targeting mutant K-Ras have been largely unsuccessful, thus creating a demand for novel therapeutic approaches. Common to the aforementioned cancers is expression of the Insulin-Like Growth Factor 2 mRNA Binding Protein 1 (IMP-1). IMP-1 is part of the VICKZ family of RNA-binding proteins known for post-transcriptional regulation, including its ability to shield its mRNA substrates from decay. One notable substrate of IMP-1 is the K-Ras mRNA, which was identified using UV-crosslinking experiments. Downregulation of IMP-1 through siRNA treatment led to a reduction of K-Ras protein by 60% in SW480 colorectal cancer cells and consequently reduced cell proliferation. The studies suggest a relationship between IMP-1 binding, mRNA turnover and the resultant protein expression of target substrates. To study the IMP-1-mRNA relationship and to help find inhibitors of such interaction, we developed a fluorescence polarization (FP) method that employed 45-nt K-Ras and 39-nt CD44 fluorescein-labeled RNA probes. Using full-length IMP1, truncated and point-mutated IMP1, we showed that the FP method faithfully recapitulated the relative affinity of recombinant proteins for both K-Ras and CD44 RNAs. The FP method was then used to find inhibitors of the IMP-1-K-Ras mRNA interaction and to elucidate potential domain target of inhibitors. We found that oligonucleotides SM6 and SM7, but not SM2, could significantly reduce the FP units, consistent with the results from electrophoretic mobility shift assay. In addition, the molecule UNBC143 was found to decrease FP units in a dose-dependent manner, suggesting its ability to inhibit IMP-1-K-Ras RNA interaction. The SM oligonucleotides and UNBC143 were also assessed on K-Ras expression in SW480 human colon cancer cells. These experiments and their results will be discussed in further detail upon presentation. Citation Format: Victor Liu, Chuyi Wang, Chow Lee. Investigating novel inhibitors of the IMP-1-KRAS mRNA interaction [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A43.

MiR-132 Regulates PTSD-like Behaviors in Rats Following Single-Prolonged Stress Through Fragile X-Related Protein 1.

Fragile X-related protein 1 (FXR1) is a member of the fragile X family of RNA-binding proteins, which regulates a number of neurological and neuropsychiatric disorders such as fragile X syndrome, and is expected as a novel therapeutic target for some psychiatric diseases. However, it is unknown how FXR1 changes and functions in post-traumatic stress disorder (PTSD), a common mental disorder related to trauma and stressor. In this study, we characterized the expression pattern of FXR1 in the pathophysiological process of PTSD and further investigated the possible mechanism underlying these changes by finding an upstream regulator, namely miRNA-132 (miR-132). Furthermore, we verified whether miR-132 silence had an effect on the PTSD-like behaviors of single prolonged stress (SPS) rats through open field test, forced swimming test, and water maze test. At last, we examined the expression levels of PSD95 and synapsin I in the hippocampus, which was one of the key brain regions associated with PTSD. We showed that the levels of FXR1 and fragile X mental retardation protein (FMRP), an autosomal homolog of FXR1, were decreased in the hippocampus of PTSD rats, but the levels of PSD95 and synapsin I were increased, which could be reversed by downregulation of miR-132. The results revealed that miR-132 could modulate PTSD-like behaviors in rats following SPS through regulating FXR1 and FMRP.

Interleukin-10 control of pre-miR155 maturation involves CELF2.

The anti-inflammatory cytokine interleukin-10 (IL10) is essential for attenuating inflammatory responses, which includes reducing the expression of pro-inflammatory microRNA-155 (miR155) in lipopolysaccharide (LPS) activated macrophages. miR155 enhances the expression of pro-inflammatory cytokines such as TNFα and suppresses expression of anti-inflammatory molecules such as SHIP1 and SOCS1. We previously found that IL10 interfered with the maturation of pre-miR155 to miR155. To understand the mechanism by which IL10 interferes with pre-miR155 maturation we isolated proteins that associate with pre-miR155 in response to IL10 in macrophages. We identified CELF2, a member of the CUGBP, ELAV-Like Family (CELF) family of RNA binding proteins, as protein whose association with pre-miR155 increased in IL10 treated cells. CRISPR-Cas9 mediated knockdown of CELF2 impaired IL10's ability to inhibit both miR155 expression and TNFα expression.

Fission Yeast Puf2, a Pumilio and FBF Family RNA-Binding Protein, Links Stress Granules to Processing Bodies

Stress granules (SGs) are cytoplasmic aggregates formed upon stress when untranslated messenger ribonucleoproteins accumulate in the cells. In a GFP-library screening of the fission yeast SG proteins, Puf2 of the PUF family of RNA-binding proteins was identified that is required for SG formation after deprivation of glucose. Accordingly, puf2 mutant is defective in recovery from glucose starvation with a much longer lag to re-enter the cell cycle. In keeping with these results, Puf2 contains several low-complexity and intrinsically disordered protein regions with a tendency to form aggregates and, when over-expressed, represses translation to induce aggregation of poly(A) binding protein Pabp, the signature constituent of SGs. Intriguingly, over-expression of Puf2 also enhances the structure of processing bodies (PBs), another type of cytoplasmic RNA granules, a complex of factors involved in mRNA degradation. We demonstrate, in this study, a function of the fission yeast PB in SG formation and Puf2 may provide the link of these two structures.

Generation of An Endogenous FGFR2-BICC1 Gene Fusion/58 Megabase Inversion Using Single-Plasmid CRISPR/Cas9 Editing in Biliary Cells.

Fibroblast growth factor receptor 2 (FGFR2) gene fusions are bona fide oncogenic drivers in 10–15% of intrahepatic cholangiocarcinoma (CCA), yet currently there are no cell lines publically available to study endogenous FGFR2 gene fusions. The ability of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to generate large yet precise chromosomal rearrangements has presented the possibility of engineering endogenous gene fusions for downstream studies. In this technical report, we describe the generation of an endogenous FGFR2–Bicaudal family RNA binding protein 1 (BICC1) fusion in multiple independent cholangiocarcinoma and immortalized liver cell lines using CRISPR. BICC1 is the most common FGFR2 fusion partner in CCA, and the fusion arises as a consequence of a 58-megabase-sized inversion on chromosome 10. We replicated this inversion to generate a fusion product that is identical to that seen in many human CCA. Our results demonstrate the feasibility of generating large megabase-scale inversions that faithfully reproduce human cancer aberrations.

A tumor-specific modulation of heterogeneous ribonucleoprotein A0 promotes excessive mitosis and growth in colorectal cancer cells

RNA regulation mediating RNA-binding proteins (RBPs) have been shown to be related to the maintenance of homeostasis as well as cancer progression. However, the tumor-associated functions as well as the detailed mechanisms underlying the anti-tumor effects of most RBPs have yet to be explored. We herein report that the phosphorylated heterogeneous ribonucleoprotein (hnRNP) A0 promotes mitosis through the RAS-associated protein 3 GTPase-activating protein catalytic subunit 1 (RAB3GAP1)-Zeste white 10 interactor (ZWINT1) cascade. The downregulation assay of 20 representative hnRNPs, a major family of RNA-binding proteins, in colorectal cancer cells revealed that hnRNPA0 is a strong regulator of cancer cell growth. The tumor promotive function of hnRNPA0 was confirmed in gastrointestinal cancer cells, including pancreatic, esophageal, and gastric cancer cells, but not in non-cancerous cells. Flow cytometry and Western blotting analyses revealed that hnRNPA0 inhibited the apoptosis through the maintenance of G2/M phase promotion in colorectal cancer cells. A comprehensive analysis of mRNAs regulated by hnRNP A0 and immunostaining revealed that mitotic events were regulated by the hnRNPA0-RAB3GAP1 mRNA-mediated ZWINT-1 stabilization in colorectal cancer cells, but not in non-tumorous cells. The interaction of hnRNP A0 with mRNAs was dramatically changed by the deactivation of its phosphorylation site in cancer cells, but not in non-tumorous cells. Therefore, the tumor-specific biological functions characterized by the abnormal phosphorylation of RBPs are considered to be an attractive target for tumor treatment.

Distribution, organization an evolutionary history of La and LARPs in eukaryotes

ABSTRACT The fate of any cellular RNA is largely influenced by the nature and diversity of its interactions with various RNA-binding proteins (RBPs) leading to the formation of a biologically significant ribonucleoprotein (RNP) complex. La motif-containing proteins (composed of genuine La and La-related proteins (LARPs)) represent an evolutionary conserved family of RBPs that encompass a large range of crucial functions, involving coding and non-coding RNAs. In this work, we provide data that extend our previous knowledge on the distribution, organization and evolutionary history of this important protein family. Using a repertoire of 345 La motif-containing proteins from 135 species representing all major eukaryotic lineages, we were able to pinpoint many lineage-specific variations in the structural organization of La and LARPs and propose new evolutive scenarios to explain their modern genomic distribution.

Unraveling a new player in multiple sclerosis pathogenesis: The RNA-binding protein HuR

BackgroundELAV-like proteins are a small family of RNA-binding proteins that are fundamental players in post-transcriptional mechanisms and are involved in the pathogenesis of neurologic and psychiatric disorders. HuR, the ubiquitously expressed member of the family, is also implicated in sustaining inflammation and inflammatory diseases, supporting the production of pro-inflammatory cytokines. Inflammation plays a central role in Multiple Sclerosis (MS), which represents the most common cause of permanent physical disability in young adults. MS is a chronic autoimmune disease affecting the Central Nervous System, with a complex aetiology involving genetic, environmental and epigenetic factors. No data are available on the potential entanglement of HuR in MS pathogenesis in patients. In the present work, we aimed at exploring HuR protein levels in peripheral blood mononuclear cells (PBMCs) from MS patients, compared to healthy controls. To further elucidate the possible involvement of HuR in MS, we also investigated the relationship between this specific RNA-binding protein and HSP70-2 protein, also considering the HSP70-2 rs1061581 polymorphism, given that HSP70-2 mRNA has been reported as a HuR target and this specific polymorphism to be associated with MS risk. MethodsAlleles and genotypes for HSP70-2 rs1061581 polymorphism were assessed, by using a Polymerase Chain Reaction-Restriction Fragment Length Polymorphism, followed by digestion with restriction enzyme, in MS patients and healthy controls. PBMCs from a subgroup of patients and controls were used to evaluate HuR and HSP70-2 protein content by Western blot. ResultsPBMCs from 52 MS patients had a lower HuR and higher HSP70-2 protein content compared to 43 healthy controls. An increase of 100 units of HuR significantly decreased the risk of developing MS by 9.8% (OR: 0.902, 95% CI: 0.83–0.98), controlling for HSP70-2 protein expression, HSP70-2 rs1061581 genotype, age and sex. Moreover, holding HuR levels, an increase of 100 units of HSP70-2 protein significantly increased the MS risk by 18.1% (OR: 1.181, 95% CI: 1.03–1.36) and the genetic susceptibility of developing MS for HSP70-2 rs1061581 GG carriers is confirmed. Of interest, MS patients with a moderate to severe form of MS (MSSS ≥ 3) showed a trend towards a reduction of HuR protein levels compared to patients with mild disease severity (MSSS < 3). ConclusionsHuR protein levels are reduced in MS patients compared to healthy subjects, and the protein amount may continue to decline with disease progression, suggesting a putative role of this RNA-binding protein. Moreover, our results suggest that MS pathology may have disrupted the link between HuR and its target transcript HSP70-2. It will be important to further explore the exact role of HuR in MS, considering the complex interplay with other RNA-binding factors and target mRNAs.

Evolution of the Small Family of Alternative Splicing Modulators Nuclear Speckle RNA-binding Proteins in Plants.

RNA-Binding Protein 1 (RBP1) was first identified as a protein partner of the long noncoding RNA (lncRNA) ENOD40 in Medicago truncatula, involved in symbiotic nodule development. RBP1 is localized in nuclear speckles and can be relocalized to the cytoplasm by the interaction with ENOD40. The two closest homologs to RBP1 in Arabidopsis thaliana were called Nuclear Speckle RNA-binding proteins (NSRs) and characterized as alternative splicing modulators of specific mRNAs. They can recognize in vivo the lncRNA ALTERNATIVE SPLICING COMPETITOR (ASCO) among other lncRNAs, regulating lateral root formation. Here, we performed a phylogenetic analysis of NSR/RBP proteins tracking the roots of the family to the Embryophytes. Strikingly, eudicots faced a reductive trend of NSR/RBP proteins in comparison with other groups of flowering plants. In Medicago truncatula and Lotus japonicus, their expression profile during nodulation and in specific regions of the symbiotic nodule was compared to that of the lncRNA ENOD40, as well as to changes in alternative splicing. This hinted at distinct and specific roles of each member during nodulation, likely modulating the population of alternatively spliced transcripts. Our results establish the basis to guide future exploration of NSR/RBP function in alternative splicing regulation in different developmental contexts along the plant lineage.

Diverse Roles of PUF Proteins in Germline Stem and Progenitor Cell Development in C. elegans.

Stem cell development depends on post-transcriptional regulation mediated by RNA-binding proteins (RBPs) (Zhang et al., 1997; Forbes and Lehmann, 1998; Okano et al., 2005; Ratti et al., 2006; Kwon et al., 2013). Pumilio and FBF (PUF) family RBPs are highly conserved post-transcriptional regulators that are critical for stem cell maintenance (Wickens et al., 2002; Quenault et al., 2011). The RNA-binding domains of PUF proteins recognize a family of related sequence motifs in the target mRNAs, yet individual PUF proteins have clearly distinct biological functions (Lu et al., 2009; Wang et al., 2018). The C. elegans germline is a simple and powerful model system for analyzing regulation of stem cell development. Studies in C. elegans uncovered specific physiological roles for PUFs expressed in the germline stem cells ranging from control of proliferation and differentiation to regulation of the sperm/oocyte decision. Importantly, recent studies started to illuminate the mechanisms behind PUF functional divergence. This review summarizes the many roles of PUF-8, FBF-1, and FBF-2 in germline stem and progenitor cells (SPCs) and discusses the factors accounting for their distinct biological functions. PUF proteins are conserved in evolution, and insights into PUF-mediated regulation provided by the C. elegans model system are likely relevant for other organisms.

BOPC1 Enantiomers Preparation and HuR Interaction Study. From Molecular Modeling to a Curious DEEP-STD NMR Application.

The Hu family of RNA-binding proteins plays a crucial role in post-transcriptional processes; indeed, Hu-RNA complexes are involved in various dysfunctions (i.e., inflammation, neurodegeneration, and cancer) and have been recently proposed as promising therapeutic targets. Intrigued by this concept, our research efforts aim at identifying small molecules able to modulate HuR-RNA interactions, with a focus on subtype HuR, upregulated and dysregulated in several cancers. By applying structure-based design, we had already identified racemic trans-BOPC1 as promising HuR binder. In this Letter, we accomplished the enantio-resolution, the assignment of the absolute configuration, and the recognition study with HuR of enantiomerically pure trans-BOPC1. For the first time, we apply DEEP (differential epitope mapping)-STD NMR to study the interaction of BOPC1 with HuR and compare its enantiomers, gaining information on ligand orientation and amino acids involved in the interaction, and thus increasing focus on the in silico binding site model.

Formation of S. pombe Erh1 homodimer mediates gametogenic gene silencing and meiosis progression

Timely and accurate expression of the genetic information relies on the integration of environmental cues and the activation of regulatory networks involving transcriptional and post-transcriptional mechanisms. In fission yeast, meiosis-specific transcripts are selectively targeted for degradation during mitosis by the EMC complex, composed of Erh1, the ortholog of human ERH, and the YTH family RNA-binding protein Mmi1. Here, we present the crystal structure of Erh1 and show that it assembles as a homodimer. Mutations of amino acid residues to disrupt Erh1 homodimer formation result in loss-of-function phenotypes, similar to erh1∆ cells: expression of meiotic genes is derepressed in mitotic cells and meiosis progression is severely compromised. Interestingly, formation of Erh1 homodimer is dispensable for interaction with Mmi1, suggesting that only fully assembled EMC complexes consisting of two Mmi1 molecules bridged by an Erh1 dimer are functionally competent. We also show that Erh1 does not contribute to Mmi1-dependent down-regulation of the meiosis regulator Mei2, supporting the notion that Mmi1 performs additional functions beyond EMC. Overall, our results provide a structural basis for the assembly of the EMC complex and highlight its biological relevance in gametogenic gene silencing and meiosis progression.

The RNA-binding protein QKI-7 recruits the poly(A) polymerase GLD-2 for 3′ adenylation and selective stabilization of microRNA-122

MicroRNA-122 (miR-122) is highly expressed in hepatocytes, where it plays an important role in regulating cholesterol and fatty acid metabolism, and it is also a host factor required for hepatitis C virus replication. miR-122 is selectively stabilized by 3' adenylation mediated by the cytoplasmic poly(A) polymerase GLD-2 (also known as PAPD4 or TENT2). However, it is unclear how GLD-2 specifically stabilizes miR-122. Here, we show that QKI7 KH domain-containing RNA binding (QKI-7), one of three isoforms of the QKI proteins, which are members of the signal transduction and activation of RNA (STAR) family of RNA-binding proteins, is involved in miR-122 stabilization. QKI down-regulation specifically decreased the steady-state level of mature miR-122, but did not affect the pre-miR-122 level. We also found that QKI-7 uses its C-terminal region to interact with GLD-2 and its QUA2 domain to associate with the RNA-induced silencing complex protein Argonaute 2 (Ago2), indicating that the GLD-2-QKI-7 interaction recruits GLD-2 to Ago2. QKI-7 exhibited specific affinity to miR-122 and significantly promoted GLD-2-mediated 3' adenylation of miR-122 in vitro Taken together, our findings indicate that miR-122 binds Ago2-interacting QKI-7, which recruits GLD-2 for 3' adenylation and stabilization of miR-122.

Identification of soybean trans-factors associated with plastid RNA editing sites.

RNA editing is a posttranscriptional process that changes nucleotide sequences, among which cytosine-to-uracil by a deamination reaction can revert non-neutral codon mutations. Pentatricopeptide repeat (PPR) proteins comprise a family of RNA-binding proteins, with members acting as editing trans-factors that recognize specific RNA cis-elements and perform the deamination reaction. PPR proteins are classified into P and PLS subfamilies. In this work, we have designed RNA biotinylated probes based in soybean plastid RNA editing sites to perform trans-factor specific protein isolation. Soybean cis-elements from these three different RNA probes show differences in respect to other species. Pulldown samples were submitted to mass spectrometry for protein identification. Among detected proteins, five corresponded to PPR proteins. More than one PPR protein, with distinct functional domains, was pulled down with each one of the RNA probes. Comparison of the soybean PPR proteins to Arabidopsis allowed identification of the closest homologous. Differential gene expression analysis demonstrated that the PPR locus Glyma.02G174500 doubled its expression under salt stress, which correlates with the increase of its potential rps14 editing. The present study represents the first identification of RNA editing trans-factors in soybean. Data also indicated that potential multiple trans-factors should interact with RNA cis-elements to perform the RNA editing.

Interplay between the RNA binding-protein Musashi and developmental signaling pathways.

Musashi comprises an evolutionarily conserved family of RNA-binding proteins (RBP) that regulate cell fate decisions during embryonic development and play key roles in the maintenance of self-renewal and differentiation of stem cells and adult tissues. More recently, several studies have shown that any dysregulation of MSI1 and MSI2 can lead to cellular dysfunctions promoting tissue instability and tumorigenesis. Moreover, several reports have characterized many molecular interactions between members of the Musashi family with ligands and receptors of the signaling pathways responsible for controlling normal embryonic development: Notch, Transforming Growth Factor Beta (TGF-β), Wingless (Wnt) and Hedgehog Signaling (Hh); all of which, when altered, are strongly associated with cancer onset and progression, especially in pediatric tumors. In this context, the present review aims to compile possible cross-talks between Musashi proteins and members of the above cited molecular pathways for which dysregulation plays important roles during carcinogenesis and may be modulated by these RBP.