Oligo dT Research Articles

Identification of RNA-binding domains of RNA-binding proteins in cultured cells on a system-wide scale with RBDmap.

This protocol is an extension to: Nat. Protoc. 8, 491-500 (2013); doi:10.1038/nprot.2013.020; published online 14 February 2013RBDmap is a method for identifying, in a proteome-wide manner, the regions of RNA-binding proteins (RBPs) engaged in native interactions with RNA. In brief, cells are irradiated with UV light to induce protein-RNA cross-links. Following stringent denaturing washes, the resulting covalently linked protein-RNA complexes are purified with oligo(dT) magnetic beads. After elution, RBPs are subjected to partial proteolysis, in which the protein regions still bound to the RNA and those released to the supernatant are separated by a second oligo(dT) selection. After sample preparation and mass-spectrometric analysis, peptide intensity ratios between the RNA-bound and released fractions are used to determine the RNA-binding regions. As a Protocol Extension, this article describes an adaptation of an existing Protocol and offers additional applications. The earlier protocol (for the RNA interactome capture method) describes how to identify the active RBPs in cultured cells, whereas this Protocol Extension also enables the identification of the RNA-binding domains of RBPs. The experimental workflow takes 1 week plus 2 additional weeks for proteomics and data analysis. Notably, RBDmap presents numerous advantages over classic methods for determining RNA-binding domains: it produces proteome-wide, high-resolution maps of the protein regions contacting the RNA in a physiological context and can be adapted to different biological systems and conditions. Because RBDmap relies on the isolation of polyadenylated RNA via oligo(dT), it will not provide RNA-binding information on proteins interacting exclusively with nonpolyadenylated transcripts. Applied to HeLa cells, RBDmap uncovered 1,174 RNA-binding sites in 529 proteins, many of which were previously unknown.

Evaluation of reference genes for reverse transcription quantitative real-time PCR (RT-qPCR) studies in Silene vulgaris considering the method of cDNA preparation.

Accurate gene expression measurements are essential in studies of both crop and wild plants. Reverse transcription quantitative real-time PCR (RT-qPCR) has become a preferred tool for gene expression estimation. A selection of suitable reference genes for the normalization of transcript levels is an essential prerequisite of accurate RT-qPCR results. We evaluated the expression stability of eight candidate reference genes across roots, leaves, flower buds and pollen of Silene vulgaris (bladder campion), a model plant for the study of gynodioecy. As random priming of cDNA is recommended for the study of organellar transcripts and poly(A) selection is indicated for nuclear transcripts, we estimated gene expression with both random-primed and oligo(dT)-primed cDNA. Accordingly, we determined reference genes that perform well with oligo(dT)- and random-primed cDNA, making it possible to estimate levels of nucleus-derived transcripts in the same cDNA samples as used for organellar transcripts, a key benefit in studies of cyto-nuclear interactions. Gene expression variance was estimated by RefFinder, which integrates four different analytical tools. The SvACT and SvGAPDH genes were the most stable candidates across various organs of S. vulgaris, regardless of whether pollen was included or not.

Whole genome sequence phylogenetic analysis of four Mexican rabies viruses isolated from cattle

Phylogenetic analysis of the rabies virus in molecular epidemiology has been traditionally performed on partial sequences of the genome, such as the N, G, and P genes; however, that approach raises concerns about the discriminatory power compared to whole genome sequencing. In this study we characterized four strains of the rabies virus isolated from cattle in Querétaro, Mexico by comparing the whole genome sequence to that of strains from the American, European and Asian continents. Four cattle brain samples positive to rabies and characterized as AgV11, genotype 1, were used in the study. A cDNA sequence was generated by reverse transcription PCR (RT-PCR) using oligo dT. cDNA samples were sequenced in an Illumina NextSeq 500 platform. The phylogenetic analysis was performed with MEGA 6.0. Minimum evolution phylogenetic trees were constructed with the Neighbor-Joining method and bootstrapped with 1000 replicates. Three large and seven small clusters were formed with the 26 sequences used. The largest cluster grouped strains from different species in South America: Brazil, and the French Guyana. The second cluster grouped five strains from Mexico. A Mexican strain reported in a different study was highly related to our four strains, suggesting common source of infection. The phylogenetic analysis shows that the type of host is different for the different regions in the American Continent; rabies is more related to bats. It was concluded that the rabies virus in central Mexico is genetically stable and that it is transmitted by the vampire bat Desmodus rotundus.

Creation of Recombinant Antibodies: Using 5'-RACE to Amplify Immunoglobulin Sequences.

Modern molecular biology techniques have been applied to the production of therapeutic antibodies. Nonetheless, recombinant antibodies remain the exception rather than the rule for the antibodies used in most research and diagnostic applications. The lack of penetration of recombinant antibodies into the research arena can be attributed largely to the fact that the Ig gene families are large and the variable region gene segments are, indeed, variable, precluding the use of polymerase chain reaction (PCR) with two simple primers to amplify the heavy and light chain gene segments. Because of the complexity of the V gene family and the number of possible sequences for amplification, there may be a distinct advantage to using a PCR method that does not require a specific 5' primer to amplify the gene segment. 5'-RACE is just such a method. In the original 5'-RACE method, mRNA served as a template for cDNA synthesis using either oligo(dT) priming or a gene-specific primer. Terminal deoxynucleotide transferase (TdT) was used to tail the first strand with a region of known sequence, such as poly(A). Once tailed, the second strand can be amplified using poly(T). This 5'-RACE protocol uses the TdT activity of reverse transcriptase, which allows nontemplated addition of nucleotides to the end of the nascently made cDNA first strand.

Creation of Recombinant Antibodies: Using Degenerate Oligonucleotides to Amplify Heavy- and Light-Chain Sequences.

Recombinant antibodies can be modified to suit the application: Changes in isotype, format (e.g., scFv, Fab, bispecific antibodies), and specificity can be made once the heavy- and light-chain sequences are available. However, Ig gene families are large and the variable region gene segments are, indeed, variable, precluding the use of polymerase chain reaction (PCR) with two simple primers to amplify the heavy and light chain gene segments. A wide variety of approaches have been taken to obviate the complexity of the variable region gene segments, including using "universal" or degenerate primers. In this method, mRNA is obtained from B cells as a source of heavy- and light-chain sequences. cDNA is prepared using purified mRNA and either oligo(dT) or a C-region 3' primer. The cDNA negative strand is complementary to the coding sequence of the heavy or light chain. This approach can aid in the design of a variable region primer or in selection of the appropriate degenerate primer for a specific variable gene subfamily.

CspB of an arctic bacterium, Polaribacter irgensii KOPRI 22228, confers extraordinary freeze-tolerance.

Freezing temperatures are a major challenge for life at the poles. Decreased membrane fluidity, uninvited secondary structure formation in nucleic acids, and protein cold-denaturation all occur at cold temperatures. Organisms adapted to polar regions possess distinct mechanisms that enable them to survive in extremely cold environments. Among the cold-induced proteins, cold shock protein (Csp) family proteins are the most prominent. A gene coding for a Csp-family protein, cspB, was cloned from an arctic bacterium, Polaribacter irgensii KOPRI 22228, and overexpression of cspB greatly increased the freeze-survival rates of Escherichia coli hosts, to a greater level than any previously reported Csp. It also suppressed the cold-sensitivity of an E. coli csp-quadruple deletion strain, BX04. Sequence analysis showed that this protein consists of a unique domain at its N-terminal end and a well conserved cold shock domain at its C-terminal end. The most common mechanism of Csp function in cold adaption is melting of the secondary structures in RNA and DNA molecules, thus facilitating transcription and translation at low temperatures. P. irgensii CspB bound to oligo(dT)-cellulose resins, suggesting single-stranded nucleic acid-binding activity. The unprecedented level of freeze-tolerance conferred by P. irgensii CspB suggests a crucial role for this protein in survival in polar environments.

MRNA Interactome Capture from Plant Protoplasts.

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional gene regulation (PTGR) of messenger RNAs (mRNAs). In the past few years, a number of mRNA-bound proteomes from yeast and mammalian cell lines have been successfully isolated through the use of a novel method called "mRNA interactome capture," which allows for the identification of mRNA-binding proteins (mRBPs) directly from a physiological environment. The method is composed of in vivo ultraviolet (UV) crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo(dT) beads, and the subsequent identification of the crosslinked proteins by mass spectrometry (MS). Very recently, by applying the same method, several plant mRNA-bound proteomes have been reported simultaneously from different Arabidopsis tissue sources: etiolated seedlings, leaf tissue, leaf mesophyll protoplasts, and cultured root cells. Here, we present the optimized mRNA interactome capture method for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type that serves as a versatile tool for experiments that include various cellular assays. The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the mRNA-bound proteome obtained from a medium-scale experiment (107 cells), RBPs noted to have RNA-binding capacity were found to be overrepresented, and many novel RBPs were identified. The experiment can be scaled up (109 cells), and the optimized method can be applied to other plant cell types and species to broadly isolate, catalog, and compare mRNA-bound proteomes in plants.

MRNA interactome capture in mammalian cells.

Throughout their entire life cycle, mRNAs are associated with RNA-binding proteins (RBPs), forming ribonucleoprotein (RNP) complexes with highly dynamic compositions. Their interplay is one key to control gene regulatory mechanisms from mRNA synthesis to decay. To assay the global scope of RNA-protein interactions, we and others have published a method combining crosslinking with highly stringent oligo(dT) affinity purification to enrich proteins associated with polyadenylated RNA (poly(A)+ RNA). Identification of the poly(A)+ RNA-bound proteome (also: mRNA interactome capture) has by now been applied to a diversity of cell lines and model organisms, uncovering comprehensive repertoires of RBPs and hundreds of novel RBP candidates. In addition to determining the RBP catalog in a given biological system, mRNA interactome capture allows the examination of changes in protein-mRNA interactions in response to internal and external stimuli, altered cellular programs and disease.

Characterization Of Expression Levels Of Genes Involved In Adipogenesis And Inflammation In Congenic Mice Carrying Obesity and Hyperlipidemia QTL on Chromosome 1

Obesity is an international health crisis and is considered the leading cause of preventable death. The development of obesity is likely due to interactions of susceptibility genes with obesity‐promoting environments such as high fat diets (HFD). These interactions lead to an expansion of adipose tissue, through an increase in adipocyte size and/or number. Adipose tissue inflammation is a well‐recognized characteristic of obesity. Previously, we identified quantitative trait loci (QTL) linked to obesity and hyperlipidemia on mouse chromosome (Chr) 1 in an intercross of obese and diabetic TALLYHO/Jng (TH) mice with C57BL/6 (B6) mice. Subsequently, we established a congenic mouse strain carrying the Chr1 QTL, 128 Mb in size, derived from TH on a B6 background (B6.TH‐Chr1–128Mb). The congenic mice were found to be more susceptible to diet‐induced obesity and hyperlipidemia than their B6 counterparts. The purpose of this study was to characterize the congenic mice by examining mRNA and protein levels of adipocyte differentiation and inflammation markers in white adipose tissue and liver. B6.TH‐Chr1–128Mb congenic and B6 mice were weaned onto chow and HFD and maintained until 16 weeks of age. Total RNA was isolated from adipose tissue and reverse‐transcribed with SUPERSCRIPT RT using oligo dT as primer to synthesize first‐strand cDNA. mRNA levels of peroxisome proliferator‐activated receptor gamma (Pparg), CCAAT/enhancer binding protein beta (Cebpb), preadipocyte factor 1 (Pref‐1), and Interleukin 6 (Il‐6) genes were measured by real‐time quantitative RT‐PCR. For protein levels, adipose tissue and liver were lysed in RIPA buffer and lysates subjected to SDA‐PAGE and Western blotting. Blots were probed with specific antibodies against PPARG and IL‐6, and detection of immunoreactive bands performed using enhanced chemiluminescence. Data analysis was conducted by ANOVA with GraphPad Prism 7. Congenic mice had a 2‐fold increase in gene expression level of Pparg, a major regulator of adipogenesis and lipogenesis, in adipose tissue compared to B6 mice on chow. HFD significantly increased the gene expression level of Pparg in both B6 and congenic mice, with greater degree in congenic mice. This trend was also the case for the protein expression level of PPARG in adipose tissue, as well as in liver, but it did not reach statistical significance. There were no significant differences in gene expression levels of Pref‐1, the most upstream repressor to adipocyte differentiation, and Cebpb, a major transcription factor during an early stage of adipogenesis, in adipose tissue between congenic and B6 mice for either diet. The gene expression level of Il‐6, a proinflammatory marker, was increased by HFD in both congenic and B6 mice, without genotype difference. A similar trend was also shown in IL‐6 protein levels in adipose tissue, as well as in liver, but it did not reach statistical significance. Collectively, we demonstrated that gene expression levels of Pparg were significantly upregulated in adipose tissue of B6.TH‐Chr1–128Mb congenic mice on chow and HFD.Support or Funding InformationThis work was supported by NASA WVSGC Graduate Fellowship to JKP and in part by NIH/NIDDK Grant R01DK077202 and institutional start‐up funding from Marshall University to JHK.

Prostaglandin synthesis enzymes' gene transcription in bitches with endometritis.

Endometritis is a major cause of infertility in many domestic species. However, until now the pathogenesis of the endometritis in the bitch is unclear. The aim of this study was to evaluate the gene transcription pattern of prostaglandin (PG) synthesis enzymes (cyclooxygenase [COX2], PTGES-1 and PGFS) in the endometrium of bitches with or without endometritis. Thirty mixed breed bitches in dioestrus, aged between 1 and 5years, and weighing between 10 and 30kg were used. After ovariohysterectomy (OVX), uterine biopsy samples were collected from the middle part of both horns. Then, endometrial epithelium was collected using the cytobrush method and mRNA analysis was performed by real-time RT-PCR. Data were analysed with Kruskal-Wallis anova using the sas® software. Uterine condition was identified by endometrial biopsies (normal endometria [n=11; NE], acute endometritis [n=10; AE] and chronic endometritis [n=9; CE]). The COX2, PTGES-1 and PGFS/AKR1C3 mRNA expression in bitches with and without endometritis was similar. Except for PGFS/AKR1C3, gene transcription of COX2 and PTGES-1 was significantly increased in AE compared with CE. In addition, COX2 gene transcription was significantly increased in AE compared with NE. In contrast, no differences were found for COX2, PTGES-1 and PGFS/AKR1C3 mRNA expression in the samples of NE compared with CE.

Long term stability of Oligo (dT) 25 magnetic beads for the expression analysis of Euglena gracilis for long term space projects

The unicellular freshwater flagellate Euglena gracilis has a highly developed sensory system. The cells use different stimuli such as light and gravity to orient themselves in the surrounding medium to find areas for optimal growth. Due to the ability to produce oxygen and consume carbon dioxide, Euglena is a suitable candidate for life support systems. Participation in a long-term space experiment would allow for the analysis of changes and adaptations to the new environment, and this could bring new insights into the mechanism of perception of gravity and the associated signal transduction chain. For a molecular analysis of transcription patterns, an automated system is necessary, capable of performing all steps from taking a sample, processing it and generating data. One of the developmental steps is to find long-term stable reagents and materials and test them for stability at higher-than-recommended temperature conditions during extended storage time. We investigated the usability of magnetic beads in an Euglena specific lysis buffer after addition of the RNA stabilizer Dithiothreitol over 360 days and the lysis buffer with the stabilizer alone over 455 days at the expected storage temperature of 19 °C. We can claim that the stability is not impaired at all after an incubation period of over one year. This might be an interesting result for researchers who have to work under non-standard lab conditions, as in biological or medicinal fieldwork.

Anti-tumour research of recombinant BCG using BZLF1 and hGM-CSF fusion genes

The random primer Oligo(dT)15 was used in RT-PCR to obtain cDNA from the human granulocyte macrophage colony stimulating factor (hGM-CSF) gene and the Epstein-Barr virus (EBV) gene BZLF1. Then, the sequence splicing overlap extension method was used to obtain a GCBF fusion gene containing a linker sequence that encoded the polypeptide (Gly4Ser)3. The GCBF fusion gene was inserted into pMV261, which was then transformed into competent E. coli DH5 alpha cells, and positive cells were selected based on kanamycin resistance on LB plates. The recombinant plasmid pMVBZLF1 was extracted from E. coli, and BCG (Bacillus Calmette-Guérin) was transformed into competent cells. According to the RT-PCR results, the target genes hGM-CSF and BZLF1 were 461bp and 788bp in size, which was in agreement with the expected values. Construction of the recombinant plasmid by double enzyme digestion, amplification, sequencing and Western blotting confirmed that the GCBF fusion gene (1204bp) was correctly inserted into pMV261, successfully transformed into BCG competent cells, and properly expressed.After mice were injected with rBCG (recombinant BCG), antibody levels were detected using ELISA, and spleen cells were obtained and the killing rates of specific CTLs by rBCG were detected using a CTL assay kit. Then, the influence of rBCG on tumour cells was analysed in C57BL/6 mice. We found that rBCG-secreting cytokines hybridized with hGM-CSF and BZLF1 antibodies and that the rBCG vaccine stimulated antibody production in C57BL/6 mice. The specific cytotoxic effects of the spleen cells from the rBCG group on EB virus-positive tumour cells was significantly different from the cytotoxic effects of the control group cells (P<0.01). CD8+ T and CD4+ T lymphocytes were detected in the tumour tissues of the rBCG group mice by flow cytometry, indicating that CD8+ T and CD4+ T lymphocytes infiltrated into the tumour tissue in the mice. Morphological observations of the tumour sections from the rBCG-immunized mice showed the infiltration of lymphocytes into the tumour tissues. The average rBCG tumour volume was less than the average tumour volume of the control group. Thus, rBCG may inhibit the growth of EB virus-positive tumour cells in mice.

Comprehensive Identification of mRNA-Binding Proteins of Leishmania donovani by Interactome Capture.

Leishmania are unicellular eukaryotes responsible for leishmaniasis in humans. Like other trypanosomatids, leishmania regulate protein coding gene expression almost exclusively at the post-transcriptional level with the help of RNA binding proteins (RBPs). Due to the presence of polycystronic transcription units, leishmania do not regulate RNA polymerase II-dependent transcription initiation. Recent evidence suggests that the main control points in gene expression are mRNA degradation and translation. Protein-RNA interactions are involved in every aspect of RNA biology, such as mRNA splicing, polyadenylation, localization, degradation, and translation. A detailed picture of these interactions would likely prove to be highly informative in understanding leishmania biology and virulence. We developed a strategy involving covalent UV cross-linking of RBPs to mRNA in vivo, followed by interactome capture using oligo(dT) magnetic beads to define comprehensively the mRNA interactome of growing L. donovani amastigotes. The protein mass spectrometry analysis of captured proteins identified 79 mRNA interacting proteins which withstood very stringent washing conditions. Strikingly, we found that 49 of these mRNA interacting proteins had no orthologs or homologs in the human genome. Consequently, these may represent high quality candidates for selective drug targeting leading to novel therapeutics. These results show that this unbiased, systematic strategy has the promise to be applicable to study the mRNA interactome during various biological settings such as metabolic changes, stress (low pH environment, oxidative stress and nutrient deprivation) or drug treatment.

Post‐translational modifications of Annexin A2 are linked to its association with perinuclear nonpolysomal mRNP complexes

Various post‐translational modifications (PTMs) regulate the localisation and function of the multifunctional protein Annexin A2 (AnxA2). In addition to its various tasks as a cytoskeletal‐ and membrane‐associated protein, AnxA2 can function as a trans‐acting protein binding to cis‐acting sequences of specific mRNAs. In the present study, we have examined the role of Ser25 phosphorylation in subcellular localisation of AnxA2 and its interaction with mRNP complexes. Subcellular fractionation and confocal microscopy of rat neuroendocrine PC12 cells showed that Ser25‐phosphorylated AnxA2 (pSer25AnxA2) is absent from the nucleus and mainly localised to the perinuclear region, evidently associating with both membranes and cytoskeletal elements. Perinuclear targeting of AnxA2 was abolished by inhibition of protein kinase C activity, which resulted in cortical enrichment of the protein. Although oligo(dT)‐affinity purification of mRNAs revealed that pSer25AnxA2 associates with nonpolysomal, translationally inactive mRNP complexes, it displayed only partial overlap with a marker of P‐bodies. The phosphorylated protein is present as high‐molecular‐mass forms, indicating that it contains additional covalent PTMs, apparently triggered by its Ser25 phosphorylation. The subcellular distributions of these forms clearly differ from the main form of AnxA2 and are also distinct from that of Tyr23‐phosphorylated AnxA2. Immunoprecipitation verified that these high‐molecular‐mass forms are due to ubiquitination and/or sumoylation. Moreover, these results indicate that Ser25 phosphorylation and ubiquitin/SUMO1 conjugation of AnxA2 promote its association with nonpolysomal mRNAs, providing evidence of a possible mechanism to sequester a subpopulation of mRNAs in a translationally inactive and transport competent form at a distinct subcellular localisation.

Staphylococcus Aureus Elicit an Increased Expression of TLR9 Gene to the Innate Immune Response in Bovine Mastitis

Mastitis in dairy cattle caused by S. aureus infection may eventually turn into a chronic disease with a lifelong persistence of the bacteria. TLR9 plays a crucial role in cell survival through recognition of various bacterial components. This study was carried out to assess the expression of TLR9 gene in spontaneous bovine sub-clinical and clinical mastitis caused by S. aureus. RNA isolated from milk somatic cells was converted as cDNA using oligo (dT) primers. Quantitation of mRNA of TLR9 was analysed by RT-qPCR system. The mRNA expression of TLR9 gene in sub-clinical mastitis was higher (3.71 fold) than clinical case (1.15 fold) when compared to normal bovine case. The expression of TLR9 gene at high level during sub-clinical stage of infection, may be subsided itself without precipitating into clinical mastitis. The results presented would facilitate deeper insight into the specific regulatory mechanisms of TLR9 mediated signalling cascades to elucidate host immune response against bovine mastitis.

FIRST REPORT OF CHILLI VEINAL MOTTLE VIRUS IN TOMATO IN PAKISTAN

Chilli veinal mottle virus (ChiVMV, genus Potyvirus ) infects several solanaceous species worldwide (Zhao et al ., 2014). In April 2014, a total of 20 tomato leaf samples with symptoms of mosaic, necrosis and mottling were collected from the research farm of PMAS-Arid Agriculture University Rawalpindi. All disease samples were screened for the presence of potyvirus infection by indirect plate-trapped antigen (PTA)-ELISA using “Poty group test” kit (Bioreba AG, Switzerland). Only 12 samples were positive for potyvirus infection. These samples were further tested for Potato virus Y (PVY), Pepper veinal mottle virus (PVMV) (Bioreba AG) and ChiVMV (Loewe Biochemicals, Germany) using virus specific DAS- ELISA. Of 12 potyvirus confirmed samples, two were positive for ChiVMV and remaining ten were positive for PVY. No reaction was observed with antibodies against PVMV. The ChiVMV ELISA-positive samples were further confirmed by RT-PCR using ChiVMV specific primers Poty3 and Oligo(dT) (Tsai et al ., 2008). Both PCR amplicons were purified using QIAquick® PCR purification kit (Qiagen) and subsequently sequenced in both orientations . A product 759 nucleotides in size (partial CP gene including 3’UTR) was obtained (both clones were 100% identical) and the sequence of ChiVMV isolate tomato (AARTPK) was deposited in GenBank (Accession No. KT876048). BLASTn revealed >90% sequence identity with ChiVMV isolates from India (JN624776, JN692501), China (HQ218936, KF738253, KC711055, JX088636) and Pakistan (KJ472764). This destructive virus has been previously reported from chilli pepper in Pakistan (Shah et al ., 2009). To the best of our knowledge, this is the first confirmed report of ChiVMV from tomato in Pakistan.

Opioid precursor protein isoform is targeted to the cell nuclei in the human brain

BackgroundNeuropeptide precursors are traditionally viewed as proteins giving rise to small neuropeptide molecules. Prodynorphin (PDYN) is the precursor protein to dynorphins, endogenous ligands for the κ-opioid receptor. Alternative mRNA splicing of neuropeptide genes may regulate cell- and tissue-specific neuropeptide expression and produce novel protein isoforms. We here searched for novel PDYN mRNA and their protein product in the human brain. MethodsNovel PDYN transcripts were identified using nested PCR amplification of oligo(dT) selected full-length capped mRNA. Gene expression was analyzed by qRT-PCR, PDYN protein by western blotting and confocal imaging, dynorphin peptides by radioimmunoassay. Neuronal nuclei were isolated using fluorescence-activated nuclei sorting (FANS) from postmortem human striatal tissue. Immunofluorescence staining and confocal microscopy was performed for human caudate nucleus. ResultsTwo novel human PDYN mRNA splicing variants were identified. Expression of one of them was confined to the striatum where its levels constituted up to 30% of total PDYN mRNA. This transcript may be translated into ∆SP-PDYN protein lacking 13 N-terminal amino acids, a fragment of signal peptide (SP). ∆SP-PDYN was not processed to mature dynorphins and surprisingly, was targeted to the cell nuclei in a model cellular system. The endogenous PDYN protein was identified in the cell nuclei in human striatum by western blotting of isolated neuronal nuclei, and by confocal imaging. Conclusions and general significanceHigh levels of alternatively spliced ∆SP-PDYN mRNA and nuclear localization of PDYN protein suggests a nuclear function for this isoform of the opioid peptide precursor in human striatum.

A versatile tandem RNA isolation procedure to capture in vivo formed mRNA-protein complexes

We describe a tandem RNA isolation procedure (TRIP) that enables purification of in vivo formed messenger ribonucleoprotein (mRNP) complexes. The procedure relies on the purification of polyadenylated mRNAs with oligo(dT) beads from cellular extracts, followed by the capture of specific mRNAs with 3'-biotinylated 2'-O-methylated antisense RNA oligonucleotides, which are recovered with streptavidin beads. TRIP was applied to isolate in vivo crosslinked mRNP complexes from yeast, nematodes and human cells for subsequent analysis of RNAs and bound proteins. The method provides a basis for adaptation to other types of polyadenylated RNAs, enabling the comprehensive identification of bound proteins/RNAs, and the investigation of dynamic rearrangement of mRNPs imposed by cellular or environmental cues.

Cloning, expression, and purification of Chlamydomonas reinhardtii CC-503 sedoheptulose 1,7-bisphosphatase in Escherichia coli

ABSTRACTSedoheptulose 1,7-bisphosphatase (SBPase), a nuclear-encoded chloroplastic enzyme, is an important rate-limiting enzyme of the carbon fixation cycle (Calvin cycle). SBPase is unique to only photosynthetic organisms and is involved in the regeneration of ribulose-1,5-bisphosphate. SBPases from several sources have been studied for their induction and regulation. However, SBPase from Chlamydomonas reinhardtii CC-503, the widely studied model microalga, has not been isolated and functionally confirmed to date. In this study, the full-length cDNA for SBPase was isolated from C. reinhardtii CC-503 using anchored oligo(dT)24VGN primer for reverse transcription. The SBPase cDNA was cloned into pET28a expression vector for the production of 6X His-tagged protein in Escherichia coli BL21 (DE3) strain. Although initially most of the enzyme was obtained as insoluble protein aggregates, solubilization of protein was improved by optimization of protein induction with respect to growth temperature and isopropyl β-D-1-thiogalactopyranoside concentrations. The induced protein was purified by immobilized metal affinity chromatography using nickel–nitrilotriacetic acid resin in a phosphate-free buffer leading to an accurate SBPase activity measurement. The present study demonstrates, for the first time, successful cloning of C. reinhardtii CC-503 SBPase in E. coli leading to the expression of a functionally active enzyme.

Chaperonin-containing T-complex Protein 1 Subunit ζ Serves as an Autoantigen Recognized by Human Vδ2 γδ T Cells in Autoimmune Diseases

Human γδ T cells recognize conserved endogenous and stress-induced antigens typically associated with autoimmune diseases. However, the role of γδ T cells in autoimmune diseases is not clear. Few autoimmune disease-related antigens recognized by T cell receptor (TCR) γδ have been defined. In this study, we compared Vδ2 TCR complementarity-determining region 3 (CDR3) between systemic lupus erythematosus (SLE) patients and healthy donors. Results show that CDR3 length distribution differed significantly and displayed oligoclonal characteristics in SLE patients when compared with healthy donors. We found no difference in the frequency of Jδ gene fragment usage between these two groups. According to the dominant CDR3δ sequences in SLE patients, synthesized SL2 peptides specifically bound to human renal proximal tubular epithelial cell line HK-2; SL2-Vm, a mutant V sequence of SL2, did not bind. We identified the putative protein ligand chaperonin-containing T-complex protein 1 subunit ζ (CCT6A) using SL2 as a probe in HK-2 cell protein extracts by affinity chromatography and liquid chromatography-electrospray ionization-tandem mass spectrometry analysis. We found CCT6A expression on the surface of HK-2 cells. Cytotoxicity of only Vδ2 γδ T cells to HK-2 cells was blocked by anti-CCT6A antibody. Finally, we note that CCT6A concentration was significantly increased in plasma of SLE and rheumatoid arthritis patients. These data suggest that CCT6A is a novel autoantigen recognized by Vδ2 γδ T cells, which deepens our understanding of mechanisms in autoimmune diseases.