RNA Ligase-mediated Rapid Amplification Of cDNA Ends Research Articles

Discovery and genomic characterization of three double-stranded RNA viruses coinfecting Conidiobolus taihushanensis.

Conidiobolus sensu lato, a genus within the family Ancylistaceae, encompasses a diverse range of fungal species that are widely distributed in plant debris and soil. In this study, we identified three double-stranded RNA (dsRNA) viruses coinfecting a strain of Conidiobolus taihushanensis. These viruses were identified as Conidiobolus taihushanensis totivirus 1 (CtTV1), Conidiobolus nonsegmented RNA virus 1-2 (CNRV1-2), and Conidiobolus taihushanensis virus 1 (CtV1). Through high-throughput sequencing and RNA-ligase-mediated rapid amplification of cDNA ends (RLM-RACE), we determined their complete genome sequences. The genome of CtTV1 is 6,921 nucleotides in length, containing two open reading frames (ORFs). ORF1 encodes a 1,124-amino-acid capsid protein (CP) with a molecular weight of 125.07 kDa, and ORF2 encodes a 780-amino-acid RNA-dependent RNA polymerase (RdRp) with a molecular weight of 88.05 kDa. CNRV1-2, approximately 3.0 kb in length, also contains two ORFs, which are predicted to encode a 186-amino-acid hypothetical protein (HP) and a 758-amino-acid RdRp. CtV1 has a smaller genome consisting of 3,081 base pairs (bp) with two ORFs: one encoding a 244-amino-acid HP (26.85 kDa) and the other encoding a 707-amino-acid RdRp (80.64 kDa). Phylogenetic analysis based on RdRp sequences revealed that CtTV1 shows the highest similarity to Phytophthora pluvialis RNA virus 1, with 38.79% sequence identity, and clusters with members of the family Orthototiviridae, and it is most closely related to Utsjoki toti-like virus. In contrast, CtV1 formed a unique branch and might represent a new genus. The genome sequence of CNRV1-2 is 99.74% identical to that of the previously described Conidiobolus non-segmented RNA virus 1 (CNRV1). Our findings indicate that CtTV1 and CtV1 are distinct novel viruses, while CNRV1-2 appears to be a variant of CNRV1. This study enhances our understanding of the genetic diversity and evolutionary relationships among mycoviruses associated with C. taihushanensis.

Preparation of HSV-IgM human-mouse chimeric antibody and development of stable recombinant cell line

In order to achieve large-scale production of HSV-IgM (HSV1, HSV2) human-mouse chimeric antibody in vitro, the gene sequence of the corresponding hybridoma cell was harvested by RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) technique to clone the chimeric antibody into eukaryotic expression vectors, and express the target proteins in CHO-S cells. At the same time, the screening process of stable cell lines was optimized, and the pressure conditions of pool construction stage and monoclonal screening stage were explored. Finally, the target protein was purified by protein L affinity purification method and the biological activity was detected. The recombinant IgM antibodies, HSV1 and HSV2, weighted at 899 kDa and 909 kDa respectively, were prepared. The optimal screening pressure was 20P200M (the first phase of pressure) and 50P1000M (the second phase of pressure). The final titer for the monoclonal expression of HSV1-IgM and HSV2-IgM was 1 620 mg/L and 623 mg/L, respectively. This study may facilitate the development of quality control products of HSV1 and HSV2 IgM series recombinant antibodies as well as efficient expression of IgM subtype antibodies in vitro.

Novel RNA viruses from the native range of Hymenoscyphus fraxineus, the causal fungal agent of ash dieback

The native Japanese population of the fungus Hymenoscyphus fraxineus, the causal agent of ash dieback in Europe, was screened for viruses using a high-throughput sequencing method. Five RNA viruses were detected in 116 fungal isolates sequenced via Illumina RNA-seq platform, with an overall virus prevalence of 11.2%. The viruses were completely sequenced by RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE) followed by Sanger sequencing. The sequences appear to represent new species from three established families (Mito-, Endorna- and Partitiviridae), one recognized genus (Botybirnavirus) and a negative-sense single-stranded RNA virus in the order Bunyavirales from the proposed family “Mybuviridae”. The highest prevalence was found for the mitovirus (7.8%), that had two genomic forms (linear and circular), while the other viruses were detected each in one isolate. Co-infection of a mitovirus and an endornavirus was also observed in one of the infected isolates. Here we describe the molecular characterization of the identified viruses. This study expands the diversity of viruses in H. fraxineus and provides the basis for investigating the virus-mediated control of ash dieback in Europe.

Characterization and regulatory mechanism analysis of VvmiR156a-VvAGL80 pair during grapevine flowering and parthenocarpy process induced by gibberellin.

MicroRNA156 (miR156) is an important conserved miRNA family in plants. Recently, we revealed VvmiR156a could involve in the modulation of gibberellin (GA)-mediated flower and berry development process of grapevine (Vitis vinifera L.). However, how to manipulate this process is unclear. For this, we used the GA-induced grapevine parthenocarpy system to investigate the regulatory roles of VvmiR156a during this process. Here, we cloned the mature and precursor sequences of VvmiR156a in Wink grape and identified its potential target gene VvAGL80, which belongs to the MADS-box gene family. Moreover, using RNA ligase-mediated 5' rapid amplification of cDNA ends (RLM-RACE) and poly(A)polymerase-mediated 3' rapid amplification of cDNA (PPM-RACE) technologies, it confirmed that VvAGL80 was the true target gene of VvmiR159a. Analysis of promoter cis-elements and β-glucuronidase (GUS) staining showed that both VvmiR156a and VvAGL80 contained GA-responsive elements and could respond to GA treatments. Quantitative real-time-polymerase chain reaction (qRT-PCR) analysis exhibited the VvmiR156a and VvAGL80 showed opposite expression trends during grapevine flower and berry development, indicating that VvmiR156a negatively regulated the expression of VvAGL80 during this process. After GA treatment, the expression of miR156 in flowers was downregulated significantly, while that of VvAGL80 was upregulated, thereby accelerating grapevine flowering. Furthermore, GA treatment enhanced the negative regulation of VvmiR156a on VvAGL80 in seed, especially at the seed-coat hardening stage, which was the key period of seed growth and development. Our findings enriched the knowledge of the regulatory mechanism of the miRNA-mediated grapevine parthenocarpy process.

Fusarium graminearum DICER-like-dependent sRNAs are required for the suppression of host immune genes and full virulence

In filamentous fungi, gene silencing by RNA interference (RNAi) shapes many biological processes, including pathogenicity. Recently, fungal small RNAs (sRNAs) have been shown to act as effectors that disrupt gene activity in interacting plant hosts, thereby undermining their defence responses. We show here that the devastating mycotoxin-producing ascomycete Fusarium graminearum (Fg) utilizes DICER-like (DCL)-dependent sRNAs to target defence genes in two Poaceae hosts, barley (Hordeum vulgare, Hv) and Brachypodium distachyon (Bd). We identified 104 Fg-sRNAs with sequence homology to host genes that were repressed during interactions of Fg and Hv, while they accumulated in plants infected by the DCL double knock-out (dKO) mutant PH1-dcl1/2. The strength of target gene expression correlated with the abundance of the corresponding Fg-sRNA. Specifically, the abundance of three tRNA-derived fragments (tRFs) targeting immunity-related Ethylene overproducer 1-like 1 (HvEOL1) and three Poaceae orthologues of Arabidopsis thaliana BRI1-associated receptor kinase 1 (HvBAK1, HvSERK2 and BdSERK2) was dependent on fungal DCL. Additionally, RNA-ligase-mediated Rapid Amplification of cDNA Ends (RLM-RACE) identified infection-specific degradation products for the three barley gene transcripts, consistent with the possibility that tRFs contribute to fungal virulence via targeted gene silencing.

Molecular characterization of a new endornavirus inhabiting the ectomycorrhizal fungus Hygrophorus penarioides.

Viruses hosted by uncultivated fungi have been poorly studied. We carried out studies to characterize a large dsRNA segment (~20 kbp) detected in the basidiomycetous, ectomycorrhizal fungus Hygrophorus penarioides. The dsRNA was gel-purified and its randomly amplified cDNA fragments were used for high throughput sequencing (HTS). Reads were de novo assembled and BLASTx analysis revealed sequence similarity to viruses of the family Endornaviridae. The 5' and 3' terminal sequences of the dsRNA segment were determined by performing RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE). The full-length cDNA sequence of the putative endornavirus comprises 16,785 nt and contains a single, long open reading frame which encodes for a polyprotein of 5522 aa with conserved domains for cysteine-rich region, helicase, glycosyltransferase, and RNA-dependent RNA polymerase. The virus was named Hygrophorus penarioides endornavirus 1 (HpEnV1). A BLASTp search performed using the polyprotein sequence revealed that the most closely related, fully sequenced endornavirus to HpEnV1 is Ceratobasidium endornavirus B.

Genome-wide identification and characterization of microRNAs by small RNA sequencing for low nitrogen stress in potato.

Nitrogen is an important nutrient for plant growth and tuber quality of potato. Since potato crop requires high dose of N, improving nitrogen use efficiency (NUE) of plant is an inevitable approach to minimize N fertilization. The aim of this study was to identify and characterize microRNAs (miRNAs) by small RNA sequencing in potato plants grown in aeroponic under two contrasting N (high and low) regimes. A total of 119 conserved miRNAs belonging to 41 miRNAs families, and 1002 putative novel miRNAs were identified. From total, 52 and 54 conserved miRNAs, and 404 and 628 putative novel miRNAs were differentially expressed in roots and shoots, respectively under low N stress. Of total 34,135 predicted targets, the gene ontology (GO) analysis indicated that maximum targets belong to biological process followed by molecular function and cellular component. Eexpression levels of the selected miRNAs and targets were validated by real time-quantitative polymerase chain reaction (RT-qPCR) analysis. Two predicted targets of potential miRNAs (miR397 and miR398) were validated by 5’ RLM-RACE (RNA ligase mediated rapid amplification of cDNA ends). In general, predicted targets are associated with stress-related, kinase, transporters and transcription factors such as universal stress protein, heat shock protein, salt-tolerance protein, calmodulin binding protein, serine-threonine protein kinsae, Cdk10/11- cyclin dependent kinase, amino acid transporter, nitrate transporter, sugar transporter, transcription factor, F-box family protein, and zinc finger protein etc. Our study highlights that miR397 and miR398 play crucial role in potato during low N stress management. Moreover, study provides insights to modulate miRNAs and their predicted targets to develop N-use efficient potato using transgenic/genome-editing tools in future.

Role of phasiRNAs from two distinct phasing frames of GhMYB2 loci in cis- gene regulation in the cotton genome

BackgroundPhased small interfering RNA (phasiRNA) is primarily derived from the 22-nt miRNA targeting loci. GhMYB2, a gene with potential roles in cotton fiber cell fate determination, is a target gene of miR828 and miR858 in the generation of phasiRNAs.ResultsIn the presented work, through the evaluation of phasing scores and phasiRNA distribution pattern, we found that phasiRNAs from GhMYB2 were derived from the 3′ cleavage fragments of 22-nt miR828 and 21-nt miR858 respectively. These two miRNA targeting sites initiated two phasing frames on transcripts of one locus. By means of RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), we further demonstrated that phasiRNAs derived from the two phasing frames played a role in cis-regulation of GhMYB2. The phasiRNAs derived from GhMYB2 were expressed in the somatic tissues, especially in anther and hypocotyl. We further employed our previous small RNA sequencing data as well as the degradome data of cotton fiber bearing ovules, anthers, hypocotyls and embryogenic calli tissues published in public databases, to validate the expression, phasing pattern and functions of phasiRNAs.ConclusionsThe presenting research provide insights of the molecular mechanism of phasiRNAs in regulation of GhMYB2 loci.

Dynamic changes of miR166s at both the transcriptional and post-transcriptional levels during somatic embryogenesis in Lilium

miR166s are involved in fundamentally important biological processes in plants and exhibit functional redundancy and specificity. However, the regulatory role of miR166-mediated somatic embryogenesis (SE) in plants remains unclear. Our previous results preliminarily identified four miR166 family members that have diverse expression profiles during SE in Lilium pumilum DC. Fisch. and in Lilium davidii var. unicolor. In the present study, phylogenetic inference indicated that pre-miR166s are relatively diverse within a species but are closely related among different Lilium species, suggesting that plant miRNA precursors are complex. The predicted targets of LMBR1, IDD1 and PHB were identified by RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), and we dynamically monitored miR166s at both the transcriptional and post-transcriptional levels via qRT-PCR. The results indicated that the expression of miR166 precursors and targets was negatively regulated by miR166 s. Moreover, the expression profiles of miR166 family members differed between the two lily species, and the expression levels of miR166 family members also varied among different members of the same species. Lpu-miR166a was expressed at low levels in the embryonic callus and embryo maturation stages, but lpu-miR166b, lda-miR166a and lda-miR166b were expressed at high levels in the torpedo and cotyledon stages. The first gene studied in plant SE, LMBR1, was expressed at high levels during the embryonic callus and late SE stages, suggesting that this gene may be crucial for embryonic cell formation and embryo germination.

Cloning and characterization of a tyrosine decarboxylase involved in the biosynthesis of galanthamine in Lycoris aurea.

BackgroundGalanthamine, one kind of Amaryllidaceae alkaloid extracted from the Lycoris species, is used in the treatment of Alzheimer’s disease. In regards to medical and economic importance, the biosynthesis and regulatory mechanism of the secondary metabolites in Lycoris remain uninvestigated.MethodsBLAST was used to identify the sequence of tyrosine decarboxylase in the transcriptome of Lycoris aurea (L’Hér) Herb. The enzyme activity of this TYDC was determined by using heterologous expressed protein in the Escherichia coli cells. The related productive contents of tyramine were detected using High Performance Liquid Chromatography (HPLC). According to the available micro RNA sequencing profiles and degradome database of L. aurea, microRNA396 were isolated, which targets to LaTYDC1 and RNA Ligase-Mediated-Rapid Amplification of cDNA Ends (RLM-RACE) were used to confirm the cleavage. The expression levels of miR396 and LaTYDC1 were measured using a quantitative real-time polymerase chain reaction (qRT-PCR).ResultsLaTYDC1 was mainly expressed in root, bulb, leaf and flower fitting the models for galanthamine accumulation. This decarboxylase efficiently catalyzes tyrosine to tyramine conversion. Under methyl jasmonate (MeJA) treatment, the expression of LaTYDC1 and the content of tyramine sharply increase. The use of RLM-RACE confirms that miR396 promotes the degradation of LaTYDC1 mRNA. Under MeJA treatment, the expression of miR396 was suppressed while the expression level of LaTYDC1 sharply increased. Following the increase of the miR396 transcriptional level, LaTYDC1 was significantly repressed.ConclusionLaTYDC1 participates in the biosynthesis of galanthamine, and is regulated by miR396. This finding also provides genetic strategy for improving the yield of galanthamine in the future.

Characterization and primary functional analysis of Pinus densata miR171

The miR171 is a conserved microRNA (miRNA) family and has been shown to participate in plant growth and development. However, the precise function of miR171 in Pinus densata remains largely unclear. Mature miR171 sequence comparison reveals high similarity between Arabidopsis thaliana and P. densata and the pre-miR171 could fold into a characteristic stem-loop hairpin structure. Genes encoding GRAS (GAI-RGA-SCR) family transcription factors and actin binding protein were identified as targets of pde-miR171 using a modified RNA ligase mediated 5’ rapid amplification of cDNA ends (RLM-RACE). Furthermore, the interaction between pde-miR171 and Arabidopsis SCL6 (SCARECROW-LIKE6) was further validated through transient co-expression of both genes in Nicotiana benthamiana leaves. Next, results of real-time quantitative PCR demonstrated that the expression of pde-miR171 was significantly up-regulated in miR171-overexpressing plants than in wild-type plants, which was inversely correlated with the expression of Arabidopsis SCL6 genes. In addition, overexpression of pde-miR171 in Arabidopsis induced larger leaves and earlier flowering under long-day conditions compared with the wild type. The findings presented here suggest that miR171 derived from a P. densata precursor together with its target gene SCL6 may play important roles in the regulation of primary root growth, leaf shape, and flowering time in plants.

Identification of miRNAs and target genes regulating catechin biosynthesis in tea (Camellia sinensis)

MicroRNAs (miRNAs) are endogenous non-protein-coding small RNAs that play crucial and versatile regulatory roles in plants. Using a computational identification method, we identified 55 conserved miRNAs in tea (Camellia sinensis) by aligning miRNA sequences of different plant species with the transcriptome library of tea strain 1005. We then used quantitative real-time PCR (qRT-PCR) to analyze the expression of 31 identified miRNAs in tea leaves of different ages, thereby verifying the existence of these miRNAs and confirming the reliability of the computational identification method. We predicted which miRNAs were involved in catechin synthesis using psRNAtarget Software based on conserved miRNAs and catechin synthesis pathway-related genes. Then, we used RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) to obtain seven miRNAs cleaving eight catechin synthesis pathway-related genes including chalcone synthase (CHS), chalcone isomerase (CHI), dihydroflavonol 4-reductase (DFR), anthocyanidin reductase (ANR), leucoanthocyanidin reductase (LAR), and flavanone 3-hydroxylase (F3H). An expression analysis of miRNAs and target genes revealed that miR529d and miR156g-3p were negatively correlated with their targets CHI and F3H, respectively. The expression of other miRNAs was not significantly related to their target genes in the catechin synthesis pathway. The RLM-RACE results suggest that catechin synthesis is regulated by miRNAs that can cleave genes involved in catechin synthesis.

Down-regulation of genes coding for core RNAi components and disease resistance proteins via corresponding microRNAs might be correlated with successful Soybean mosaic virus infection in soybean.

Plants protect themselves from virus infections by several different defence mechanisms. RNA interference (RNAi) is one prominent antiviral mechanism, which requires the participation of AGO (Argonaute) and Dicer/DCL (Dicer-like) proteins. Effector-triggered immunity (ETI) is an antiviral mechanism mediated by resistance (R) genes, most of which encode nucleotide-binding site-leucine-rich repeat (NBS-LRR) family proteins. MicroRNAs (miRNAs) play important regulatory roles in plants, including the regulation of host defences. Soybean mosaic virus (SMV) is the most common virus in soybean and, in this work, we identified dozens of SMV-responsive miRNAs by microarray analysis in an SMV-susceptible soybean line. Amongst the up-regulated miRNAs, miR168a, miR403a, miR162b and miR1515a predictively regulate the expression of AGO1, AGO2, DCL1 and DCL2, respectively, and miR1507a, miR1507c and miR482a putatively regulate the expression of several NBS-LRR family disease resistance genes. The regulation of target gene expression by these seven miRNAs was validated by both transient expression assays and RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) experiments. Transcript levels for AGO1, DCL1, DCL2 and five NBS-LRR family genes were repressed at different time points after SMV infection, whereas the corresponding miRNA levels were up-regulated at these same time points. Furthermore, inhibition of miR1507a, miR1507c, miR482a, miR168a and miR1515a by short tandem target mimic (STTM) technology compromised SMV infection efficiency in soybean. Our results imply that SMV can counteract soybean defence responses by the down-regulation of several RNAi pathway genes and NBS-LRR family resistance genes via the induction of the accumulation of their corresponding miRNA levels.

Molecular characterization of a novel ssRNA ourmia-like virus from the rice blast fungus Magnaporthe oryzae.

In this study we characterize a novel positive and single stranded RNA (ssRNA) mycovirus isolated from the rice field isolate of Magnaporthe oryzae Guy11. The ssRNA contains a single open reading frame (ORF) of 2,373 nucleotides in length and encodes an RNA-dependent RNA polymerase (RdRp) closely related to ourmiaviruses (plant viruses) and ourmia-like mycoviruses. Accordingly, we name this virus Magnaporthe oryzae ourmia-like virus 1 (MOLV1). Although phylogenetic analysis suggests that MOLV1 is closely related to ourmia and ourmia-like viruses, it has some features never reported before within the Ourmiavirus genus. 3' RLM-RACE (RNA ligase-mediated rapid amplification of cDNA ends) and extension poly(A) tests (ePAT) suggest that the MOLV1 genome contains a poly(A) tail whereas the three cytosine and the three guanine residues present in 5' and 3' untranslated regions (UTRs) of ourmia viruses are not observed in the MOLV1 sequence. The discovery of this novel viral genome supports the hypothesis that plant pathogenic fungi may have acquired this type of viruses from their host plants.

Study on Expression Modes and Cleavage Role of miR156b/c/d and its Target Gene Vv-SPL9 During the Whole Growth Stage of Grapevine.

miR156 regulates the expression of its target SPL (PROMOTER BINDING-LIKE) genes during flower and fruit development, diverse developmental stage transitions, especially from vegetative to reproductive growth phases, by cleaving the target mRNA SPL of one plant-specific transcription factor. However, systematic reports on grapevine have yet to be presented. Here, the precise sequence of miR156 (vvi-miR156b/c/d) in grapevine "Takatsuma" was cloned with a previously cloned grapevine SPL (Vv-SPL9). Expression profiles in 18 grapevine tissues were identified through stem-loop RT-PCR. The interaction mode between vvi-miR156b/c/d and Vv-SPL9 was further validated by detecting the cleavage site and cleavage products of 3'- and 5'-ends via an integrated approach of 5'-RLM-RACE (RNA ligase-mediated 5'-rapid amplification of cDNA ends), 3'-PPM-RACE (poly(A) polymerase-mediated 3'-rapid amplification of cDNA ends), and qRT-PCR (real time reverse transcriptase-polymerase chain reaction). The variation in their cleavage roles in the whole growth stage of grapevine was also systematically investigated. Results showed that vvi-miR156b/c/d exhibited typical temporal-spatial-specific expression levels. The expression levels were higher in vegetative organs, such as leaf, than in reproductive organs, such as tendrils, flowers, and berries. A significant variation was observed during vegetative-to-reproductive transition. The expression patterns of Vv-SPL9 showed the opposite trends with those of vvi-miR156b. We confirmed that the cleavage site was at the 10th site of vvi-miR156b/c/d complementary to Vv-SPL9 in "Takatsuma" grapevine. We also identified the temporal-spatial variation of the cleavage products. This variation can indicate the regulatory function of miR156 on SPL in grapevines. Our findings provide further insights into the functions of vvi-miR156b/c/d and its target Vv-SPL9, and also help enrich our knowledge of small RNA-mediated regulation in grapevine.

Characterization and expression analysis of conserved miRNAs and their targets in Pinus densata

MicroRNAs (miRNAs) are small non-coding RNAs that play crucial regulatory roles in diverse developmental processes via cleavage or translational inhibition of their target mRNAs. Although a growing number of miRNAs and their targets have been predicted and discovered via experimentation in many plants, little is known about conserved miRNAs and their target genes in Pinus densata. In the present study, the conserved miRNAs, miR171 and miR482, from Pinus densata were characterized. Analysis of miR171 and miR482 reveal that these miRNAs were highly conserved in other plant species. In addition, the precursors of miR171 and miR482 were validated by real time-PCR and sequencing. Using real-time quantitative PCR, miR171 and miR482 as well as their corresponding targets were found to be differentially expressed in needles, stems, and roots of Pinus densata. Furthermore two target genes, one GRAS family transcription factor protein gene and one nucleotide-binding site leucine-rich repeat (NBS-LRR) resistance protein gene, were experimentally verified to be the targets of pde-miR171 and pde-miR482, respectively, using RNA ligase-mediated 5’-rapid amplification of cDNA ends (RLM-RACE).

Regulation of miR394 in Response to Fusarium oxysporum f. sp. cepae (FOC) Infection in Garlic (Allium sativum L).

MicroRNAs (miRNAs) are a class of post-transcriptional regulators that negatively regulate gene expression through target mRNA cleavage or translational inhibition and play important roles in plant development and stress response. In the present study, six conserved miRNAs from garlic (Allium sativum L.) were analyzed to identify differentially expressed miRNAs in response to Fusarium oxysporum f. sp. cepae (FOC) infection. Stem-loop RT-PCR revealed that miR394 is significantly induced in garlic seedlings post-treatment with FOC for 72 h. The induction of miR394 expression during FOC infection was restricted to the basal stem plate tissue, the primary site of infection. Garlic miR394 was also upregulated by exogenous application of jasmonic acid. Two putative targets of miR394 encoding F-box domain and cytochrome P450 (CYP450) family proteins were predicted and verified using 5′ RLM-RACE (RNA ligase mediated rapid amplification of cDNA ends) assay. Quantitative RT-PCR showed that the transcript levels of the predicted targets were significantly reduced in garlic plants exposed to FOC. When garlic cultivars with variable sensitivity to FOC were exposed to the pathogen, an upregulation of miR394 and down regulation of the targets were observed in both varieties. However, the expression pattern was delayed in the resistant genotypes. These results suggest that miR394 functions in negative modulation of FOC resistance and the difference in timing and levels of expression in variable genotypes could be examined as markers for selection of FOC resistant garlic cultivars.

Antibody targeting facilitates effective intratumoral siRNA nanoparticle delivery to HER2-overexpressing cancer cells.

The therapeutic potential of RNA interference (RNAi) has been limited by inefficient delivery of short interfering RNA (siRNA). Tumor-specific recognition can be effectively achieved by antibodies directed against highly expressed cancer cell surface receptors. We investigated the utility of linking an internalizing streptavidin-conjugated HER2 antibody to an endosome-disruptive biotinylated polymeric nanocarrier to improve the functional cytoplasmic delivery of siRNA in breast and ovarian cancer cells in vitro and in an intraperitoneal ovarian cancer xenograft model in vivo, yielding an 80% reduction of target mRNA and protein levels with sustained repression for at least 96 hours. RNAi-mediated site specific cleavage of target mRNA was demonstrated using the 5′ RLM-RACE (RNA ligase mediated-rapid amplification of cDNA ends) assay. Mice bearing intraperitoneal human ovarian tumor xenografts demonstrated increased tumor accumulation of Cy5.5 fluorescently labeled siRNA and 70% target gene suppression after treatment with HER2 antibody-directed siRNA nanocarriers. Detection of the expected mRNA cleavage product by 5′ RLM-RACE assay confirmed that suppression occurs via the expected RNAi pathway. Delivery of siRNA via antibody-directed endosomolytic nanoparticles may be a promising strategy for cancer therapy.

Biochemical properties of Bacillus Calmette Guerin ribonuclease III.

Double-stranded RNA (dsRNA) is discovered to participate in the regulation of gene expression in both bacterial and eukaryotic cells. Members of ribonuclease III (RNase III) family recognize RNA motifs and cleave substrates at specific sites in a divalent-metal-ion-dependent manner. In this study, we find the RNase III from Bacillus Calmette Guerin (BCG-RNase III) cleaves small hairpin RNA based on the conserved stem structure associated with Mycobacterium 16S ribosomal RNA precursor at specific sites which are not determined. To evaluate the influence of remnant endogenous ribonucleases from expression host on RNA cleavage assays for RNase III, we use E44A and D48A mutant of the enzyme to perform RNA cleavage assays and find that remnant ribonucleases have no effect on cleavage assays. The RNA cleavage activity of the enzyme can be supported by Mg(2+), Mn(2+), and Co(2+) and enhanced with the increasing salt concentration. The catalytic activity of the enzyme is exhibited when the temperature of the reaction buffer ranges from 30 to 55 °C and the pH of the buffer from 7.0 to 10.0. Two major cleavage sites in RNA substrate are identified using RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE).

Identification of a New Subclass of ALK Negative Anaplastic Large Cell Lymphoma Expressing Aberrant Levels of ERBB4 Transcripts

Anaplastic Large Cell Lymphoma (ALCL) is a clinical and biological heterogeneous disease including the ALK+ and ALK- systemic forms. While ALK+ ALCL are molecularly characterized and can be readily diagnosed, no specific markers and molecular events leading to ALK- ALCL transformation have been identified so far.To discover biomarkers and/or genes potentially involved in ALK- ALCL pathogenesis, we applied the Cancer Outlier Profile Analysis (COPA) algorithm to a large gene expression profiling (GEP) data set including 249 cases of T-NHLs and normal T-cells. Among the top outliers, ERBB4 and COL29A1 genes were exclusively expressed in a subset of ALK- ALCL cases, and resulted highly correlated. Differential analysis comparing the expression profiles of ERBB4/COL29A1+ to ALK+ ALCL patients identified 48 genes up- and 37 down-regulated (qval < 0.0076). The most significantly over-represented functional category included transcriptional regulators. Gene-set-enrichment-analysis (GSEA) indicated that ERBB4/COL29A1+ samples shared activation of specific pathways such as inflammatory response, TNF-NF-κB, JAK-STAT, cytokine, and angiogenesis. RNA sequencing and 5’RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE) identified two ERBB4 truncated transcripts (referred as I20ΔERBB4 and I12ΔERBB4), displaying Transcription Starting Sites (TSS) in intron 12 and 20, respectively. On the contrary, full length COL29A1 mRNA was expressed in ERBB4/COL29A1+ samples. RT-qPCR analysis performed on 170 T-NHL samples (51 PCTL-NOS, 44 ALK+ ALCL, and 75 ALK- ALCL) highlighted specific expression of ERBB4 aberrant transcripts in 25% of ALK- ALCL (18 out of 75). ERBB4+ patients showed higher expression of I20ΔERBB4 as compared to I12ΔERBB4 transcript. ERBB4 expression at protein level was confirmed by immunohistochemistry and Western Blotting on selected cases. Inspection of intronic regions spanning two hypothetical TSS revealed the presence of Human Endogenous Retrovirus (HERV) Long Terminal Repeats (LTR) displaying promoter activity, as demonstrated by luciferase assays.In conclusion, we defined a new subclass of ALK- ALCL characterized by ectopic expression of ERBB4 and COL29A1 genes. To the best of our knowledge, ERBB4 truncated transcripts carrying intronic 5’UTR have never been described before. Further studies are required to address whether the expression of ERBB4 aberrant transcripts may contribute to the ALCL transformation and lymphoma maintenance. This information might lead to more rational therapeutic approaches for ERBB4+ ALCL patients. DisclosuresNo relevant conflicts of interest to declare.