Steady-state mRNA Levels Research Articles

Fatty Acid Composition and Fatty Acid Associated Gene-Expression in Gilthead Sea Bream (Sparus aurata) are Affected by Low-Fish Oil Diets, Dietary Resveratrol, and Holding Temperature.

To sustainably produce marine fish with a high lipid quality rich in omega-3 fatty acids, alternative sources of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are being identified. Moreover, the use of bioactive compounds that would stimulate the in vivo fatty acid synthesis, such as resveratrol (RV), would reduce the dependence on fish oil in aquafeeds. Gilthead sea bream (Sparus aurata) were fed four experimental diets combining two fish oil levels (6% dry matter (DM); 2% DM) with or without 0.15% DM resveratrol supplementation (F6, F2, F6 + RV, F2 + RV) for two months. Additionally, the fish were challenged either at 19 °C or 23 °C. A higher water temperature promoted their feed intake and growth, resulting in an increased crude lipid content irrespective of dietary treatment. The fatty acid composition of different tissues was significantly affected by the holding temperature and dietary fish oil level. The dietary RV significantly affected the hepatic EPA and DHA content of fish held at 19 °C. The observed effect of RV may be partly explained by alterations of the mRNA steady-state levels of ∆6-desaturase and β-oxidation-related genes. Besides the relevant results concerning RV-mediated regulation of fatty acid synthesis in marine fish, further studies need to be conducted to clarify the potential value of RV to enhance fillet lipid quality.

Flavonoids as putative modulators of Δ4‐, Δ5‐, and Δ6‐desaturases: Studies in cultured hepatocytes, myocytes, and adipocytes

This study was conducted to screen flavonoids for affecting expression of desaturases involved in omega-3 fatty acid synthesis and ceramide (CER) metabolism. To this end, cultured HepG2 hepatocytes, C2C12 myocytes, and 3T3-L1 adipocytes were treated with nontoxic concentrations of 12 selected flavonoids and expression of Δ4-, Δ5-, and Δ6-desaturases (DEGS1, FADS1, and FADS2, respectively) was determined. The flavonoids tested were more cytotoxic to HepG2 and 3T3-L1 than to C2C12 cells. In HepG2 cells, FADS1 was induced by quercetin and FADS2 expression was increased by daidzein, genistein, and pratensein treatment. DEGS1 was increased by apigenin, luteolin, orobol, and quercetin administration. In differentiated C2C12 cells, substances had no inducing effect or even lowered target gene expression. Pratensein induced both FADS1 and FADS2 in differentiated 3T3-L1 cells and DEGS1 was increased by treatment with apigenin, genistein, luteolin, orobol, and quercetin. In conclusion, pratensein may be an interesting test compound for further studies in vitro and in vivo on omega-3 synthesis since it induces its rate-limiting enzyme FADS2. Apigenin, luteolin, orobol, and quercetin induced DEGS1 and thereby possibly synthesis of proapoptotic CER in malignant HepG2 cells and 3T3-L1. In contrast, in benign C2C12 cells, they did not elevate mRNA steady state levels of DEGS1. That may partly explain the higher resistance of C2C12 cells against flavonoids compared to the other cell lines. By affecting tumor cells and nontumor cells differently, these flavonoids may be promising substances for further research regarding anticancer properties. © 2018 BioFactors, 44(5):485-495, 2018.

Dietary Buglossoides arvensis Oil as a Potential Candidate to Substitute Fish Oil in Rainbow Trout Diets.

The utilization of vegetable oils in salmonid diets substantially decreased the body content of omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA), and thus the product quality for human consumption. Therefore, new ingredients for aquaculture feeds are needed that maximize the deposition of health-promoting n-3 LC-PUFA. This study investigated Buglossoides arvensis (Ahiflower) oil, a plant oil rich in alpha-linolenic acid (18:3n-3, ALA) and stearidonic acid (18:4n-3, SDA), as a source of n-3 fatty acids in rainbow trout (Oncorhynchus mykiss) nutrition. Rainbow trout (87.4 ± 0.6 g) were fed for 56 days. The oils of the control diet (FV) were substituted by Ahiflower oil at 33%, 66%, and 100% (A33, A66, A100). Dietary Ahiflower oil increased the final body weights of fish. mRNA steady state levels of fatty acyl desaturase 2a (delta-6) (fads2a(d6)) and 2b (delta-5) (fads2b(d5)) as well as carnitine palmitoyl transferase 1 a (cpt1a) were not altered by dietary treatments. In contrast, cpt1c mRNA steady state levels were significantly downregulated in samples of fish fed A66 and A100. Significantly higher eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) levels were found in the liver and significantly higher EPA levels in the fillet of rainbow trout of A66 and A100 compared to FV. The content of DHA in fillets of fish fed Ahiflower oil was not significantly different to fish fed FV. Thus, high dietary amounts of Ahiflower oil can compensate for reduced dietary EPA and DHA levels.

Dysregulation of human NEFM and NEFH mRNA stability by ALS-linked miRNAs

Neurofilaments (NFs) are the most abundant cytoskeletal component of vertebrate myelinated axons. NFs function by determining axonal caliber, promoting axonal growth and forming a 3-dimensional lattice that supports the organization of cytoplasmic organelles. The stoichiometry of NF protein subunits (NFL, NFM and NFH) has to be tightly controlled to avoid the formation of NF neuronal cytoplasmic inclusions (NCIs), axonal degeneration and neuronal death, all pathological hallmarks of amyotrophic lateral sclerosis (ALS). The post-transcriptional control of NF transcripts is critical for regulating normal levels of NF proteins. Previously, we showed that miRNAs that are dysregulated in ALS spinal cord regulate the levels of NEFL mRNA. In order to complete the understanding of altered NF expression in ALS, in this study we have investigated the regulation of NEFM and NEFH mRNA levels by miRNAs. We observed that a small group of ALS-linked miRNAs that are expressed in human spinal motor neurons directly regulate NEFM and NEFH transcript levels in a manner that is associated with an increase in NFM and NFH protein levels in ALS spinal cord homogenates. In concert with previous observations demonstrating the suppression of NEFL mRNA steady state levels in ALS, these observations provide support for the hypothesis that the dysregulation of miRNAs in spinal motor neurons in ALS fundamentally alters the stoichiometry of NF expression, leading to the formation of pathological NCIs.

The C-Type Lectin Domain Gene Family in Aedes aegypti and Their Role in Arbovirus Infection

Several medically important flaviviruses that are transmitted by mosquitoes have been shown to bind to the C-type lectin fold that is present in either vertebrate or invertebrate proteins. While in some cases this interaction is part of a neutralizing anti-viral immune response, many reports have implicated this as critical for successful virus entry. Despite the establishment of mosquito C-type lectin domain containing proteins (CTLDcps) as known host factors in assisting the infectious process for flaviviruses, little is known about the structural characteristics of these proteins and their relationships to each other. In this report, we describe the manual annotation and structural characterization of 52 Aedes aegypti CTLDcps. Using existing RNAseq data, we establish that these genes can be subdivided into two classes: those highly conserved with expression primarily in development (embryo/early larvae) and those with no clear orthologs with expression primarily in late larvae/pupae or adults. The latter group contained all CTLDcps that are regulated by the Toll/Imd immune pathways, all known microbiome-regulating CTLDcps, and almost all CTLDcps that are implicated as flavivirus host factors in A. aegypti. Finally, we attempt to synthesize results from multiple conflicting gene expression profiling experiments in terms of how flavivirus infection changes steady-state levels of mRNA encoding CTLDcps.

Post-transcriptional regulation of Pabpn1 by the RNA binding protein HuR.

RNA processing is critical for proper spatial and temporal control of gene expression. The ubiquitous nuclear polyadenosine RNA binding protein, PABPN1, post-transcriptionally regulates multiple steps of gene expression. Mutations in the PABPN1 gene expanding an N-terminal alanine tract in the PABPN1 protein from 10 alanines to 11–18 alanines cause the muscle-specific disease oculopharyngeal muscular dystrophy (OPMD), which affects eyelid, pharynx, and proximal limb muscles. Previous work revealed that the Pabpn1 transcript is unstable, contributing to low steady-state Pabpn1 mRNA and protein levels in vivo, specifically in skeletal muscle, with even lower levels in muscles affected in OPMD. Thus, low levels of PABPN1 protein could predispose specific tissues to pathology in OPMD. However, no studies have defined the mechanisms that regulate Pabpn1 expression. Here, we define multiple cis-regulatory elements and a trans-acting factor, HuR, which regulate Pabpn1 expression specifically in mature muscle in vitro and in vivo. We exploit multiple models including C2C12 myotubes, primary muscle cells, and mice to determine that HuR decreases Pabpn1 expression. Overall, we have uncovered a mechanism in mature muscle that negatively regulates Pabpn1 expression in vitro and in vivo, which could provide insight to future studies investigating therapeutic strategies for OPMD treatment.

SRSF3 promotes pluripotency through Nanog mRNA export and coordination of the pluripotency gene expression program.

The establishment and maintenance of pluripotency depend on precise coordination of gene expression. We establish serine-arginine-rich splicing factor 3 (SRSF3) as an essential regulator of RNAs encoding key components of the mouse pluripotency circuitry, SRSF3 ablation resulting in the loss of pluripotency and its overexpression enhancing reprogramming. Strikingly, SRSF3 binds to the core pluripotency transcription factor Nanog mRNA to facilitate its nucleo-cytoplasmic export independent of splicing. In the absence of SRSF3 binding, Nanog mRNA is sequestered in the nucleus and protein levels are severely downregulated. Moreover, SRSF3 controls the alternative splicing of the export factor Nxf1 and RNA regulators with established roles in pluripotency, and the steady-state levels of mRNAs encoding chromatin modifiers. Our investigation links molecular events to cellular functions by demonstrating how SRSF3 regulates the pluripotency genes and uncovers SRSF3-RNA interactions as a critical means to coordinate gene expression during reprogramming, stem cell self-renewal and early development.

External Beam Radiation Therapy Enhances Mesenchymal Stem Cell-Mediated Sodium-Iodide Symporter Gene Delivery.

The tumor-homing properties of mesenchymal stem cells (MSC) have led to their development as delivery vehicles for the targeted delivery of therapeutic genes such as the sodium-iodide symporter (NIS) to solid tumors. External beam radiation therapy may represent an ideal setting for the application of engineered MSC-based gene therapy, as tumor irradiation may enhance MSC recruitment into irradiated tumors through the increased production of select factors linked to MSC migration. In the present study, the irradiation of human liver cancer cells (HuH7; 1-10 Gy) showed a strong dose-dependent increase in steady-state mRNA levels of CXCL8, CXCL12, FGF2, PDGFB, TGFB1, THBS1, and VEGF (0-48 h), which was verified for most factors at the protein level (after 48 h). Radiation effects on directed MSC migration were tested in vitro using a live cell tracking migration assay and supernatants from control and irradiated HuH7 cells. A robust increase in mean forward migration index, mean center of mass, and mean directionality of MSCs toward supernatants was seen from irradiated as compared to non-irradiated tumor cells. Transferability of this effect to other tumor sources was demonstrated using the human breast adenocarcinoma cell line (MDA-MB-231), which showed a similar behavior to radiation as seen with HuH7 cells in quantitative polymerase chain reaction and migration assay. To evaluate this in a more physiologic in vivo setting, subcutaneously growing HuH7 xenograft tumors were irradiated with 0, 2, or 5 Gy followed by CMV-NIS-MSC application 24 h later. Tumoral iodide uptake was monitored using 123I-scintigraphy. The results showed increased tumor-specific dose-dependent accumulation of radioiodide in irradiated tumors. The results demonstrate that external beam radiation therapy enhances the migratory capacity of MSCs and may thus increase the therapeutic efficacy of MSC-mediated NIS radionuclide therapy.

Polysome Profiling in <em>Leishmania</em>, Human Cells and Mouse Testis

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

Polysome Profiling in Leishmania, Human Cells and Mouse Testis.

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

Regulation and Functional Studies of Tribbles homolog 2 (TRIB2) in Ovarian Granulosa Cells

Tribbles homolog (TRIB) 1, 2 and 3 represent atypical members of the serine/threonine kinase superfamily and are homologs of Drosophila tribbles. TRIB2 mRNA is rapidly induced by mitogens, has a short half‐life, and is expressed in a cell‐specific manner. We previously identified TRIB2 as a differentially expressed gene in granulosa cells (GC) of bovine preovulatory follicles. This study aimed to further investigate TRIB2 mRNA and protein regulation, to identify its binding partners, and study its function in GC of bovine dominant follicles. Granulosa cells were obtained from follicles at different developmental stages: small follicles (SF: 2–4 mm), dominant follicles (DF) at day 5 of the estrous cycle (day 0 = day of the estrous), ovulatory follicles (OF) 24 h following injection of an ovulatory dose of hCG, and corpus luteum (CL) at day 5 of the estrous cycle. RT‐qPCR analyses showed greatest expression of TRIB2 in GC of DF, while the weakest expression was in OF (P < 0.0001). Temporal expression of TRIB2 mRNA was further studied in follicular walls (granulosa and theca cells) obtained from ovulatory follicles recovered at 0, 6, 12, 18 and 24 h after hCG injection. There was a 20‐ and 166‐fold reduction of TRIB2 steady‐state mRNA levels in follicular walls, respectively at 6 and 24 hours post‐hCG as compared to 0 hour (P < 0.001). Additionally, we have generated specific anti‐TRIB2 polyclonal antibodies that confirmed, in western blot analyses, TRIB2 downregulation by LH/hCG at the protein level. Yeast two‐hybrid screening of a DF‐cDNA library as well as functional studies using the CRISPR‐Cas9 approach are currently being used to identify TRIB2 binding partners and its function in GC. These results support a physiologically relevant role of TRIB2 in follicular development and GC function.Support or Funding InformationResearch supported by internal funds from Université de Montréal to KN and NSERC of Canada grant # 104199 to JGL.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Silencing of HaAce1 gene by host-delivered artificial microRNA disrupts growth and development of Helicoverpa armigera.

The polyphagous insect-pest, Helicoverpa armigera, is a serious threat to a number of economically important crops. Chemical application and/or cultivation of Bt transgenic crops are the two strategies available now for insect-pest management. However, environmental pollution and long-term sustainability are major concerns against these two options. RNAi is now considered as a promising technology to complement Bt to tackle insect-pests menace. In this study, we report host-delivered silencing of HaAce1 gene, encoding the predominant isoform of H. armigera acetylcholinesterase, by an artificial microRNA, HaAce1-amiR1. Arabidopsis pre-miRNA164b was modified by replacing miR164b/miR164b* sequences with HaAce1-amiR1/HaAce1-amiR1* sequences. The recombinant HaAce1-preamiRNA1 was put under the control of CaMV 35S promoter and NOS terminator of plant binary vector pBI121, and the resultant vector cassette was used for tobacco transformation. Two transgenic tobacco lines expressing HaAce1-amiR1 was used for detached leaf insect feeding bioassays. Larval mortality of 25% and adult deformity of 20% were observed in transgenic treated insect group over that control tobacco treated insect group. The reduction in the steady-state level of HaAce1 mRNA was 70–80% in the defective adults compared to control. Our results demonstrate promise for host-delivered amiRNA-mediated silencing of HaAce1 gene for H. armigera management.

Effect of multiple low-dose PGF2α injections on the mature corpus luteum in non-pregnant bitches

This study investigated molecular regulation in the canine corpus luteums/corpora lutea (CL) following multiple low-dose prostaglandin F2 alpha (PGF2α) injections in non-pregnant bitches around 30–35 days after ovulation. The CL were obtained by ovariohysterectomy 1 h after the last PGF2α injection. The subjects were divided into the following groups: control (no PGF2α injection, n = 4), one PGF2α injection (injection at 0 h, 1PGF, n = 4), two PGF2α injection (injection at 0 and 8 h, 2PGF, n = 4), and three PGF2α injection (injection at 0, 8 and 24 h, 3PGF, n = 4). In the 1PGF group, the steady-state mRNA levels of an immediate early gene (NR4A1) and immune system-related genes (MCP-1 and IL-8) increased. NR4A1 was localized in luteal and endothelial cells. In contrast, MCP-1 was localized in the luteal tissue between the luteal and endothelial cells. LHCGR, CYP11A1, and StAR mRNA expression decreased after the second PGF2α injection. FASLG increased only after the third PGF2α injection. The mRNA levels of PTGFR, PGT, and PTGS2 decreased as the number of PGF2α injections increased. Immunohistochemistry showed a decrease in StAR protein density as the number of PGF2α injections increased. BAX and CASP3 mRNA expression levels were similar among the groups. Serum progesterone (P4) levels decreased dramatically after the PGF2α injections but were still higher than the basal level at the end of the study. In conclusion, repeated low-dose PGF2α injections could induce luteolytic mechanisms in the CL of non-pregnant bitches. Furthermore, it can be concluded that, in non-pregnant bitches, some aspects of the molecular regulation of luteolysis in the CL are similar to some aspects of such regulation in other domestic animals.

Synthetic Adeno-Associated Viral Vector Efficiently Targets Mouse and Nonhuman Primate Retina In Vivo.

Gene therapy is a promising approach in the treatment of inherited and common complex disorders of the retina. Preclinical and clinical studies have validated the use of adeno-associated viral vectors (AAV) as a safe and efficient delivery vehicle for gene transfer. Retinal pigment epithelium and rods-and to a lesser extent, cone photoreceptors-can be efficiently targeted with AAV. Other retinal cell types however are more challenging targets. The aim of this study was to characterize the transduction profile and efficiency of in silico designed, synthetic Anc80 AAVs for retinal gene transfer. Three Anc80 variants were evaluated for retinal targeting in mice and primates following subretinal delivery. In the murine retina Anc80L65 demonstrated high level of retinal pigment epithelium and photoreceptor targeting with comparable cone photoreceptor affinity compared to other AAVs. Remarkably, Anc80L65 enhanced transduction kinetics with visible expression as early as day 1 and steady state mRNA levels at day 3. Inner retinal tropism of Anc80 variants demonstrated distinct transduction patterns of Müller glia, retinal ganglion cells and inner nuclear layer neurons. Finally, murine findings with Anc80L65 qualitatively translated to the Rhesus macaque in terms of cell targets, levels and onset of expression. Our findings support the use of Anc80L65 for therapeutic subretinal gene delivery.

Regulation of Luteinizing Hormone Receptor mRNA Expression in the Ovary: The Role of miR-122.

The expression of luteinizing hormone receptor (LHR) in the mammalian ovary is regulated in response to changes in the secretion of follicle-stimulating hormone and luteinizing hormone by the anterior pituitary, at least in part, through posttranscriptional mechanisms. The steady-state levels of LHR mRNA are maintained by controlling its rate of degradation by an RNA-binding protein designated as LHR mRNA-binding protein (LRBP). LRBP forms a complex with LHR mRNA and targets it for degradation in the p bodies. miR-122, an 18 nucleotide noncoding RNA, regulates the expression of LRBP. Thus, the levels of miR-122 determine the cellular levels of LHR mRNA expression. This phenomenon has been examined during the induction of LHR mRNA expression that occurs during follicle maturation in response to rising levels of FSH. In this situation, miR-122 and LRBP levels decrease as LHR mRNA expression undergoes downregulation in response to preovulatory LH surge. miR-122 expression as well as LRBP levels show robust increases. The mechanism of induction of LRBP by miR-122 has also been discussed.

Selection of optimal reference genes for quantitative RT-PCR transcript abundance analysis in white clover (Trifolium repens L.).

Quantitative reverse transcription PCR (qRT-PCR) is a widely used method for transcript abundance analyses in plants. Relative quantification by qRT-PCR requires the use of a stably expressed reference gene. There are many 'housekeeping' genes reported in different plant species that are used as reference genes. However, it is important that the steady-state mRNA levels of these housekeeping genes are confirmed across different conditions and tissues in each species studied. Prior to this study, no comprehensive work had been performed in identifying optimal reference genes in white clover (Trifolium repens L.). To identify suitable reference genes in white clover, we analysed the transcript abundance stability of seven candidate genes in two organs (leaves and stolons) across two treatments (water-limited and well-watered). ΔCt, NormFinder and ANOVA tests were carried out to evaluate the mRNA level stability of candidate reference genes. According to the ΔCt results, the genes with the most stable mRNA levels were EF1α and ACT11. When stability among groups was evaluated by NormFinder, UBQ was the most stable across all organs and treatments. By multiple criteria, EF1α, followed by ACT11 and UBQ, was the most stably-expressed gene across organs and treatments, and each of these are recommended as reference genes for transcript abundance studies in white clover.

NDP52 activates nuclear myosin VI to enhance RNA polymerase II transcription

Myosin VI (MVI) has been found to be overexpressed in ovarian, breast and prostate cancers. Moreover, it has been shown to play a role in regulating cell proliferation and migration, and to interact with RNA Polymerase II (RNAPII). Here, we find that backfolding of MVI regulates its ability to bind DNA and that a putative transcription co-activator NDP52 relieves the auto-inhibition of MVI to enable DNA binding. Additionally, we show that the MVI–NDP52 complex binds RNAPII, which is critical for transcription, and that depletion of NDP52 or MVI reduces steady-state mRNA levels. Lastly, we demonstrate that MVI directly interacts with nuclear receptors to drive expression of target genes, thereby suggesting a link to cell proliferation and migration. Overall, we suggest MVI may function as an auxiliary motor to drive transcription.

A MADS-box transcription factor of ‘Kuerlexiangli’(Pyrus sinkiangensis Yu) PsJOINTLESS gene functions in floral organ abscission

MADS-box proteins have been implicated in many biological processes. However, plant MADS-box proteins functioning in floral organ abscission and the underlying physiological mechanisms remain poorly understood. Here, we report the identification and functional characterization of PsJOINTLESS isolated from ‘Kuerlexiangli’. PsJOINTLESS had a complete open reading frame of 672bp, encoding a 224 amino acid peptide, and shared high sequence identities with MADS-box from other plants. PsJOINTLESS was subcellularly targeted to the nucleus, supporting its role as a transcription factor. Expression levels of PsJOINTLESS in the calyx tube were strongly induced by calyx abscission treatment at 6d after full bloom. Overexpression of PsJOINTLESS in tomato enhanced the rate of pedicel abscission rate. Of special note, the transgenic plants increased the abscission zone cell layer compared with wild type. Furthermore, the tomato transgenic lines showed thinner flower pedicels, more cell number and small pedicel cell size. The cellulase activity in pedicel abscission zone of transgenic plants was higher than that of wild type. In addition, steady-state mRNA levels of five cell wall hydrolase genes coding for either functional or regulatory proteins were induced to higher levels in the transgenic lines. These results clearly demonstrate that PsJOINTLESS may affect pedicel abscission zone development.

The Mitochondrion-Targeted PENTATRICOPEPTIDE REPEAT78 Protein Is Required for nad5 Mature mRNA Stability and Seed Development in Maize

Pentatricopepetide repeat (PPR) proteins are a large family of RNA-binding proteins involved in RNA metabolism in plant organelles. Although many PPR proteins have been functionally studied, few of them are identified with a function in mitochondrial RNA stability. By using a reverse genetic approach, we characterized the role of the mitochondrion-targeted PPR78 protein in nad5 mature mRNA stability and maize (Zea mays) seed development. Loss of PPR78 function leads to a dramatic reduction in the steady-state level of mitochondrial nad5 mature mRNA, blocks the assembly of complex I in the electron transport chain, and causes an arrest in embryogenesis and endosperm development. Characterization of a second strong allele confirms the function of PPR78 in nad5 mRNA accumulation and maize seed development. The generation of mature nad5 requires the assembly of three distinct precursor RNAs via trans-splicing reactions, and the accumulation of nad5T1 precursor is reduced in the ppr78 mutants. However, it is the instability of mature nad5 rather than nad5T1 causing loss of the full-length nad5 transcript, and degradation of nad5 losing both translation start and stop codons is enriched in the mutant. Our data imply the assembly of mature nad5 mRNA precedes the protection of PPR78.

Long non-coding RNA SeT and miR-155 regulate the Tnfα gene allelic expression profile.

It is becoming increasingly appreciated that the non-coding genome may have a great impact on the regulation of chromatin structure and gene expression. The innate immune response can be mediated upon lipopolysaccharide stimulation of macrophages which leads to immediate transcriptional activation of early responsive genes including tumor necrosis factor alpha (Tnfα). The functional role of non-coding RNAs, such as lncRNAs and microRNAs, on the transcriptional activation of proinflammatory genes and the subsequent regulation of the innate immune response is still lacking mechanistic insights. In this study we wanted to unravel the functional role of the lncRNA SeT, which is encoded from the murine Tnfα gene locus, and miR-155 on the transcriptional regulation of the Tnfα gene. We utilized genetically modified mice harboring either a deletion of the SeT promoter elements or the mature miR-155 and studied the response of macrophages to lipopolysaccharide (LPS) stimulation. We found that decreased expression of the lncRNA SeT in murine primary macrophages resulted in increased mortality of mice challenged with LPS, which was corroborated by increased Tnfα steady state mRNA levels and a higher frequency of biallelically expressing macrophages. On the contrary, miR-155 deletion resulted in reduced Tnfα mRNA levels supported by a lower frequency of biallelically expressing macrophages upon stimulation with LPS. In both cases, in the absence of either lncRNA SeT or miR-155 we observed a deregulation of the Tnfα allele homologous pairing, previously shown to regulate the switch from mono- to bi-allelic gene expression. Although lncRNA SeT was not found to be a direct target of miR-155 its stability was increased upon miR-155 deletion. This study suggests a role of the non-coding genome in mediating Tnfα mRNA dosage control based on the regulation of homologous pairing of gene alleles and their subsequent biallelic expression.