Double-stranded RNA Binding Research Articles

Staufen-1 Interacts with the Human Endogenous Retrovirus Family HERV-K(HML-2) Rec and Gag Proteins and Increases Virion Production

The human endogenous retrovirus family HERV-K(HML-2) Rec protein is an RNA transport factor that enhances nuclear export of intron-containing retroviral transcripts. Using the yeast two-hybrid approach, we have newly identified human Staufen-1 as a Rec-interacting protein. The interaction was confirmed by coimmunoprecipitation experiments, and the relevant site in Staufen-1 has been mapped to double-stranded RNA binding domain 4 (RBD4). Staufen-1 is in several aspects functionally related to retroviral RNA transport proteins. It binds mRNAs and targets its ribonuclear cargo to polysomes for efficient translation. We observed an accumulation of Staufen-1 in the nucleus of Rec-expressing cells and colocalization in the nucleoli as well as in the cytoplasm. Overexpression of Staufen-1 resulted in a 5-fold enhancement in nuclear export and/or translation of unspliced HERV-K(HML-2) viral RNAs in the presence of Rec and its Rec-responsive element (RcRE) binding site together with a clear increase in virus production. Staufen-1 was previously shown to interact with the Gag protein of HIV-1, promoting Gag oligomerization and RNA encapsidation. We demonstrate here that Staufen-1 also binds to the Gag protein of HERV-K(HML-2). Under stress conditions, Rec colocalizes with Staufen-1 in stress granules in cells that express viral RNA but not in mRNA-decay-related processing bodies. Our results suggest a new role for Staufen-1 as a cellular Rec and HERV-K(HML-2) Gag cofactor.

Development of a specific siRNA delivery system into HeLa cells using an IgG-binding fusion protein

Stable carriers are required for delivering siRNA to cells. The use of polyethyleneimine (PEI) as gene carrier has been researched extensively; however, it does not provide sufficient protection from RNase degradation and is not suitable for targeted siRNA delivery to specific cells. In this study, two repeats of Fc binding domain of protein G (C2) were used to introduce a specific antibody to PEI-based carrier of siRNA. In addition, we used the double-stranded RNA binding domain (DRBD) that can bind to siRNA. The complex, consisting of PEI, siRNA and constructed fusion protein, TrxC2DRBD including C2 and DRBD domains, could protect siRNA from RNase degradation. Furthermore, cell specific siRNA delivery into HeLa cells could be performed by the complex fusion with specific antibodies via C2 domain.

Complementation of Hyponastic Leaves1 by Double-Strand RNA-Binding Domains of Dicer-Like1 in Nuclear Dicing Bodies

MicroRNAs (miRNAs) are a class of small regulatory RNAs that are found in almost all of the eukaryotes. Arabidopsis (Arabidopsis thaliana) miRNAs are processed from primary miRNAs (pri-miRNAs), mainly by the ribonuclease III-like enzyme DICER-LIKE1 (DCL1) and its specific partner, HYPONASTIC LEAVES1 (HYL1), a double-strand RNA-binding protein, both of which contain two double-strand RNA-binding domains (dsRBDs). These dsRBDs are essential for miRNA processing, but the functions of them are not clear. Here, we report that the two dsRBDs of DCL1 (DCL1-D1D2), and to some extent the second dsRBD (DCL1-D2), complement the hyl1 mutant, but not the first dsRBD of DCL1 (DCL1-D1). DCL1-D1 is diffusely distributed throughout the nucleoplasm, whereas DCL1-D2 and DCL1-D1D2 concentrate in nuclear dicing bodies in which DCL1 and HYL1 colocalize. We show further that protein-protein interaction is mainly mediated by DCL1-D2, while DCL1-D1 plays a major role in binding of pri-miRNAs. These results suggest parallel roles between C-terminal dsRBDs of DCL1 and N-terminal dsRBDs of HYL1 and support a model in which Arabidopsis pri-miRNAs are recruited to dicing bodies through functionally divergent dsRBDs of microprocessor for accurate processing of plant pri-miRNAs.

Antiviral RNA Silencing Initiated in the Absence of RDE-4, a Double-Stranded RNA Binding Protein, in Caenorhabditis elegans

Small interfering RNAs (siRNAs) processed from double-stranded RNA (dsRNA) of virus origins mediate potent antiviral defense through a process referred to as RNA interference (RNAi) or RNA silencing in diverse organisms. In the simple invertebrate Caenorhabditis elegans, the RNAi process is initiated by a single Dicer, which partners with the dsRNA binding protein RDE-4 to process dsRNA into viral siRNAs (viRNAs). Notably, in C. elegans this RNA-directed viral immunity (RDVI) also requires a number of worm-specific genes for its full antiviral potential. One such gene is rsd-2 (RNAi spreading defective 2), which was implicated in RDVI in our previous studies. In the current study, we first established an antiviral role by showing that rsd-2 null mutants permitted higher levels of viral RNA accumulation, and that this enhanced viral susceptibility was reversed by ectopic expression of RSD-2. We then examined the relationship of rsd-2 with other known components of RNAi pathways and established that rsd-2 functions in a novel pathway that is independent of rde-4 but likely requires the RNA-dependent RNA polymerase RRF-1, suggesting a critical role for RSD-2 in secondary viRNA biogenesis, likely through coordinated action with RRF-1. Together, these results suggest that RDVI in the single-Dicer organism C. elegans depends on the collective actions of both RDE-4-dependent and RDE-4-independent mechanisms to produce RNAi-inducing viRNAs. Our study reveals, for the first time, a novel siRNA-producing mechanism in C. elegans that bypasses the need for a dsRNA-binding protein.

The double-stranded RNA binding domain of human Dicer functions as a nuclear localization signal

Dicer is a key player in microRNA (miRNA) and RNA interference (RNAi) pathways, processing miRNA precursors and double-stranded RNA into ∼21-nt-long products ultimately triggering sequence-dependent gene silencing. Although processing of substrates in vertebrate cells occurs in the cytoplasm, there is growing evidence suggesting Dicer is also present and functional in the nucleus. To address this possibility, we searched for a nuclear localization signal (NLS) in human Dicer and identified its C-terminal double-stranded RNA binding domain (dsRBD) as harboring NLS activity. We show that the dsRBD-NLS can mediate nuclear import of a reporter protein via interaction with importins β, 7, and 8. In the context of full-length Dicer, the dsRBD-NLS is masked. However, duplication of the dsRBD localizes the full-length protein to the nucleus. Furthermore, deletion of the N-terminal helicase domain results in partial accumulation of Dicer in the nucleus upon leptomycin B treatment, indicating that CRM1 contributes to nuclear export of Dicer. Finally, we demonstrate that human Dicer has the ability to shuttle between the nucleus and the cytoplasm. We conclude that Dicer is a shuttling protein whose steady-state localization is cytoplasmic.

The integrative expression and co-expression analysis of the AGO gene family in rice

Argonautes (AGOs) play crucial roles in RNAi and related pathways in several species and regulate plant growth and development. However the investigation in rice argonautes (OsAGOs) remains elusive. Here we focused on the expression pattern and co-expression profiles of OsAGO genes. Microarray-based and qRT-PCR expression profiling of 19 OsAGO genes indicated that most OsAGOs expressed specifically and preferentially during stages of reproductive development, and exhibited preferential up-regulation in panicle stages. Six OsAGO genes showed specific up/down-regulation in response to Gibberellin A3 (GA3), Kinetin (KT), or 1-Naphthaleneacetic acid (NAA) treatments. And three OsAGOs presented specific up-regulation in response to light and dark treatments. Ten OsAGOs were co-expressed with Dicer-like (DCL), Double-stranded RNA Binding (DRB) and RNA-dependent RNA polymerase (RDR) genes, which were related with RNA processing including RNAi pathways. Twelve OsAGOs were correlated with 17 kinds of transcription factors involving diverse functions. Four OsAGOs RNAi plants were constructed, the expression level of co-expression genes, including DCL3, DRB2, RDR4 etc., were changed while OsAGOs were down-regulated in RNAi lines, providing experimental evidence for co-expression networks. The results provide new insights in understanding the biological pathways of OsAGO genes, as well as in selecting the candidate genes involved in RNA silencing mechanisms.

The Interactomes of Influenza Virus NS1 and NS2 Proteins Identify New Host Factors and Provide Insights for ADAR1 Playing a Supportive Role in Virus Replication

Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1) appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.

Comparative sequence analysis of double stranded RNA binding protein encoding gene of parapoxviruses from Indian camels

The dsRNA binding protein (RBP) encoding gene of parapoxviruses (PPVs) from the Dromedary camels, inhabitating different geographical region of Rajasthan, India were amplified by polymerase chain reaction using the primers of pseudocowpoxvirus (PCPV) from Finnish reindeer and cloned into pGEM-T for sequence analysis. Analysis of RBP encoding gene revealed that PPV DNA from Bikaner shared 98.3% and 76.6% sequence identity at the amino acid level, with Pali and Udaipur PPV DNA, respectively. Reference strains of Bovine papular stomatitis virus (BPSV) and PCPV (reindeer PCPV and human PCPV) shared 52.8% and 86.9% amino acid identity with RBP gene of camel PPVs from Bikaner, respectively. But different strains of orf virus (ORFV) from different geographical areas of the world shared 69.5–71.7% amino acid identity with RBP gene of camel PPVs from Bikaner. These findings indicate that the camel PPVs described are closely related to bovine PPV (PCPV) in comparison to caprine and ovine PPV (ORFV).

β-glucan supplemented diets induce high and broad expression levels of TLR3 what explains protection conferred by these additives against viral infections in fish

We have previously observed that in common carp (Cyprinus carpio), administration of β-glucan (MacroGard™) as feed additive leads to a lower expression of pro-inflammatory cytokines suggesting that this immunostimulant may be preventing an acute and potentially dangerous response to infection. However, in general, mechanisms to detect and eliminate pathogens must also be induced in order to achieve an efficient clearance of the infection. Protection against viral diseases acquired through β-glucan-supplemented feed has been extensively reported for several experimental models in fish but the underlining mechanisms are still unknown. Thus, in order to better characterize the antiviral action induced by β-glucans in fish, MacroGardTM was administered daily to common carp in the form of supplemented commercial food pellets. Carp were fed for a period of 25 days prior to intra-peritoneal injection with polyinosinic:polycytidylic acid (poly I:C), a well-known double-stranded RNA mimic that triggers a type-I interferon (IFN) response and a set of immune related genes, including Mx, were analysed by real-time PCR in liver, spleen, head-kidney and mid-gut. Results obtained confirmed that treatment with β-glucan alone generally down-regulated the mRNA expression of pro-inflammatory cytokines when compared to untreated fish, while Mx gene expression remained stable. A similar expression pattern was observed for cytokines in samples obtained from β-glucan fed fish 24 h after injection with poly I:C. However, poly I:C injection markedly increased Mx gene expression but mainly in the group fed with β-glucan. Toll-like receptor 3 (TLR3) is the candidate pattern recognition receptor possibly responsible also in fish for the binding of viral double-stranded RNA and triggering of a type-I IFN response. Through a carp genome data mining, two sequences for carp TLR3 were retrieved (ccTLR3.1 and ccTLR3.2) and characterized. Constitutive gene expression of both genes was detected by real-time PCR in cDNA of all analysed carp organs. Strikingly, 25 days after β-glucan supplementation, very high levels of ccTLR3.2 gene expression were observed in all analysed organs, with the exception of liver. This suggests that β-glucan-mediated protection against viral diseases could be the result of a general induction of ccTLR3.2 gene expression in several organs in carp.

MiRNA Biogenesis Enzyme Drosha Is Required for Vascular Smooth Muscle Cell Survival

miRNA biogenesis enzyme Drosha cleaves double-stranded primary miRNA by interacting with double-stranded RNA binding protein DGCR8 and processes primary miRNA into precursor miRNA to participate in the miRNA biogenesis pathway. The role of Drosha in vascular smooth muscle cells (VSMCs) has not been well addressed. We generated Drosha conditional knockout (cKO) mice by crossing VSMC-specific Cre mice, SM22-Cre, with Drosha loxp/loxp mice. Disruption of Drosha in VSMCs resulted in embryonic lethality at E14.5 with severe liver hemorrhage in mutant embryos. No obvious developmental delay was observed in Drosha cKO embryos. The vascular structure was absent in the yolk sac of Drosha homozygotes at E14.5. Loss of Drosha reduced VSMC proliferation in vitro and in vivo. The VSMC differentiation marker genes, including αSMA, SM22, and CNN1, and endothelial cell marker CD31 were significantly downregulated in Drosha cKO mice compared to controls. ERK1/2 mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/AKT were attenuated in VSMCs in vitro and in vivo. Disruption of Drosha in VSMCs of mice leads to the dysregulation of miRNA expression. Using bioinformatics approach, the interactions between dysregulated miRNAs and their target genes were analyzed. Our data demonstrated that Drosha is required for VSMC survival by targeting multiple signaling pathways.

Double-stranded RNA binding by the human cytomegalovirus PKR antagonist TRS1

Protein Kinase R (PKR) inhibits translation initiation following double-stranded RNA (dsRNA) binding and thereby represses viral replication. Human cytomegalovirus (HCMV) encodes two noncanonical dsRNA binding proteins, IRS1 and TRS1, and the expression of at least one of these PKR antagonists is essential for HCMV replication. In this study, we investigated the role of dsRNA binding by TRS1 in PKR inhibition. We found that purified TRS1 binds specifically to dsRNA with an affinity lower than that of PKR. Point mutants in the TRS1 dsRNA binding domain that were deficient in rescuing the replication of vaccinia virus lacking its PKR antagonist E3L were unable to bind to dsRNA but retained the ability bind to PKR. Thus TRS1 binding to dsRNA and to PKR are separable. Overall, our results are most consistent with a model in which TRS1 binds simultaneously to both dsRNA and PKR to inhibit PKR activation.

Abstract 2882: Growth promoting effects of the Toll-like receptor 3 in Head and Neck carcinoma cells .

Abstract We have previously reported an intense expression of the Toll-Like receptor 3 (TLR3) in malignant cells from EBV-associated nasopharyngeal carcinomas and other Head and Neck carcinomas (Vérillaud et al., Infectious Agents and Cancer, in press). TLR3 is involved in innate immune response triggered by the binding of double-stranded RNA (dsRNA) of viral origin. Its consistent expression by malignant epithelial cells suggests that it has some functions in cell proliferation and survival. The aim of this work was to investigate these functions using 3 cell lines derived from Head and Neck squamous cell carcinomas : CNE1 (EBV-negative nasopharyngeal carcinoma), SQ20B (laryngeal carcinoma) and Fadu (hypopharyngeal carcinoma). Cell clones conditionally knocked-down for TLR-3 were derived from each parental cell line by stable transfection of a vector encoding a microRNA targeting TLR3 transcripts and whose expression is regulated by tetracycline. Stimulation of TLR3 in parental cells was done using small concentrations (250 ng/ml) of the synthetic TLR3-ligand poly(A/U). Modest but consistent growth enhancing effects were observed in deficient culture conditions marked by low concentrations of foetal calf serum and/or low amounts of nutrients and/or hypoxia. The influence of TLR3 on growth characteristics was investigated in more details using the CNE1 cells, both parental and conditionally knocked-down for TLR3. We found that under low-serum culture conditions, cell doubling time was shortened when TLR3 was stimulated by poly(A:U). On the opposite, knocking-down TLR3 resulted in a slower growth. To better understand the mechanism underlying the growth promoting effects of TLR3 in CNE1 cells, we assessed the metabolic changes induced by poly(A/U) stimulation or TLR3 silencing . Using the Seahorse Bioscience XF Glycolysis Stress Test Kit ® we found that the glycolytic capacity of CNE1 was significantly increased under stimulation by poly(A:U), whereas it was decreased under silencing of TLR3. Finally, we investigated the impact of TLR3-stimulation on the expression of HIF1-alpha which is a key transcription factor for cell adaptation to hypoxia and a major regulator of glucid metabolism. Western blot analysis showed that HIF1-alpha could be detected even in normoxic conditions when CNE1 cells were treated by poly(A:U) for 24 hours in low serum conditions. On the other hand, knocking-down TLR3 in CNE1 cells resulted in a decrease in the cellular concentration of HIF1-alpha under hypoxia. Overall, these results suggest that TLR3-stimulation induces metabolic changes favorable to cell growth in deficient culture conditions. Future investigations will intend to investigate: 1) the in vivo functions of TLR-3 using xenografted CNE1 cells; 2) the contribution of HIF1-alpha to the metabolic changes resulting from TLR3 stimulation; 3) the nature and origin of the endogenous ligands of TLR3. Citation Format: Benjamin Vérillaud, Mélanie Gressette, Philippe Herman, Anne-Sophie Jimenez, Pierre Busson. Growth promoting effects of the Toll-like receptor 3 in Head and Neck carcinoma cells . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2882. doi:10.1158/1538-7445.AM2013-2882

Staufen1 dimerizes through a conserved motif and a degenerate dsRNA-binding domain to promote mRNA decay

Staufen (STAU)1-mediated mRNA decay (SMD) degrades mammalian-cell mRNAs that bind the double-stranded (ds)RNA-binding protein STAU1 in their 3′-untranslated region. We report a new motif, which typifies STAU homologs from all vertebrate classes, that is responsible for human (h)STAU1 homodimerization. Our crystal structure and mutagenesis analyses reveal that this motif, now named the Staufen-swapping motif (SSM), and dsRNA-binding domain 5 (‘RBD’5) mediate protein dimerization: the two SSM α-helices of one molecule interact primarily through a hydrophobic patch with the two ‘RBD’5 α-helices of a second molecule. ‘RBD’5 adopts the canonical α-β-β-β-α fold of a functional RBD, but it lacks residues and features needed to bind duplex RNA. In cells, SSM-mediated hSTAU1 dimerization increases the efficiency of SMD by augmenting hSTAU1 binding to the ATP-dependent RNA helicase hUPF1. Dimerization regulates keratinocyte-mediated wound-healing and, undoubtedly, many other cellular processes.

Recognition of viral RNA stem–loops by the tandem double-stranded RNA binding domains of PKR

In humans, the double-stranded RNA (dsRNA)-activated protein kinase (PKR) is expressed in late stages of the innate immune response to viral infection by the interferon pathway. PKR consists of tandem dsRNA binding motifs (dsRBMs) connected via a flexible linker to a Ser/Thr kinase domain. Upon interaction with viral dsRNA, PKR is converted into a catalytically active enzyme capable of phosphorylating a number of target proteins that often results in host cell translational repression. A number of high-resolution structural studies involving individual dsRBMs from proteins other than PKR have highlighted the key features required for interaction with perfectly duplexed RNA substrates. However, viral dsRNA molecules are highly structured and often contain deviations from perfect A-form RNA helices. By use of small-angle X-ray scattering (SAXS), we present solution conformations of the tandem dsRBMs of PKR in complex with two imperfectly base-paired viral dsRNA stem-loops; HIV-1 TAR and adenovirus VA(I)-AS. Both individual components and complexes were purified by size exclusion chromatography and characterized by dynamic light scattering at multiple concentrations to ensure monodispersity. SAXS ab initio solution conformations of the individual components and RNA-protein complexes were determined and highlight the potential of PKR to interact with both stem and loop regions of the RNA. Excellent agreement between experimental and model-based hydrodynamic parameter determination heightens our confidence in the obtained models. Taken together, these data support and provide a framework for the existing biochemical data regarding the tolerance of imperfectly base-paired viral dsRNA by PKR.

Proteomic analysis of interleukin enhancer binding factor 3 (Ilf3) and nuclear factor 90 (NF90) interactome

Interleukin enhancer binding factor 3 (Ilf3) and Nuclear Factor 90 (NF90) are two ubiquitous proteins generated by alternative splicing from the ILF3 gene that provides each protein with a long and identical N-terminal domain of 701 amino acids and a specific C-terminal domain of 210 and 15 amino acids, respectively. They exhibit a high polymorphism due to their posttranscriptional and posttranslational modifications. Ilf3 and NF90 functions remain unclear although they have been described as RNA binding proteins but have been implicated in a large scale of cellular phenomena depending on the nature of their interacting partners, the composition of their protein complexes and their subcellular localization. In order to better understand the functions of Ilf3 and NF90, we have investigated their protein partners by an affinity chromatography approach. In this report, we have identified six partners of Ilf3 and NF90 that interact with their double-stranded RNA binding motifs: hnRNP A/B, hnRNP A2/B1, hnRNP A3, hnRNP D, hnRNP Q and PSF. These hnRNP are known to be implicated in mRNA stabilization, transport and/or translation regulation whereas PSF is a splicing factor. Furthermore, Ilf3, NF90 and most of their identified partners have been shown to be present in large complexes. Altogether, these data suggest an implication of Ilf3 and NF90 in mRNA metabolism. This work allows to establish a link between Ilf3 and NF90 functions, as RNA binding proteins, and their interacting partners implicated in these functions.

Contribution of Double-Stranded RNA and CPSF30 Binding Domains of Influenza Virus NS1 to the Inhibition of Type I Interferon Production and Activation of Human Dendritic Cells

The influenza virus nonstructural protein 1 (NS1) inhibits innate immunity by multiple mechanisms. We previously reported that NS1 is able to inhibit the production of type I interferon (IFN) and proinflammatory cytokines in human primary dendritic cells (DCs). Here, we used recombinant viruses expressing mutant NS1 from the A/Texas/36/91 and A/Puerto Rico/08/34 strains in order to analyze the contribution of different NS1 domains to its antagonist functions. We show that the polyadenylation stimulating factor 30 (CPSF30) binding function of the NS1 protein from A/Texas/36/91 influenza virus, which is absent in the A/Puerto Rico/08/34 strain, is essential for counteracting these innate immune events in DCs. However, the double-stranded RNA (dsRNA) binding domain, present in both strains, specifically inhibits the induction of type I IFN genes in infected DCs, while it is essential only for inhibition of type I IFN proteins and proinflammatory cytokine production in cells infected with influenza viruses lacking a functional CPSF30 binding domain, such as A/Puerto Rico/08/34.

ATP-independent diffusion of double-stranded RNA binding proteins

The proteins harboring double-stranded RNA binding domains (dsRBDs) play diverse functional roles such as RNA localization, splicing, editing, export, and translation, yet mechanistic basis and functional significance of dsRBDs remain unclear. To unravel this enigma, we investigated transactivation response RNA binding protein (TRBP) consisting of three dsRBDs, which functions in HIV replication, protein kinase R(PKR)-mediated immune response, and RNA silencing. Here we report an ATP-independent diffusion activity of TRBP exclusively on dsRNA in a length-dependent manner. The first two dsRBDs of TRBP are essential for diffusion, whereas the third dsRBD is dispensable. Two homologs of TRBP, PKR activator and R3D1-L, displayed the same diffusion, implying a universality of the diffusion activity among this protein family. Furthermore, a Dicer-TRBP complex on dsRNA exhibited dynamic diffusion, which was correlated with Dicer's catalytic activity. These results implicate the dsRNA-specific diffusion activity of TRBP that contributes to enhancing siRNA and miRNA processing by Dicer.

Second Double-Stranded RNA Binding Domain of Dicer-like Ribonuclease 1: Structural and Biochemical Characterization

Dicer-like ribonuclease III enzymes are involved in different paths related to RNA silencing in plants. Little is known about the structural aspects of these processes. Here we present a structural characterization of the second double-stranded RNA binding domain (dsRBD) of DCL1, which is presumed to participate in pri-micro-RNA recognition and subcellular localization of this protein. We determined the solution structure and found that it has a canonical fold but bears some variation with respect to other homologous domains. We also found that this domain binds both double-stranded RNA and double-stranded DNA, in contrast to most dsRBDs. Our characterization shows that this domain likely has functions other than substrate recognition and binding.

SON Regulates GATA-2 Through Transcriptional Control of the Mir-23a∼27a∼24-2 Cluster

Abstract 110While characterizing AML1-ETO domains important for leukemia development and identifying proteins interacting with these domains, we discovered SON as a novel AML1-ETO binding protein (PNAS, 2008, 105:17103). SON is a large, poorly characterized serine/arginine rich SR protein localized to nuclear speckles. SON has DNA, single stranded RNA, and double stranded RNA binding domains and long repeats of amino acids. Overexpression of a partial fragment of SON in a transformed cell line decreased the tumorigenic potential of the cell in nude mice and protected yeast from apoptosis. However, partially due to its large size, the SON protein has not been well characterized. Recently, we reported that SON plays an important role in RNA splicing of a specific set of cell cycle related genes: ones that possess weak splice sites (Molecular Cell, 2011, 42:185).While SON is expressed ubiquitously, its expression level is noticeably higher in hematopoietic organs/tissues and blood cells compared to other tissues, suggesting important roles of SON in the hematopoietic system. To examine whether SON expression is regulated during hematopoietic differentiation, we measured relative mRNA level of mouse Son in different stages of hematopoiesis. Son mRNA level is higher in lineage marker negative (Lin-) bone marrow cells when compared to total bone marrow cells. Macrophages showed less expression of Son, suggesting that Son is down-regulated along the hematopoietic differentiation. We further sorted Lin- cells and measured the Son level in LSK (Lin-, Sca1+, cKit+), CMP (commom myeloid progenitors), GMP (granulocyte/monocyte progenitors) and MEP (megakaryocyte/erythroid progenitors) populations. LSK cells, which precede other progenitors, showed the highest level of Son. In addition, we confirmed that SON is down-regulated during TPA-induced monocytic differentiation of U937 myeloid cells. Taken together, SON is more abundantly expressed in immature hematopoietic cells and down-regulated during differentiation.Since SON is differentially expressed during hematopoietic differentiation, we examined whether SON is involved in regulation of hematopoietic transcription factors that are key dictators of hematopoietic differentiation. Among the several transcription factors analyzed, we found that Gata-2 mRNA was consistently reduced by two different Son shRNAs in Lin- bone marrow cells. Down regulation of the GATA-2 mRNA level was further confirmed in human K562 leukemic cell line. More interestingly, while having 20∼40% reduction of mRNA level, the GATA-2 protein level is more remarkably down-regulated upon SON knockdown, resulting in 75∼90% reduction. These results indicate that upon SON knockdown, GATA-2 protein level is mainly regulated at the post-transcriptional steps.Sequence analysis of the 3’ UTR of the human GATA-2 gene predicted several candidates of targeting microRNAs. Among them, we confirmed that the mature form of miR-27a and miR-24 are up-regulated upon SON knockdown. Next, we tested the effect of over-expression of miR-27a and miR-24 on GATA-2 expression using a GATA-2 3’ UTR-containing luciferase reporter construct and demonstrated that miR-27a indeed inhibits GATA-2 mRNA level. miR-27a is a member of the miR-23a∼27a∼24-2 cluster. RTqPCR showed that primary miRNA of the miR-23a∼27a∼24 cluster is upregulated upon SON knockdown. To test whether the increase of pri-miR of this cluster is due to promoter activation, we used a reporter construct containing the promoter sequence of the miR-23a∼27a∼24-2 cluster fused to the luciferase reporter. The expression of luciferase driven by this promoter is significantly elevated upon SON knockdown, suggesting that that SON functions to repress transcription of the miR-23a-27a-24-2 cluster, thereby relieving GATA-2 mRNA from targeting by miR-27a, and contributes to maintaining the GATA-2 protein level. Taken together, our results reveal a previously unidentified function of SON in microRNA transcription and controlling the GATA-2 protein level in hematopoietic cells. Disclosures:No relevant conflicts of interest to declare.

Fast-Forward Genetics Identifies Plant CPL Phosphatases as Regulators of miRNA Processing Factor HYL1

MicroRNAs (miRNAs) are processed from primary transcripts that contain partially self-complementary foldbacks. As in animals, the core microprocessor in plants is a Dicer protein, DICER-LIKE1 (DCL1). Processing accuracy and strand selection is greatly enhanced through the RNA binding protein HYPONASTIC LEAVES 1 (HYL1) and the zinc finger protein SERRATE (SE). We have combined a luciferase-based genetic screen with whole-genome sequencing for rapid identification of new regulators of miRNA biogenesis and action. Among the first six mutants analyzed were three alleles of C-TERMINAL DOMAIN PHOSPHATASE-LIKE 1 (CPL1)/FIERY2 (FRY2). In the miRNA processing complex, SE functions as a scaffold to mediate CPL1 interaction with HYL1, which needs to be dephosphorylated for optimal activity. In the absence of CPL1, HYL1 dephosphorylation and hence accurate processing and strand selection from miRNA duplexes are compromised. Our findings thus define a new regulatory step in plant miRNA biogenesis.