The extreme N-terminal domain of a hordeivirus TGB1 movement protein mediates its localization to the nucleolus and interaction with fibrillarin

The hordeiviral movement protein encoded by the first gene of the triple gene block (TGBp1) of Poa semilatent virus (PSLV), interacts with viral genomic RNAs to form RNP particles which are considered to be a form of viral genome capable of cell-to-cell and long-distance transport in infected plants. The PSLV TGBp1 contains a C-terminal NTPase/helicase domain (HELD) and an N-terminal extension region consisting of two structurally and functionally distinct domains: an extreme N-terminal domain (NTD) and an internal domain (ID). This study demonstrates that transient expression of TGBp1 fused to GFP in Nicotiana benthamiana leaves results in faint but obvious fluorescence in the nucleolus in addition to cytosolic distribution. Mutagenesis of the basic amino acids inside the NTD clusters A ¹¹⁶KSKRKKKNKK¹²⁵ and B ¹⁷⁵KKATKKESKKQTK¹⁸⁷ reveals that these clusters are indispensable for nuclear and nucleolar targeting of PSLV TGBp1 and may contain nuclear and nucleolar localization signals or their elements. The PSLV TGBp1 is able to bind to fibrillarin, the major nucleolar protein (AtFib2 from Arabidopsis thaliana) in vitro. This protein–protein interaction occurs between the glycine-arginine-rich (GAR) domain of fibrillarin and the first 82 amino acid residues of TGBp1. The interaction of TGBp1 with fibrillarin is also visualized in vivo by bimolecular fluorescence complementation (BiFC) during co-expression of TGBp1 or its deletion mutants, and fibrillarin as fusions to different halves of YFP in N. benthamiana plants. The sites responsible for nuclear/nucleolar localization and fibrillarin binding, have been located within the intrinsically disordered TGBp1 NTD. These data could suggest that specific functions of hordeivirus TGBp1 may depend on its interaction with nucleolar components.     

Hormonal regulation of Drosophila microRNA let-7 and miR-125 that target innate immunity

The steroid 20-hydroxy-ecdysone (20-HE) and the sesquiterpenoid Juvenile Hormone (JH) coordinate insect life stage transitions. 20-HE exerts these effects by the sequential induction of response genes. In the nematode Caenorhabditis elegans hormones also play a role in such transitions, but notably, microRNA such as let-7 and lin-4 have likewise been found to help order developmental steps. Little is known about the corresponding function of homologous microRNA in Drosophila melanogaster, and the way microRNA might be regulated by 20-HE in the fly is ambiguous. Here we used Drosophila S2 cells to analyze the effects of 20-HE on D. melanogaster microRNA let-7 and miR-125, the homolog of lin-4. The induction by 20-HE of let-7 and miR-125 in S2 cells is inhibited by RNai knockdown of the ecdysone receptor and, as previously shown, by knockdown of its cofactor broad-complex C. To help resolve the currently ambiguous role of 20-HE in the control of microRNa, we show that nanomolar concentrations of 20-HE primes cells to subsequently express microRNa when exposed to micromolar levels of 20-HE. We then explore the role microRNa plays in the established relationship between 20-HE and the induction of innate immunity. We show that the 3′UTR of the antimicrobial peptide diptericin has a let-7 binding site and that let-7 represses translation from this site. We conclude that 20-HE facilitates the initial expression of innate immunity while it simultaneously induces negative regulation via microRNa control of antimicrobial peptide translation.Key words: microRNA, ecdysone, innate immunity, let-7, diptericin, inflammation     

Let-7g induces granulosa cell apoptosis by targeting MAP3K1 in the porcine ovary

Follicular atresia mainly results from apoptosis of granulosa cells (GCs). Our previous microRNA array data indicated that the miRNA let-7g level increases significantly during porcine ovary follicular atresia. It is uncertain if GCs apoptosis is mediated by microRNA let-7g. In this study, the expression levels of the apoptosis-associated genes CASP3, BAX and BIM were significantly upregulated when let-7g mimic was transfected into porcine GCs, and the anti-apoptotic genes BCL-2 and MCL-1 were significantly downregulated. The apoptosis rate was measured by flow cytometry, and our results indicated that let-7g significantly enhanced GCs apoptosis. In further studies, we found that overexpression of let-7g induced the expression of FoxO1 in GCs and led to nuclear accumulation of dephosphorylated FoxO1. In addition, the effect of let-7g on FoxO1 expression and dephosphorylation resulted from repression of the expression of the MAP3K1 gene in porcine GCs. The site on MAP3K1 mRNA targeted by let-7g was confirmed by luciferase reporter assay. The anti-apoptotic effect of MAP3K1 was validated by silencing MAP3K1 using small interfering RNA technology. In conclusion, our data indicate that let-7g induces porcine GCs apoptosis by inhibiting the MAP3K1 gene, which promotes FoxO1 expression and dephosphorylation with nuclear accumulation.     

NMR structure of the let-7 miRNA interacting with the site LCS1 of lin-41 mRNA from Caenorhabditis elegans

We have determined the 3D structure of a 34-nt RNA construct, herein named LCS1co, which mimics the interaction of let-7 microRNA (miRNA) to one of its complementary binding sites, LCS1, in the 3′-untranslated region of lin-41 mRNA by solution-state NMR spectroscopy. let-7 miRNAs control the timing of development of the nematode Caenorhabditis elegans and are highly conserved in mammals. The sequence and structure of the two conserved let-7 complementary sites, LCS1 and LCS2, in the 3′-untranslated region of lin-41 mRNA are important for a proper downregulation of lin-41. The high-resolution NMR structure reveals details of the binding of let-7 miRNA to lin-41 mRNA which involves formation of a complex with non-canonical structural elements within the seed region. LCS1co exhibits a stem-loop structure with two stems, an asymmetric internal loop and an adenine bulge. Comparison with the NMR solution-state structure of the let-7:lin-41 complex involving the LCS2-binding site shows that conformational freedom of the asymmetric internal loop of LCS1co correlates with a smaller bend between the upper and lower stems in comparison to the well-defined asymmetric loop of LCS2co.     

RNA-binding protein GLD-1/quaking genetically interacts with the mir-35 and the let-7 miRNA pathways in Caenorhabditis elegans

Messenger RNA translation is regulated by RNA-binding proteins and small non-coding RNAs called microRNAs. Even though we know the majority of RNA-binding proteins and microRNAs that regulate messenger RNA expression, evidence of interactions between the two remain elusive. The role of the RNA-binding protein GLD-1 as a translational repressor is well studied during Caenorhabditis elegans germline development and maintenance. Possible functions of GLD-1 during somatic development and the mechanism of how GLD-1 acts as a translational repressor are not known. Its human homologue, quaking (QKI), is essential for embryonic development. Here, we report that the RNA-binding protein GLD-1 in C. elegans affects multiple microRNA pathways and interacts with proteins required for microRNA function. Using genome-wide RNAi screening, we found that nhl-2 and vig-1, two known modulators of miRNA function, genetically interact with GLD-1. gld-1 mutations enhance multiple phenotypes conferred by mir-35 and let-7 family mutants during somatic development. We used stable isotope labelling with amino acids in cell culture to globally analyse the changes in the proteome conferred by let-7 and gld-1 during animal development. We identified the histone mRNA-binding protein CDL-1 to be, in part, responsible for the phenotypes observed in let-7 and gld-1 mutants. The link between GLD-1 and miRNA-mediated gene regulation is further supported by its biochemical interaction with ALG-1, CGH-1 and PAB-1, proteins implicated in miRNA regulation. Overall, we have uncovered genetic and biochemical interactions between GLD-1 and miRNA pathways.     

Ribosomal protein RPS-14 modulates let-7 microRNA function in Caenorhabditis elegans

The let-7 microRNA (miRNA) regulates developmental timing at the larval-to-adult transition in Caenorhabditis elegans. Dysregulation of let-7 results in irregular hypodermal and vulval development. Disrupted let-7 function is also a feature of human lung cancer. However, little is known about the mechanism and co-factors of let-7. Here we demonstrate that ribosomal protein RPS-14 is able to modulate let-7 function in C. elegans. The RPS-14 protein co-immunoprecipitated with the nematode Argonaute homolog, ALG-1. Reduction of rps-14 gene expression by RNAi suppressed the aberrant vulva and hypodermis development phenotypes of let-7(n2853) mutant animals and the mis-regulation of a reporter bearing the lin-41 3′UTR, a well established let-7 target. Our results indicate an interactive relationship between let-7 miRNA function and ribosomal protein RPS-14 in regulation of terminal differentiation that may help in understanding the mechanism of translational control by miRNAs.     

Cadmium can induce alterations in the cellular localization and expression of three major nucleolar proteins in root tip cells of Vicia faba L

Aims

The findings of our earlier investigations indicated that cadmium (Cd) could induce some nucleolar materials containing the argyrophilic proteins scattered in the nuclei and located in cytoplasm in the root tip cells of Allium sative and Vicia faba. However, what kinds of nucleolar proteins are affected has not been reported up to now. In order to further confirm the cytological effects of Cd on nucleolus and nucleolar proteins, alterations in the cellular localization and expression of three major nucleolar proteins: nucleophosmin, fibrillarin and nucleolin were investigated under the treatment with Cd in the root tip cells of V. faba in this study.

Methods

Silver methods, Indirect immunofluorescent microscopy; Western blotting.

Results

The obvious toxic effects on nucleoli in the cells were observed after Cd treatment. Nucleolar proteins, nucleophosmin, fibrillarin, and nucleolin in root tip cells exposed to Cd (50 μM) for 48 h were translocated from nucleolus to nucleoplasm and cytoplasm and expression of the three major nucleolar proteins was relatively higher when compared with control.     

Decrease of Let-7f in Low-Dose Metronomic Paclitaxel Chemotherapy Contributed to Upregulation of Thrombospondin-1 in Breast Cancer

Low-dose metronomic (LDM) paclitaxel therapy displayed a stronger anti-angiogenic activity on breast tumors with fewer side effects. Upregulation of anti-angiogenic factor Thrombospondin-1 (TSP-1) accords for therapeutic potency of LDM paclitaxel, but its molecular mechanism has not been elucidated yet. microRNAs (miRNAs) have emerged as new important regulators of tumor growth and metastasis. Here, we hypothesize that miRNAs are involved in TSP-1 overexpression in paclitaxel LDM therapy of breast tumors. The miRNA profile of tumor tissues from control, LDM and MTD groups in 4T1 mouse breast cancer model was detected by microarray, and then verified by quantitative real-time PCR (qRT-PCR). Luciferase assay and western blot were employed to explore the mechanisms of miRNAs involved in this process. We found that let-7f, let-7a, miR-19b and miR-340-5p were reduced by >2 fold, and miR-543* and miR-684 were upregulated by at least 50% in paclitaxel LDM therapy. qRT-PCR verification revealed that let-7f level was reduced most significantly in LDM therapy. Computational prediction using TargetScan and miRanda suggested THBS1 which encodes TSP-1 as a potential target for let-7f. Luciferase activity assay further confirmed that let-7f may bind to 3''UTR of THBS1 gene and inhibit its activity. Moreover, forced expression of let-7f led to a decrease of TSP-1 at both mRNA and protein levels in MCF-7 cells. Contrastly, let-7f inhibition induced an increased expression of THBS1 mRNA and TSP-1 protein, but did not affect the proliferation and apoptosis of MCF-7 cells. Paclitaxel LDM therapy led to a decrease of let-7f and the elevation of TSP-1 protein expression in MCF-7 cells, while overexpression of let-7f may abolish LDM-induced the upregulation of TSP-1 in MCF-7 cells. In summary, let-7f inhibition contributed to the upregulation of TSP-1 in paclitaxel LDM therapy, independently of proliferation, cell cycle arrest and apoptosis of breast cancer. This study indicates let-7f as a potential therapeutic target for breast tumor.     

Post-transcriptional fine-tuning of COP9 signalosome subunit biosynthesis is regulated by the c-Myc/Lin28B/let-7 pathway

The COP9 signalosome (CSN) complex controls protein degradation via the ubiquitin (Ub) proteasome system (UPS) in eukaryotes. In mammalian cells, the multimeric CSN is composed of eight subunits (CSN1 - CSN8). It regulates cullin-RING Ub ligases (CRLs), which target essential regulatory proteins for ubiquitination and subsequent degradation. Thereby, the CSN cooperates with the UPS in a variety of essential cellular functions, including DNA repair, cell cycle and differentiation. Although functions of the CSN have been elucidated, mechanisms and regulatory principles of its de novo formation are completely unknown. Here, we show that there is a fundamental mechanism that allows a coordinated expression of all CSN subunits, a prerequisite for CSN assembly. CSN subunit mRNAs are targets of miRNAs of the let-7 family suppressing CSN subunit expression in human cells. Factors that reduce or block let-7 miRNAs induce the coordinated expression of CSN subunits. For instance, over-expression of CSN1 specifically traps let-7a-1 miRNA and elevates CSN subunit levels by two- to fourfold in a coordinated manner. CSN subunit expression is also increased by specific miRNA inhibitors or by interferon (IFN)-mediated induction of STAT1 and c-Myc reducing levels of let-7 miRNAs. Activation of STAT1 by IFNα or IFNγ induces c-Myc, which increases CSN subunit expression via the Lin28B/let-7 regulatory pathway. By contrast, a let-7a-1 mimic reduces CSN subunit expression. Our data show that let-7 miRNAs control the fine-tuning and coordinated expression of subunits for CSN de novo formation, presumably a general regulatory principle for other Zomes complexes as well.     

Control of Nuclear and Nucleolar Localization of Nuclear Inclusion Protein a of Picorna-Like Potato virus A in Nicotiana Species

The multifunctional nuclear inclusion protein a (NIa) of potyviruses (genus Potyvirus; Potyviridae) accumulates in the nucleus of virus-infected cells for unknown reasons. In this study, two regions in the viral genome-linked protein (VPg) domain of NIa in Potato virus A (PVA) were found to constitute nuclear and nucleolar localization signals (NLS) in plant cells (Nicotiana spp). Amino acid substitutions in both NLS I (residues 4 to 9) and NLS II (residues 41 to 50) prevented nuclear localization, whereas mutations in either single NLS did not. Mutations in either NLS, however, prevented nucleolar localization and prevented or diminished virus replication in protoplasts, accumulation in infected plant tissues, and/or systemic movement in plants. One NLS mutant was partially complemented by the wild-type VPg expressed in transgenic plants. Furthermore, NLS I controlled NIa accumulation in Cajal bodies. The VPg domain interacted with fibrillarin, a nucleolar protein, and depletion of fibrillarin reduced PVA accumulation. Overexpression of VPg in leaf tissues interfered with cosuppression of gene expression (i.e., RNA silencing), whereas NLS I and NLS II mutants, which exhibited reduced nuclear and nucleolar localization, showed no such activity. These results demonstrate that some of the most essential viral functions required for completion of the infection cycle are tightly linked to regulation of the NIa nuclear and nucleolar localization.     

microRNA-mediated translation repression through GYF-1 and IFE-4 in C. elegans development

microRNA (miRNA)-mediated gene silencing is enacted through the recruitment of effector proteins that direct translational repression or degradation of mRNA targets, but the relative importance of their activities for animal development remains unknown. Our concerted proteomic surveys identified the uncharacterized GYF-domain encoding protein GYF-1 and its direct interaction with IFE-4, the ortholog of the mammalian translation repressor 4EHP, as key miRNA effector proteins in Caenorhabditis elegans. Recruitment of GYF-1 protein to mRNA reporters in vitro or in vivo leads to potent translation repression without affecting the poly(A) tail or impinging on mRNA stability. Loss of gyf-1 is synthetic lethal with hypomorphic alleles of embryonic miR-35–42 and larval (L4) let-7 miRNAs, which is phenocopied through engineered mutations in gyf-1 that abolish interaction with IFE-4. GYF-1/4EHP function is cascade-specific, as loss of gyf-1 had no noticeable impact on the functions of other miRNAs, including lin-4 and lsy-6. Overall, our findings reveal the first direct effector of miRNA-mediated translational repression in C. elegans and its physiological importance for the function of several, but likely not all miRNAs.