microRNA-1388-5p inhibits NF-κB signaling pathway in miiuy croaker through targeting IRAK1.

Innate immune response is an important response mechanism for the host to achieve self-protection, and it plays an important role in identifying pathogens and resisting pathogen invasion. Growing evidences have shown that microRNA functions as a crucial regulator involved in the host innate immune response. In this study, the regulations of miR-1388-5p to regulate NF-κB signaling pathways via targeting the IRAK1 gene was studied in miiuy croaker. First, through bioinformatics software prediction, we found that IRAK1 is the direct target of miR-1388-5p, and then the prediction results were verified by using dual-luciferase assays. Next, we found that both miR-1388-5p mimics and pre-miR-1388 plasmids inhibit IRAK1 expression by complementing the seed sequence in the 3'-untranslated region (3'-UTR) of IRAK1. Finally, we observed that miR-1388-5p could negatively regulate NF-κB pathways through targeting IRAK1. These results provide new insights into the function of miR-1388-5p in fish innate immunity, meanwhile enriching miRNA-mediated regulatory networks.

Pioneer translation products as an alternative source for MHC-I antigenic peptides.

The notion that alternative peptide substrates can be processed and presented to the MHC class I pathway has opened for new aspects on how the immune system detects infected or damaged cells. Recent works show that antigenic peptides are derived from intron sequences in pre-mRNAs target for the nonsense-mediated degradation pathway. Introns are spliced out co-transcriptionally suggesting that such pioneer translation products (PTPs) are synthesized on the nascent RNAs in the nuclear compartment to ensure that the first peptides to emerge from an mRNA are destined for the class I pathway. This illustrates an independent translation event during mRNA maturation that give rise to specific peptide products with a specific function in the immune system. The characterization of the translation apparatus responsible for PTP synthesis will pave the way for understanding how PTP production is regulated in different tissues under different conditions and will help designing new vaccine strategies.

Targeted deletion of the gene encoding the La autoantigen (Sjögren's syndrome antigen B) in B cells or the frontal brain causes extensive tissue loss.

La antigen (Sjögren's syndrome antigen B) is a phosphoprotein associated with nascent precursor tRNAs and other RNAs, and it is targeted by autoantibodies in patients with Sjögren's syndrome, systemic lupus erythematosus, and neonatal lupus. Increased levels of La are associated with leukemias and other cancers, and various viruses usurp La to promote their replication. Yeast cells (Saccharomyces cerevisiae and Schizosaccharomyces pombe) genetically depleted of La grow and proliferate, whereas deletion from mice causes early embryonic lethality, raising the question of whether La is required by mammalian cells generally or only to surpass a developmental stage. We developed a conditional La allele and used it in mice that express Cre recombinase in either B cell progenitors or the forebrain. B cell Mb1(Cre) La-deleted mice produce no B cells. Consistent with αCamKII Cre, which induces deletion in hippocampal CA1 cells in the third postnatal week and later throughout the neocortex, brains develop normally in La-deleted mice until ∼5 weeks and then lose a large amount of forebrain cells and mass, with evidence of altered pre-tRNA processing. The data indicate that La is required not only in proliferating cells but also in nondividing postmitotic cells. Thus, La is essential in different cell types and required for normal development of various tissue types.

HnRNP A1/A2 and SF2/ASF regulate alternative splicing of interferon regulatory factor-3 and affect immunomodulatory functions in human non-small cell lung cancer cells.

Heterogeneous nuclear ribonucleoparticule A1/A2 (hnRNP A1/A2) and splicing factor 2/alternative splicing factor (SF2/ASF) are pivotal for precursor messenger RNA (pre-mRNA) splicing. Interferon regulatory factor-3 (IRF-3) plays critical roles in host defense against viral and microbial infection. Truncated IRF-3 proteins resulting from alternative splicing have been identified and characterized as functional antagonists to full-length IRF-3. In this study, we examined the molecular mechanism for splicing regulation of IRF-3 pre-mRNA and first reported the regulatory effect of hnRNP A1/A2 and SF2/ASF on IRF-3 splicing and activation. RNA interference-mediated depletion of hnRNP A1/A2 or SF2/ASF in human non-small cell lung cancer (NSCLC) cells increased exclusion of exons 2 and 3 of IRF-3 gene and reduced expression levels of IRF-3 protein and IRF-3 downstream effector molecules interferon-beta and CXCL10/IP-10. In addition, direct binding of hnRNP A1 and SF2/ASF to specific binding motifs in IRF-3 intron 1 was confirmed by RNA electrophoretic mobility shift assay. Subsequent minigene splicing assay showed that IRF-3 minigenes with mutated hnRNPA 1/A2 or SF2/ASF binding motifs increased exclusion of exons 2 and 3. Moreover, knockdown of hnRNP A1/A2 or SF2/ASF in NSCLC cells reinforced phytohemagglutinin-induced tumor necrosis factor-alpha release by peripheral blood mononuclear cells (PBMC) but suppressed that of interleukin-10 in NSCLC/PBMC co-cultures. Taken together, our results suggest that specific knockdown for hnRNP A1/A2 or SF2/ASF increase exclusion of exons 2 and 3 of IRF-3 pre-mRNA and influence immunomodulatory functions of human NSCLC cells.

Immune-related microRNAs are abundant in breast milk exosomes.

Breast milk is a complex liquid rich in immunological components that affect the development of the infant's immune system. Exosomes are membranous vesicles of endocytic origin that are found in various body fluids and that can mediate intercellular communication. MicroRNAs (miRNAs), a well-defined group of non-coding small RNAs, are packaged inside exosomes in human breast milk. Here, we identified 602 unique miRNAs originating from 452 miRNA precursors (pre-miRNAs) in human breast milk exosomes using deep sequencing technology. We found that, out of 87 well-characterized immune-related pre-miRNAs, 59 (67.82%) are presented and enriched in breast milk exosomes (P < 10(-16), χ(2) test). In addition, compared with exogenous synthetic miRNAs, these endogenous immune-related miRNAs are more resistant to relatively harsh conditions. It is, therefore, tempting to speculate that these exosomal miRNAs are transferred from the mother's milk to the infant via the digestive tract, and that they play a critical role in the development of the infant immune system.

An in vitro-transcribed-mRNA polyepitope construct encoding 32 distinct HLA class I-restricted epitopes from CMV, EBV, and Influenza for use as a functional control in human immune monitoring studies.

Interest and activity in the areas of clinical immunotherapy and therapeutic vaccines are growing dramatically, thus there is a pressing need to develop robust tools for assessment of vaccine-induced immunity. CD8+ T cell immunity against specific antigens is normally measured by either flow cytometry using MHC tetramer reagents or via biological assays such as intracellular cytokine staining or ELISPOT after stimulation with specific peptide epitopes. However, these methodologies depend on precise knowledge of HLA-restricted epitopes combined with HLA typing of subjects. As an alternative approach, electroporation of antigen presenting cells (APC) with in vitro-transcribed mRNA (IVT-mRNA) encoding the antigen of interest bypasses the requirements for HLA typing and knowledge of specific epitopes. A current limitation of the IVT-mRNA technique is the lack of robust positive control RNAs to verify the efficacy of electroporation and to ensure that the electroporated APC retain the ability to stimulate T cells. Herein we describe an IVT-mRNA construct wherein all 32 HLA class I-restricted epitopes of the widely used CEF (Cytomegalovirus, Epstein-Barr Virus and Influenza Virus) positive control peptide pool have been genetically spliced together to generate a single polyepitope construct. Each epitope is flanked by three amino- and three carboxy-terminal amino acids from the original parent protein to facilitate proteolytic processing by the proteasome. Using cells obtained from a panel of normal healthy donors and cancer patients we report that dendritic cells, CD40-activated B cells, PHA blasts, and even tumor cells can be transfected with CEF polyepitope IVT-mRNA and can elicit robust CEF-specific responses from autologous T cells, as measured by IFN-gamma ELISPOT. Moreover, the response elicited by CEF IVT-mRNA-transfected APC was similar in magnitude to the response elicited by the complete pool of CEF minimal peptide epitopes, implying that the polyepitope parent protein encoded by the CEF mRNA was efficiently processed into individual epitopes by the proteolytic machinery of the APC. In summary, the CEF polyepitope IVT-mRNA described herein comprises a robust positive control for immunomonitoring studies requiring IVT-mRNA transfection and potentially provides a unique tool for assessing MHC class I processing regardless of HLA haplotype.

The anti-inflammatory activity of the polyphenol resveratrol may be partially related to inhibition of tumour necrosis factor-alpha (TNF-alpha) pre-mRNA splicing.

The present study shows for the first time that the polyphenol resveratrol (RESV) blocks processing of tumour necrosis factor-alpha (TNF-alpha) pre-mRNA in mature mRNA. This study was carried out in turbot (Psetta maxima (L.)), a fish species that we are using to evaluate the effects of RESV on the inflammatory response in vertebrates. Treatment of turbot head kidney leucocytes with polysaccharides from the seaweed Ulva rigida (ulvan) resulted in an increase in TNF-alpha expression. RESV did not inhibit transcription but almost completely inhibited the production of mRNA in ulvan-induced cells and caused a notable increase in the level of unspliced TNF-alpha pre-mRNA. RESV also induced accumulation of IL-1beta pre-mRNA at the expense of mature mRNA, although the effects on IL-1beta were less evident than those on TNF-alpha. However, the housekeeping gene was not affected by RESV. We also evaluated the effects of RESV in vivo under an inflammatory stimulus and found an inhibitory effect on TNF-alpha and IL-1beta pre-mRNA splicing in turbot head kidney at 24 and 48h post-injection. In addition, RESV also reduced migration of cells to the peritoneal cavity under the same inflammatory stimulus. The results show that this fish species may be a useful model for analysing the effects of RESV on TNF-alpha and IL-1beta expression, and suggest that RESV could be used to decrease the levels of pro-inflammatory cytokines in vivo and to reduce inflammatory reactions in certain inflammatory diseases.

Multiple anti-interferon actions of the influenza A virus NS1 protein.

The replication and pathogenicity of influenza A virus (FLUAV) are controlled in part by the alpha/beta interferon (IFN-alpha/beta) system. This virus-host interplay is dependent on the production of IFN-alpha/beta and on the capacity of the viral nonstructural protein NS1 to counteract the IFN system. Two different mechanisms have been described for NS1, namely, blocking the activation of IFN regulatory factor 3 (IRF3) and blocking posttranscriptional processing of cellular mRNAs. Here we directly compare the abilities of NS1 gene products from three different human FLUAV (H1N1) strains to counteract the antiviral host response. We found that A/PR/8/34 NS1 has a strong capacity to inhibit IRF3 and activation of the IFN-beta promoter but is unable to suppress expression of other cellular genes. In contrast, the NS1 proteins of A/Tx/36/91 and of A/BM/1/18, the virus that caused the Spanish influenza pandemic, caused suppression of additional cellular gene expression. Thus, these NS1 proteins prevented the establishment of an IFN-induced antiviral state, allowing virus replication even in the presence of IFN. Interestingly, the block in gene expression was dependent on a newly described NS1 domain that is important for interaction with the cleavage and polyadenylation specificity factor (CPSF) component of the cellular pre-mRNA processing machinery but is not functional in A/PR/8/34 NS1. We identified the Phe-103 and Met-106 residues in NS1 as being critical for CPSF binding, together with the previously described C-terminal binding domain. Our results demonstrate the capacity of FLUAV NS1 to suppress the antiviral host defense at multiple levels and the existence of strain-specific differences that may modulate virus pathogenicity.

The La motif and the RNA recognition motifs of human La autoantigen contribute individually to RNA recognition and subcellular localization.

The human La autoantigen (hLa) protein is a predominantly nuclear phosphoprotein that contains three potential RNA binding domains referred to as the La motif and the RNA recognition motifs RRMs 1 and 2. With this report, we differentiated the contribution of its three RNA binding domains to RNA binding by combining in vitro and in vivo assays. Also, surface plasmon resonance technology was used to generate a model for the sequential contribution of the RNA binding domains to RNA binding. The results indicated that the La motif may contribute to specificity rather than affinity, whereas RRM1 is indispensable for association with pre-tRNA and hY1 RNA. Furthermore, RRM2 was not crucial for the interaction with various RNAs in vivo, although needed for full-affinity binding in vitro. Moreover, earlier studies suggest that RNA binding by hLa may direct its subcellular localization. As shown previously for RRM1, deletion of RNP2 sequence in RRM1 alters nucleolar distribution of hLa, not observed after deletion of the La motif. Here we discuss a model for precursor RNA binding based on a sequential association process mediated by RRM1 and the La motif.

Nuclear bodies and compartmentalization of pre-mRNA splicing factors in higher plants.

We studied the fine structural organization of nuclear bodies in the root meristem during germination of maize and Arabidopsis thaliana using electron microscopy (EM). Cajal bodies (CBs) were observed in quiescent embryos and germinating cells in both species. The number and distribution of CBs were investigated. To characterize the nuclear splicing domains, immunofluorescence labelling with antibodies against splicing factors (U2B" and m3G-snRNAs) and in situ hybridisation (with U1/U6 antisense probes) were performed combined with confocal microscopy. Antibodies specific to the Arabidopsis SR splicing factor atRSp31 were produced. AtRSp31 was detected in quiescent nuclei and in germinating cells. This study revealed an unexpected speckled nuclear organization of atRSp31 in root epidermal cells where micro-clusters of interchromatin granules were also observed by EM. Therefore, we examined the distribution of green fluorescent protein (GFP)-tagged atRSp31 in living cells after Agrobacterium -mediated transient expression. When expressed transiently, atRSp31-GFP exhibited a speckled distribution in leaf cells. Treatments with alpha-amanitin, okadaic acid, staurosporine or heat shock induced the speckles to reorganize. Furthermore, we generated stable Arabidopsis transgenics expressing atRSp31-GFP. The distribution of the fusion protein was identical to that of endogenous atRSp31. Three-dimensional time-lapse confocal microscopy showed that speckles were highly dynamic domains over time.

Reverse 5' caps in RNAs made in vitro by phage RNA polymerases.

We show that about one-third of the RNAs produced in vitro by viral RNA polymerases in the presence of m7GpppG dinucleotides have unusual 5' caps. In these RNAs, the initiating dinucleotide is incorporated in an orientation opposite to that expected so that the 7-methyl guanine (m7G) nucleotide is adjacent to the body of the RNA, making a "reverse" cap. The doubly methylated dinucleotide, m7GpppGm, containing a 2' O-methylated guanine (Gm) is incorporated only in the reverse orientation. Precursors of U1 snRNAs containing reverse caps are recognized by antibodies specific for the m7G cap structure. When injected into Xenopus laevis oocyte nuclei, reverse-capped pre-U1 RNAs are exported considerably more slowly than normal. Furthermore, U1 RNAs with reverse caps exhibit a striking defect in nuclear import that can be attributed to the failure of reverse caps to be hypermethylated to m2,2,7G caps. Thus, the presence of reverse-capped RNAs in RNA preparations may affect conclusions about the efficiency and extent of certain m7G cap-dependent processes.