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Paper mulberry is a valuable woody species with a good chilling tolerance. In this study, phosphoproteomic analysis, physiological measurement, and mRNA quantification were employed to explore the molecular mechanism of chilling (4 °C) tolerance in paper mulberry. After chilling for 6 h, 427 significantly changed phosphoproteins were detected in paper mulberry seedlings without obvious physiological injury. When obvious physiological injury occurred after chilling for 48 h, a total of 611 phosphoproteins were found to be significantly changed at the phosphorylation level. Several protein kinases, especially CKII, were possibly responsible for these changes according to conserved sequence analysis. The results of Gene Ontology analysis showed that phosphoproteins were mainly responsible for signal transduction, protein modification, and translation during chilling. Additionally, transport and cellular component organization were enriched after chilling for 6 and 48 h, respectively. On the basis of the protein-protein interaction network analysis, a protein kinase and phosphatases hub protein (P1959) were found to be involved in cross-talk between Ca

Food-borne pathogens have been recognized as a major cause of human infections worldwide. Their identification needs to be simpler, cheaper and more reliable than the traditional methods. Here, we constructed a low-cost paper platform for viable pathogenic bacteria detection with the naked eye. In this study, an effective isothermal amplification method was used to amplify the hlyA mRNA gene, a specific RNA marker in Listeria monocytogenes. The amplification products were applied to the paper-based platform to perform a visual test using sandwich hybridization assays. When the RNA products migrated along the platform by capillary action, the gold nanoparticles accumulated at the designated area. Under optimized experimental conditions, as little as 0.5 pg/μL genomic RNA from L. monocytogenes could be detected. It could also be used to specifically detect 20 CFU/mL L. monocytogenes from actual samples. The whole assay process, including RNA extraction, amplification, and visualization, can be completed within several hours. This method is suitable for point-of-care applications to detect food-borne pathogens, as it can overcome the false-positive results caused by amplifying nonviable L. monocytogenes. Furthermore, the results can be imaged and transformed into a two-dimensional bar code through an Android-based smart phone for further analysis or in-field food safety tracking.

We examined the utility of dried blood on filter paper for the source of cytokine messenger RNA (mRNA). Total RNA was isolated from the dried blood of mice infected with Plasmodium yoelii, and cDNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR). As a reference, we extracted total RNA from peripheral blood collected at the same time as the preparation for dried blood. There was no difference in cytokine mRNA expression between the two sources; the dried blood on filter paper and the peripheral blood. Th1 cells, Th2 cells, and monocytes/macrophages derived cytokine mRNAs in the dried blood from infected mice were detected, and the increase of some of the cytokines mRNAs after infection was also observed. These results suggested that the dried blood on filter paper is satisfactory RNA source for immunological examination in field-based studies.

To the Editor: To provide samples for neonatal mass screening, a few drops of blood are routinely collected from the heel of the newborn infant and allowed to dry on a thick filter paper called the Guthrie card.[1][1] Guthrie cards represent a valuable and comprehensive genetic repository because

Background Accurate sampling of sub-microscopic gametocytes is necessary for epidemiological studies to identify the infectious reservoir of Plasmodium falciparum. Detection of gametocyte mRNA achieves sensitive detection, but requires careful handling of samples. Filter papers can be used for collecting RNA samples, but rigorous testing of their capacity to withstand adverse storage conditions has not been fully explored. Methods Three gametocyte dilutions: 10/μL, 1.0/μL and 0.1/μL were spotted onto Whatman™ 903 Protein Saver Cards, FTA Classic Cards and 3MM filter papers that were stored under frozen, cold chain or tropical conditions for up to 13 weeks . RNA was extracted, then detected by quantitative nucleic acid sequence-based amplification (QT-NASBA) and reverse-transcriptase PCR (RT-PCR). Results Successful gametocyte detection was more frequently observed from the Whatman 903 Protein Saver Card compared to the Whatman FTA Classic Card, by both techniques (p Conclusions This study indicates the Whatman 903 Protein Saver Card is better for Pfs25 mRNA sampling compared to the Whatman FTA Classic Card, and that the Whatman 3MM filter paper may prove to be a satisfactory cheaper option for Pfs25 mRNA sampling. When appropriately dried, filter papers provide a useful approach to Pfs25 mRNA sampling, especially in settings where storage in RNA-protecting buffer is not possible.

We have previously proposed endothelin-2 (EDN2) as a granulosa cell-derived contractile signal that facilitates ovulation. Spatially, Edn2 mRNA expression is restricted to granulosa cells of periovulatory follicles. Temporally, mRNA for this contractile peptide is expressed immediately before follicle rupture. The primary objective of this study was to test the hypothesis that hypoxia mediates EDN2 expression in granulosa cells at ovulation, and if it does, to determine the region within the promoter responsible for this effect. To determine the effect of hypoxia on mRNA expression, immature mice were treated with 5 IU of PMSG followed 48 h later by 5 IU of human chorionic gonadotropin (hCG). Granulosa cells were isolated at 9 h after hCG, cultured under normal or hypoxic conditions, and the expression level of mRNA was compared. mRNA expression was increased when granulosa cells were cultured in a hypoxic environment (p < 0.05). Subsequent promoter analysis found that the 5′ upstream region of the EDN2 promoter (between –1894 bp and –1407 bp) was responsible for hypoxia-mediated changes in EDN2 expression. This promoter region contains multiple sites for potential transcriptional regulation, including that by hypoxia-inducible factor 1 (HIF-1, ACGTG) at –1297 bp. The second objective of this study was to determine whether the progesterone receptor (PR) or cyclooxygenase-2 (COX-2), two key regulators of periovulatory events, controlled EDN2 expression. To accomplish this, gonadotropin-primed mice were treated with RU-486 or indomethacin and expression of mRNA for Edn2 was determined in ovaries collected at 12 h after hCG. Treatment with RU-486 or indomethacin did not affect expression of mRNA for Edn2 (p > 0.05). Taken together, we believe that hypoxia, but not the PR or COX-2, regulate gonadotropin-induced EDN2 expression in the periovulatory follicle.

The cytoskeleton plays an important role in numerous cellular processes, including subcellular mRNA localization and translation. Several examples of mRNA localization have emerged in plant cells, and these appear to function in protein targeting, the establishment of polarity, and cell-to-cell trafficking. The identification of several cytoskeleton-associated RNA-binding proteins in plant cells has made available candidate proteins that mediate the interaction between mRNA and the cytoskeleton, and possibly play a role in mRNA localization and translational control. We propose a model that links mRNA–microtubule interactions to translational autoregulation, a process that may assist in the efficient and regulated binding of proteins to microtubules.

YopQ in Yersinia enterocolitica (YopK in Yersinia pseudotuberculosis) is a type III secreted protein required for virulence of yersiniae. In this study YopQ expression, secretion and nucleotide sequences of the corresponding yopQ gene from different yersinia strains were analysed. The cytosolic accumulation differed significantly among serotypes of Y. enterocolitica. These differences might be attributable to variations in the nucleotide sequence and their consequences on mRNA secondary structure. An mRNA signal hypothesis has been proposed for YopQ, predicting the coupling of translation and secretion via an mRNA signal. This hypothesis claims a strictly co-translational secretion of YopQ without its intracellular accumulation. The presence of YopQ in the cytosol, even with a closed secretion apparatus, is demonstrated. Moreover, post-translational secretion of YopQ could be demonstrated. These findings do not support the mRNA signal hypothesis for co-translational secretion.

As a first step toward understanding estrogen’s role in neurodevelopment, a PCR cloning strategy was used to isolate complementary DNAs encoding two distinct cytochrome P450 aromatase isoforms in adult zebrafish (Danio rerio) brain and ovary (termed P450aromB and P450aromA, respectively). Sequence and phylogenetic analysis showed that the zebrafish P450arom forms are orthologs of previously identified cyp19b and cyp19a genes in goldfish. On Northern blots, a single 4.4-kb transcript of the P450aromB subtype was identified in brain, and a 2.1-kb transcript of the P450aromA subtype in ovary, but RT-PCR showed a degree of overlapping expression. Both messenger RNA (mRNA) forms were detected in unfertilized eggs and 1.5 hpf (cleavage stage) embryos but declined by 12 hpf, indicating maternal transfer. A secondary rise in mRNAs between 12‐24 hpf indicated the onset of embryonic cyp19b and -a transcription. Both mRNA species accumulated progressively to 120 hpf (early larval stage), but the relative magnitude and pattern of change was isoform specific. Estradiol (E2, 1 mM) advanced and amplified the developmentally programmed accumulation of P450aromB mRNA, and ICI164.384 decreased expressed levels, implying blockade of an endogenous estrogen mediated regulatory component. Conversely, E2 had no effect or decreased P450aromA mRNA. The early embryonic expression of P450aromB and P450aromA isoforms, and differences in developmental programming and estrogen regulation, imply independent regulatory mechanisms and unique functions during major morphogenetic and differentiative events. (Endocrinology 142: 740 ‐750, 2001)

The present study reports the isolation of three complementary DNA (cDNA) clones encoding distinct subtypes of CRF receptors from the diploid catfish (cf) species, Ameiurus nebulosus. The first clone encodes a 446-amino acid protein (cfCRF-R1) that is highly homologous to mouse (m) CRF-R1 (93% identical). The cfCRF-R1 messenger RNA is highly expressed in the brain, and its distribution pattern correlates well with that of mammalian CRF-R1, except for weak expression in the pituitary. When transiently expressed in COS-7 cells, cfCRF-R1 bound CRF, urotensin I, and sauvagine with similar affinities. The second full-length cDNA, which was cloned from catfish heart, encodes a 406-amino acid protein that showed homology to murine CRF-R2 (88%) and when expressed in COS-7 cells preferentially bound sauvagine. The highest level of cfCRF-R2 expression was observed in the heart. The third full-length cDNA clone, which encodes a 428-amino acid protein, is structurally closer to cfCRF-R1 (85%) than to cfCRF-R2 (80%). This novel CRF receptor (cfCRF-R3) bound CRF with a 5-fold higher affinity than urotensin I and sauvagine and was expressed in the pituitary gland, urophysis, and brain. The presence of three different CRF receptors, each with distinct tissue distribution and ligand binding properties, suggests a complex CRF/urotensin I system. (Endocrinology 142: 446 ‐ 454, 2001)

Lactosylceramide synthase is an enzyme that catalyzes the transfer of galactose from UDP-Gal to glucosylceramide, and thus participates in the biosynthesis of most glycolipids in mammals. We have isolated and sequenced the cDNA clone encoding human lactosylceramide synthase. The deduced amino acid sequence of the human lactosylceramide synthase showed 94.2% identity with rat lactosylceramide synthase. Northern blotting analysis revealed that lactosylceramide synthase mRNA was expressed in various tissues, with the highest level in brain and adrenal gland.

Several lines of evidence indicate that the number of GnRH receptors (GnRH-R) and therefore, gonadotrope responsiveness to GnRH, is highly dependent upon the level of GnRH-R mRNA. To explore this aspect of regulation, we have isolated a 3.3 kb fragment encompassing the promoter region of the rat GnRH-R gene. Primer extension and RNase protection assays allowed the identification of five major transcriptional start sites located within the 110 bp region upstream of the translation start codon. Transfection experiments using the CAT reporter gene demonstrated that the 1261 bp 5′ flanking region is required to direct high efficient expression in the gonadotrope-derived α T3-1 cell line thus contrasting with mouse in which the only 500 bp proximal sequence appeared to be sufficient. Another difference between rat and mouse was apparent in the 183 bp region of the rat promoter which induced a 3-fold stimulation of thymidine kinase promoter activity in both α T3-1 and CHO cells. Subsequent deletion analysis of the region residing between −1261 and −519 revealed the presence of multiple regulatory domains that contributed to the cell-specific activity. However, despite this efficiency in the context of the wild-type promoter, they failed to induce the activity of the minimal thymidine kinase (TK) promoter in the absence of the proximal 600 bp promoter region. Accordingly, a composite TK promoter containing the entire 1.2 kb promoter induced a 10-fold increase in the activity of the TK promoter in α T3-1 cells. Taken together these data suggest that distal regulatory regions are critical and require cooperation with proximal specific-promoter elements for activating basal R-GnRH gene expression in gonadotrope cells.

In vertebrates the synthesis of ribosomal proteins is co-ordinately regulated at the translational level. The 5′-untranslated region (5′UTR) of this class of mRNAs contains conserved regions that are necessary and sufficient for translational regulation. Recently, we found that two proteins, the Xenopus laevis La autoantigen and the cellular nucleic acid binding protein (CNBP), are able to bind in vitro a pyrimidine tract at the 5′ end and a downstream region, respectively. These regions are considered the common cis-acting elements of translational regulation. It was previously observed that the binding of both these putative trans-acting factors to their RNA sequences is assisted by a protease-sensitive factor(s) that dissociates from the complex after its formation. Here we provide evidence that the requirement for an ancillary factor assisting La binding to the pyrimidine tract of ribosomal protein mRNAs is typical of this RNA, and secondly that it may involve an RNA recognition motif of the La protein not clearly characterized previously. We also show that the Ro60 autoantigen is involved in the common factor activity necessary for the binding of La and CNBP proteins to their respective sequences. In addition, our findings suggest that an RNA also participates in this process. We show that CNBP can multimerise and that it binds to the 5′UTR as a dimer. Both La and CNBP compete for the interaction with the factor, and their binding to the 5′UTR is mutually exclusive. Our results from the binding analysis of mutations in the 5′UTR, which are known to disrupt the translational control in vivo, suggest a model in which the protein interactions and the 5′UTR RNA structure may co-operate in regulating the translational fate of ribosomal protein mRNAs.

The Crithidia fasciculata KAP1 gene encodes a small basic protein (p21) associated with kinetoplast DNA. The p21 protein has a nine amino acid cleavable presequence closely related to those of several other proteins targeted to the kinetoplast and binds non-specifically to kinetoplast minicircle DNA. The p21 protein also has a calculated p I of 13 with two amino acids (lysine and alanine) accounting for more than 50% of the residues and with 25 out of 28 lysine residues contained in the C-terminal half of the protein. Immunolocalization of p21 shows that the protein is found exclusively in the kinetoplast with a localization distinctly different from the antipodal localization of kinetoplast DNA topoisomerase and DNA polymerase. The KAP1 gene is a single copy gene and the KAP1 mRNA is present at a constant level throughout the cell cycle. This highly basic protein may play a role in the condensation or segregation of the kinetoplast DNA.

Spliced leader (SL) trans-splicing generates the 5′ end of mature mRNAs through the addition of a small exon to pre-mRNAs in some flagellates (kinetoplastida and euglenoids) and metazoans (nematodes and flatworms). Although SL addition in the kinetoplastida and a subset of nematode genes serves to resolve multicistronic mRNAs into monocistronic, capped mRNAs, information regarding the functional significance of trans-splicing in flatworms is limited. We describe here the identification and characterization of a closely linked gene upstream from the trans-spliced enolase gene in the flatworm Schistosoma mansoni. This gene produces a non-trans-spliced mRNA encoding a ubiquinol binding protein, UbCRBP, that is a component of the ubiquinol-cytochrome C reductase complex. The distance between the UbCRBP polyadenylation site and the enolase trans-splice acceptor site is exceptionally short, only 54 nucleotides. Primer extension (5′ RACE), RT-PCR, and RNase mapping have identified steady state, cis-spliced RNAs which significantly overlap both the UbCRBP and enolase genes. These transcripts contain the 5′ ends of mature UbCRBP mRNAs, extend through UbCRBP, across the intergenic region, and a significant distance 3′ into the enolase gene. Interestingly, the close linkage between the UbCRBP and enolase genes is conserved in a second flatworm, Fasciola hepatica, which also trans-splices the downstream enolase gene. Taken together, the role of SL addition in resolving multicistronic transcripts in both C. elegans and the kinetoplastida, the conservation of UbCRBP/enolase gene linkage in two divergent trematodes, and the multicistronic organization of schistosome UbCRBP/enolase RNAs are consistent with the suggestion that these two genes are likely to be cotranscribed and that trans-splicing in flatworms may be associated with polycistronic transcripts.

A 3′ RACE technique was used to establish the nucleotide sequence encoding the C-terminal 379 amino acids of rat intestinal Muc3. Unlike the C-terminus of Muc2 and many secretory mucins, Muc3 contains two EGF motifs and a putative transmembrane domain. The mRNA for rat Muc3 is 7.5–8.0 kb.

BAG1 is a small heat-shock protein of Toxoplasma gondii that is specifically expressed in the cyst-forming bradyzoite stage of the parasite. Upregulation of BAG1 mRNA occurs early during the differentiation pathway from tachyzoites to bradyzoites. In order define genomic elements involved in bradyzoite-specific gene regulation, chloramphenicol acetyltransferase (CAT)-reporter gene studies were performed with 5' flanking sequences of the BAG1 gene. Tachyzoites, transiently transfected with the BAG1/cat construct, exhibited very low CAT activity (200 fold less than in parasites transfected with a tubulin promoter/cat construct). After induction of bradyzoite differentiation by alkaline pH shift, however, CAT activity increased 50 fold, demonstrating bradyzoite-specific expression of the CAT reporter gene under control of 5' flanking sequences of BAG1. Stage-specific regulation of BAG1/CAT was independent of the 3'-flanking region, since constructs containing 3'-flanking sequences of the tachyzoite-specific SAG1 gene showed identical regulation to those containing the BAG1 3'-flanking region. The kinetics of BAG1/CAT induction in stably transfected parasites is similar to the kinetics of endogenous BAG1 expression: increased CAT activity was first detected on day 3 after alkaline pH shift (20 fold) and was dramatically upregulated 250 fold on day 4. A series of deletions in the BAG1 5'-flanking sequences demonstrated that a 324 nucleotide (nt) fragment, starting 60 nt upstream of the BAG1 transcription start, is sufficient to confer stage-specific regulation on the CAT reporter. These deletion analyses demonstrate that bradyzoite-specific expression of a heterologeous reporter gene requires only minimal genomic sequences.

Publisher Summary Immobilization of the critical reactants on insoluble matrices is the means through which secondary products may be removed from a given reaction. This may enhance the rates and yields of the reaction. The oligonucleotide linked to cellulose powder is extensively used for the separation and purification of messenger RNAs (mRNAs) from other nucleic acids and proteins. Poly (U) covalently bound to diazothiophenyl paper is also used for the isolation of mRNA from total cytoplasmic RNA. In principle, complementary DNA (cDNA) is prepared directly from small amounts of mRNA trapped on oligo cellulose paper disks, using the oligo of the paper as primer for reverse transcription. The newly formed cDNA is then employed in further reactions. This procedure permits manipulation of small amounts of mRNA and cDNA; reduces the processing time of serial reactions considerably because the bound nucleic acid may be purified by rinsing in an appropriate buffer rather than by precipitation and centrifugation; and reduces losses of nucleic acid products that might otherwise occur during each of the recovery steps. The mRNA profiles of a cell may undergo significant changes in response to a variety of genetic, physiological, pathological, chemical, or environmental signals. This may involve the transcription of a large number of genes or may result in the induction or suppression of a limited number of unique genes. The method of hybridization subtraction is often used to enrich the transcripts of the affected genes for eventual cloning and characterization.

Publisher Summary This chapter discusses the biochemical manipulations of minute quantities of messenger RNA (mRNAs) and complementary DNA (cDNA) immobilized on cellulose paper discs. In principle, cDNA is prepared directly from small amounts of mRNA trapped on oligo (dT) cellulose paper disks, using the oligo (dT) of the paper as primer for reverse transcription. The newly formed cDNA, covalently linked to the disk, is then employed in further reactions. This procedure: (1) permits manipulations of very small amounts of mRNA and cDNA, (2) reduces the processing time of serial reactions considerably, because the bound nucleic acid may be purified by rinsing in an appropriate buffer rather than by precipitation and centrifugation, and (3) reduces losses of nucleic acid products that might otherwise occur during each of the recovery steps. A target mRNA of interest in a given population of mRNA can be detected or even quantitated by the dot-blot procedure, using an appropriate probe. But the extreme susceptibility of mRNA to degradation by chance contamination with RNase is a practical drawback in the storage and manipulations of RNA samples. However, once reverse transcribed and immobilized on cellulose disk, the cDNA act as stable and durable records of their corresponding mRNAs and can be repeatedly screened by the same or different probes, as needed.

The genomic RNA of coronaviruses has two unique features. Firstly, it is one of the largest stable RNAs known to exist in nature and is unquestionably the largest viral genomic RNA. The complete sequence of avian infectious bronchitis virus (IBV) RNA shows that its size is 27.6 kilobases (kb) (Boursnell et al., 1987). Although the complete sequences of other Coronavirus genomic RNAs are not yet available, preliminary data from several laboratories indicate that the genomic RNA of mouse hepatitis virus (MHV) is as long as 32 kb (Shieh and Lai, unpublished observations). The large size of these RNAs poses a theoretical quandary for the replication of Coronavirus RNA, considering the high error frequency of RNA-dependent RNA synthesis observed in some systems (Holland et al., 1982; Steinhauer and Holland, 1986). How does Coronavirus RNA replicate faithfully despite the high error frequency of RNA synthesis? It is possible that a proof-reading mechanism operates to correct unavoidable mistakes which are expect d to occur in almost every RNA molecule of this size. Secondly, Coronavirus RNA contains a leader sequence (approximately 72 nucleotides) which is repeated at the 5′-end of every subgenomic mRNA species. This structural organization appears to be similar to that of most of eukaryotic mRNAs which contain leader sequences derived by RNA splicing. Yet the Coronavirus RNA genome does not have consensus splicing signals and the virus replicates exclusively in the cytoplasm (Wilhelmson et al., 1981; Brayton et al., 1981), where there is no conventional RNA splicing machinery. Furthermore, UV transcriptional mapping studies suggest that Coronavirus mRNA species are transcribed independently (Jacobs et al., 1981), instead of being derived by cleavage of a precursor RNA. Thus, a new transcriptional mechanism must operate to transcribe Coronavirus mRNAs.

Poly A containing RNA isolated from embryonic chick calvaria was transferred from 6% formaldehyde 0.75% agarose gels to diazobenzyloxymethyl paper and the paper then hybridized to either nick translated pro alpha 1 collagen cDNA clones, pCg1 or pCg54, or to the nick translated pro alpha 2 collagen cDNA clone, pCg45. From the mobilities of the bands hybridizing most strongly to each, pro alpha 2 collagen mRNA was shown to be slightly larger than pro alpha 1 mRNA; they are 5100 and 4900 nucleotides long respectively. pCg54 also hybridized weakly to two bands of lower mobility, corresponding to RNAs 6.4 and 5.6 kb long. Neither pCg54 nor pCg45 hybridized to type II procollagen mRNA in poly A containing RNA isolated from embryonic chick sterna.

Poly(A)+ RNA was isolated from in vitro short-term-labeled total cytoplasmic RNA of Ehrlich ascites tumor cells by oligo(dT) cellulose chromatography. This poly(A)+ RNA fraction was compared with a poly(A)+ RNA fraction isolated by a new procedure which involves specific binding of poly(A)+ RNA to messenger affinity paper (mAP) and its release in hot (70 degrees C) water. In typical experiments 10-11 micrograms (2.3%) of poly(A)+ RNA can be retained from 500 micrograms of total cytoplasmic RNA per cm2 of mAP in a quick one-step procedure. The poly(A)+ RNA preparations isolated by the two methods proved to be almost identical with respect to their fraction in total cytoplasmic RNA, specific radioactivities, sucrose gradient profiles, and translation assays. Since the isolation of poly(A)+ RNA by mAP is much less time consuming than that by oligo(dT) column chromatography and since the poly(A)+ RNA can be recovered from mAP in small volumes, which avoids further loss during precipitations, it can be advantageously used for preparative isolation of poly(A)+ RNA.

The methylase METTL3 is the writer enzyme of the N6‐methyladenosine (m6A) modification of RNA. Using a structure‐based drug discovery approach, we identified a METTL3 inhibitor with potency in a biochemical assay of 280 nM, while its enantiomer is 100 times less active. We observed a dose‐dependent reduction in the m6A methylation level of mRNA in several cell lines treated with the inhibitor already after 16 h of treatment, which lasted for at least 6 days. Importantly, the prolonged incubation (up to 6 days) with the METTL3 inhibitor did not alter levels of other RNA modifications (i. e., m1A, m6Am, m7G), suggesting selectivity of the developed compound towards other RNA methyltransferases., Writer's block: We developed and characterized a small‐molecule inhibitor of m6A writer METTL3 by protein crystallography, biochemical and cellular assays. It shows high‐nanomolar potency in the biochemical assay, good selectivity against a panel of protein methyltransferases and kinases, and it reduced m6A/A ratio in mRNAs of different cell lines. In addition, we confirmed the selectivity of our compound towards other RNA methyltransferases in living cells.