Messenger RNA Can Do More for Medicine

Hybridization and sedimentation studies on “early” and “late” vaccinia messenger RNA

Synthesis of polyadenylated messenger RNA during the cell cycle of Saccharomyces cerevisiae

Tissue inhibitor of metalloproteinase-1 and tissue inhibitor of metalloproteinase-2 expression in human amnion mesenchymal and epithelial cells

Separation of ribosomal and messenger ribonucleic acid of hela cells by methyl albumin kieselguhr chromatography

The rates of polyadenylated messenger RNA and ribosomal RNA synthesis were measured in synchronously dividing cultures of fission yeast (Schizosaccharomyces pombe). Control asynchronous cultures, which had been exposed to the conditions used for preparing synchronous cultures, were investigated to check for effects of the synchronization procedure itself on RNA synthesis. After each period of DNA synthesis in synchronous culture, the rates of messenger and ribosomal RNA synthesis doubled, suggesting that gene number controls the rate of messenger and ribosomal RNA synthesis. This was confirmed by experiments with asynchronous, exponential-phase cultures in which DNA synthesis was inhibited by hydroxyurea. Both synchronous culture and hydroxyurea experiments suggested that there is a delay of 15 min (0-1 of the cell generation time) between replication of the DNA and transcription of both gene copies. A pattern of protein accumulation was calculated from changes in the rate of polyadenylated messenger RNA synthesis during synchronous culture. The simulated pattern indicates that protein is accumulated linearly, with a doubling in the rate of accumulation once per cell cycle. The simulated pattern of protein accumulation is very similar to measurements previously reported by other workers of changes in activities of 3 enzymes in synchronous cultures. It is suggested that the doubling of the rate of messenger RNA synthesis, as a consequence of the replication of the DNA once per cycle, provides the basis of a mechanism for control of the doubling of other cellular constituents during the cell cycle.

Phenotypic characterization of neurotensin messenger RNA-expressing cells in the neuroleptic-treated rat striatum: a detailed cellular co-expression study

THE possible role in morphogenesis of ribonucleic acids (RNA) produced by the cell nuclei (presumably ‘messenger’ RNA's) has been discussed recently by one of us1. In short, it was suggested that, in a relatively simple system such as the regenerating unicellular alga Acetabularia, the ‘morphogenetic substances’ of Hammerling2 could be equated to messenger (informational) RNA; however, these substances might as well be proteins, the synthesis of which is controlled by a specific RNA of nuclear origin. The situation is obviously much more complex in the case of embryonic differentiation; we have suggested1 that, in amphibian eggs, synthesis of messenger RNA does not begin before gastrulation. At that stage of development, the pre-existing polarity gradient in the distribution of ribosomal RNA would become activated by messenger RNA's produced by the nuclei: as a result, RNA and protein synthesis would proceed further along the well-known cephalocaudal and dorsoventral morphogenetic gradients.

Selective hydrolysis of barley leaf polysomal messenger RNA during the early stages of powdery mildew infection

M1 RNA, the catalytic subunit of Escherichia coli RNase P, is an essential ribozyme that processes the 5' leader sequence of tRNA precursors (ptRNAs). Using KS2003, an E. coli strain generating only low levels of M1 RNA, which showed growth defects, we examined whether M1 RNA is involved in polycistronic mRNA processing or degradation. Microarray analysis of total RNA from KS2003 revealed six polycistronic operon mRNAs (acpP-fabF, cysDNC, flgAMN, lepAB, phoPQ, and puuCBE) showing large differences in expression between the adjacent genes in the same mRNA transcript compared with the KS2001 wild type strain. Model substrates spanning an adjacent pair of genes for each polycistronic mRNA were tested for RNase P cleavage in vitro. Five model RNAs (cysNC, flgMN, lepAB, phoPQ, and puuBE) were cleaved by RNase P holoenzyme but not by M1 RNA alone. However, the cleavages occurred at non-ptRNA-like cleavage sites, with much less efficiency than the cleavage of ptRNA. Since cleavage products generated by RNase P from a polycistronic mRNA can have different in vivo stabilities, our results suggest that RNase P cleavage may lead to differential expression of each cistron.

Identification of gestationally regulated genes in rat myometrium by use of messenger ribonucleic acid differential display

Coexpression of preprotachykinin-A, alpha-calcitonin gene-related peptide, somatostatin, and neurotrophin receptor family messenger RNAs in rat dorsal root ganglion neurons.

Objectives To explore the influence of γ-ray total body irradiation (TBI) from a 60Co source induced mice thymus injury on expression levels of forkhead box protein N (Foxn) 1 and related genes, and to analyze the correlation between expression levels of Foxn1, related genes and thymus injury. Methods Sixty five male C57BL/6 mice were chosen as subjects during November and December 2014. Inclusion criteria for choosing mice: all these mice are 6-8 weeks old specific pathogen free (SPF) animals, and weighting between 18.0-21.0 g. Thirty-five mice were randomly devided into 4 groups: ① Lethal total body irradiation (L-TBI) 5 d group (n=10), mice received a total dose of 7.5 Gy γ-ray TBI from a 60Co source; ② Sublethal total body irradiation (SL-TBI) 5 d group (n=10), mice received a total dose of 5.5 Gy TBI; ③SL-TBI 24 d group (n=10), mice received a total dose of 5.5 Gy TBI; ④Control group (n=5), untreated mice were used as control. Thymus were collected on day 5 after TBI from mice in L-TBI 5 d group, SL-TBI 5 d group and control group, and on day 24 after TBI from mice in SL-TBI 24 d group. For inducing thymus injury by using gradient doses of γ-ray TBI from a 60Co source, the rest 30 mice were randomly delivered into 6 groups, including 0 Gy group (n=5), 1.0 Gy group (n=5), 2.0 Gy group (n=5), 3.0 Gy group (n=5), 4.0 Gy group (n=5) and 5.0 Gy group (n=5). Thymuses were collected on day 5 after gradient doses of TBI. The dose rate of irradiation was 0.5 Gy/min in all TBI settings of this research. The proportion of thymocyte subsets and thymus epithelial cells (TEC) were analyzed by flow cytometry (FCM), and quantitative polymerase chain reaction (PCR) was applied to detect the mRNA levels of Foxn1 and related genes. Cell counts in thymus and relative expression levels of Foxn1 and related genes in those 4 groups were compared. Correlation between mRNA levels of Foxn1 and interleukin (IL)-22, mRNA levels of Foxn1 and counts of thymocyte subsets and thymic epithelial cell (TEC) were analyzed. Results ① There were statistical significance of total cells in thymus tissue, double positive (DP) thymocytes, CD4+ single positive (SP) thymocytes and CD8+ SP thymocytes among L-TBI 5 d group, SL-TBI 5 d group, SL-TBI 24 d group and control group[averages of total cells were (32.3±6.1)×106, (39.2±8.1)×106, (187.2±20.3)×106 and (282.5±20.3)×106 respectively; median of DP thymocytes were 4.1×106, 0.5×106, 123.3×106 and 240.3×106 respectively; median of CD4+ SP thymocytes were 5.2×106, 10.4×106, 26.8×106 and 20.5×106 respectively; median of CD8+ SP thymocytes were 1.9×106, 2.4×106, 10.2×106 and 6.5×106 respectively; F/χ2=310.471, 17.863, 16.352, 16.419; P=0.000, 0.000, 0.001, 0.001]. But there were no statistical significance of double negative (DN) 2, DN3 thymocytes and TEC among 4 groups (median of DN2 and DN3 thymocytes were 2.2×106, 9.8×106, 4.8×106 and 8.1×106 respectively; media of TEC were 1.9×106, 4.6×106, 2.5×106 and 3.3×106 respectively; χ2=7.574, 7.241; P=0.056, 0.065). Comparing with control group, total cells were significantly declined in L-TBI 5 d group and SL-TBI 5 d group (P=0.000, 0.000); DP thymocytes were also significantly declined in L-TBI 5 d group and SL-TBI 5 d group (P=0.045, 0.001). Comparing with SL-TBI 5 d group, counts of total cell and DP thymocytes were increased in SL-TBI 24 d group (P=0.000, 0.045). ② There were statistical significance of mRNA relative expression levels of Foxn1 and IL-22 among L-TBI 5 d group, SL-TBI 5 d group, SL-TBI 24 d group and control group (averages relative expression levels of Foxn1 mRNA were 15.2±1.2, 10.8±1.1, 6.6±0.8 and 1.0±0.1 respectively; averages relative expression levels of IL-22 mRNA were 11.2±0.8, 7.2±1.2, 4.4±0.5 and 1.0±0.1 respectively; F=233.971, 161.067; P=0.000, 0.000). Comparing with control group, relative expression levels of Foxn1 mRNA were significantly increased in L-TBI 5 d group and SL-TBI 5 d group (P=0.000, 0.000); relative expression levels of IL-22 mRNA were also significantly increased in L-TBI 5 d group and SL-TBI 5 d group (P=0.000, 0.000). Comparing with SL-TBI 5 d group, relative expression levels of Foxn1 and IL-22 mRNA were significantly decreased in SL-TBI 24 d group (P=0.000, 0.000). Relative expression levels of Foxn1 mRNA positively correlated with relative expression levels of IL-22 mRNA (r=0.976, P<0.05). ③ After TBI of gradient doses, relative expression levels of Foxn1 mRNA of 6 gradient doses groups showed a negative correlation with the counts of DP thymocytes (r=-0.930, P<0.05). ④ There were statistical significance of relative expression levels of CC chemokine ligand (CCL) 25, delta like ligand (DLL) 4, autoimmune regulator (Aire) and bone morphogenetic protein (BMP) 4 mRNA among L-TBI 5 d group, SL-TBI 5 d group and control group (average relative expression levels of CCL25 mRNA were 11.2±1.5, 11.4±1.2 and 1.0±0.1, respectively; averages relative expression levels of DLL4 mRNA were 7.3±0.9, 6.3±0.3 and 1.0±0.1, respectively; averages relative expression levels of Aire mRNA were 5.1±1.0, 5.0±0.1 and 1.0±0.1, respectively; averages relative expression levels of BMP4 mRNA were 4.4±1.4, 3.6±0.5 and 1.0±0.1, respectively; F=148.862, 197.667, 73.911, 21.471; P=0.000, 0.000, 0.000 , 0.000). However, there were no statistical significance of relative expression levels of homeobox protein (Hox)a3 mRNA, an upstream gene of Foxn1 among these 3 groups (1.4±0.4, 0.8±0.3, 1.0±0.1; F=5.368, P=0.221). Comparing with control group, relative expression levels of CCL25, DLL4, Aire and BMP4 mRNA were significantly increased in L-TBI 5 d group (P=0.000, 0.000, 0.000); and relative expression levels of CCL25, DLL4, Aire and BMP4 mRNA were also significantly increased in SL-TBI 5 d group (P=0.000, 0.000, 0.000). Meanwhile, relative expression levels of BMP4 mRNA were increased in L-TBI 5 d group and SL-TBI 5 d group comparing to control group (P=0.000, 0.000). Conclusions γ-ray TBI from a 60Co source induces upregulation of intrathymic mRNA levels of Foxn1 and thymus function related genes, which correlates with the pathological process of thymus injury. Key words: Whn protein; Thymus gland; Whole-body irradiation; Mice

Objective To evaluate the predictive values of human papilloma virus (HPV) E6/E7 mRNA test in the prognosis of patients with cervical intraepithelial neoplasia (CIN) 1. Methods From July 2013 to October 2014, a total of 107 cases of patients who were initially diagnosed as CIN1 by pathologic results of colposcopic cervical biopsy in Department of Gynecology, Yantaishan Hospital of Yantai were selected as research subjects. All subjects received cervical ThinPrep liquid-based cytology test (TCT), HPV DNA genotyping test and HPV E6/E7 mRNA test within one month prior to initial diagnosis of CIN1. Two-year follow-ups and once every six months were conducted on all subjects, including the re-examination of TCT, HPV DNA genotyping test and HPV E6/E7 mRNA test. And cervical biopsies were conducted on suspicious lesions. According to the results of TCT, HPV DNA genotyping test, HPV E6/E7 mRNA test and colposcopic cervical biopsy at the end of follow-up, all subjects were enrolled into negative group and continuous or progressive group, respectively. Chi-square test was used to compare the positive rates of HPV E6/E7 mRNA between two groups. Mann-Whitney U rank sum test was used to compare expression levels of HPV E6/E7 mRNA between two groups. The sensitivities and specificities of these three cervical screening methods in predicting the prognosis of CIN1 were calculated respectively. Then receiver operator characteristic (ROC) curve of HPV E6/E7 mRNA expression level in predicting the prognosis of CIN1 was drawn, and the area under ROC curve (ROC-AUC) was calculated.The optimal critical value of HPV E6/E7 mRNA expression level in predicting the prognosis of CIN1 was obtained when the Youden index reaching the maximum value.And its sensitivity and specificity were calculated. This study was approved by the Ethics Committee of Human Beings in Yantaishan Hospital of Yantai and informed consent of clinical research has been signed with every subject. Results ①According to the results of TCT, HPV DNA genotyping test, HPV E6/E7 mRNA test and colposcopic cervical biopsy at the end of follow-up, all subjects were enrolled into negative group (n=63, the results of TCT, HPV DNA genotyping test and HPV E6/E7 mRNA test all were normal or the colposcopic cervical biopsy result was negative at the end of follow-up) and continuous or progressive group (n=44, the colposcopic cervical biopsy result was CIN1 or higher grade at the end of follow-up). There were no statistically significant differences between two groups in basic clinical data such as age and etc. (P>0.05). ②The positive rate of HPV E6/E7 mRNA and median expression level of HPV E6/E7 mRNA were 61.9% (39/63) and 3 738.4 copy/mL in continuous or progressive group, which were significantly higher than those in negative group (81.8%, 36/44) and 583.5 copy/mL, respectively), and both the differences were statistically significant (χ2=4.901, P=0.027; U=821.000, P<0.001). ③According to the results of two-year follow-up, the continuous or progressive rate of CIN in patients with HPV-16/18 positive was 50.8% (31/61), which was significantly higher than that in patients with HPV-16/18 negative (28.3%, 13/46), and the difference was statistically significant (χ2=5.512, P=0.019). The continuous or progressive rate of CIN in patients with HPV E6/E7 mRNA positive was 48.0% (36/75), which was significantly higher than that in patients with HPV E6/E7 mRNA negative (25.0%, 8/32), and the difference was statistically significant (χ2=4.901, P=0.027). The sensitivities of TCT, HPV DNA genotyping test and HPV E6/E7 mRNA qualitative test in predicting the prognosis of CIN1 were 50.0%, 70.5%, 81.8%, and the specificities were 66.7%, 52.4%, 38.1%, respectively. ④The results of ROC curve analysis of HPV E6/E7 mRNA expression level in predicting the prognosis of CIN1 showed that the ROC-AUC was 0.704 (95%CI: 0.601-0.806, P<0.001), and the optimal critical of value HPV E6/E7 mRNA expression level in predicting the prognosis of CIN1 was 2 724.0 copy/mL, and the sensitivity of HPV E6/E7 mRNA expression level in predicting continuous or progression of CIN was 54.5%, and the sensitivity was 81.0%. Conclusions The sensitive of HPV E6/E7 mRNA qualitative test in predicting the prognosis of CIN1 is relatively high, while the specificity of HPV E6/E7 mRNA quantitative test in predicting the prognosis of CIN1 is relatively high. HPV E6/E7 mRNA test may has clinical values in predicting the prognosis of patients with CIN1. Key words: Cervical intraepithelial neoplasia; Human papillomavirus; Papillomavirus E7 proteins; Genotyping; Prognosis; Female

Researchers Discover New Gene Regulation Mechanism:Messenger RNA "Cut and Run" scheme provides rapid stress responseCold Spring Harbor, NY, Oct. 20 -- Researchers at Cold Spring Harbor Laboratory have discovered a new kind of messenger RNA molecule that is converted from non-protein coding status to protein coding statusin response to cellular stress such as viral infection. The discovery reveals a "cut and run" mechanism that is likely to control the expression of many genes in humans and other organisms. A deeper understanding of this mechanism is predicted to have broad implications for biology and biomedical research.The central dogma of molecular biology holds that the DNA of genes is "transcribed" into messenger RNA and messenger RNA is "translated" into protein. The regulation of transcription and translation ultimately determines whether particular genes are switched on to produce protein, or switched off. Once they are made, most messenger RNA molecules are exported from the cell nucleus to the cytoplasm andare then used in the cytoplasm as templates for the production of protein.However, a few years ago, Cold Spring Harbor Laboratory scientists led by Dr. David Spector noticed that under standard growth conditions, a particular population of messenger RNA molecules lingered in the nucleus indefinitely--in structures they call "nuclear speckles"--and never reached the cytoplasm." We thought that these messenger RNAs must be doing something interesting by hanging around in the nucleus, but at the time we didn't have a way of finding out what that might be," says Spector. "Why would they be produced if they would never be used?"Then one of Spector's graduate students developed a method for purifying speckles. That allowed the researchers to identify not only the many different protein components of speckles, but also the messenger RNAs that are the basis of the new study, published in the October 21, 2005 issue of the journal Cell. The study--spearheaded by Cold Spring Harbor Laboratory postdoctoral fellow Dr. KannanganattuPrasanth--identified the first such messenger RNA: one transcribed from a mouse gene called mCAT2 that encodes a cell surface receptor." The first clue came when we found that the mCAT2 gene encodes two different kinds of messenger RNAs; the standard protein coding version that's exported to the cytoplasm as usual, and an atypical version that remains in the nucleus," says Spector. "But the big clue came when we thought about what the mCAT2 receptor does and why the mCAT2 gene would encode a messenger RNA that stays in the nucleus."The scientists learned from the work of others that the mCAT2 receptor is involved in the production of nitric oxide, and that nitric oxide production is stimulated by various stress conditions including wound healing and viral infection." That told us that when cells are stressed, maybe the atypical messenger RNA is released from the nucleus, exported to the cytoplasm, and translated into protein, thus circumventing the time-consuming process of producing new messenger RNA and providing a rapid response to viral infection or other stresses," says Spector.To test this idea, the researchers mimicked the effect of viral infection by treating cells with interferon.Sure enough, they discovered that the atypical mCAT2 messenger RNA in the nucleus was rapidly cleaved in response to interferon treatment, and that the protein coding portion of the molecule was then quicklyexported to the cytoplasm and translated into protein (see illustration). " This 'cut and run' mechanism is a completely new paradigm of gene regulation, so studying it will keep us busy for a while. But we already suspect that there is going to be a large family of genes regulated in this way," says Spector.In addition to Spector, Prasanth, and their colleagues at Cold Spring Harbor Laboratory, researchers at ISIS Pharmaceuticals (Carlsbad, CA) were involved in the study, which was funded by the National Institutes of Health (NIGMS, NCI) and the Louis Morin Charitable Trust.

Localization of messenger ribonucleic acid for adrenomedullin and adrenomedullin receptor in the human placenta in normal pregnancies and pregnancies complicated by oligohydramnios