The transcriptional cycle of HIV-1 in real-time and live cells

RNA polymerase II ( RNAPII) is a fundamental enzyme, but few studies have analyzed its activity in living cells. Using human immunodeficiency virus ( HIV) type 1 reporters, we study real- time messenger RNA ( mRNA) biogenesis by photobleaching nascent RNAs and RNAPII at specific transcription sites. Through modeling, the use of mutant polymerases, drugs, and quantitative in situ hybridization, we investigate the kinetics of the HIV- 1 transcription cycle. Initiation appears efficient because most polymerases demonstrate stable gene association. We calculate an elongation rate of approximately 1.9 kb/ min, and, surprisingly, polymerases remain at transcription sites 2.5 min longer than nascent RNAs. With a total polymerase residency time estimated at 333 s, 114 are assigned to elongation, and 63 are assigned to 3'- end processing and/ or transcript release. However, mRNAs were released seconds after polyadenylation onset, and analysis of polymerase density by chromatin immunoprecipitation suggests that they pause or lose processivity after passing the polyA site. The strengths and limitations of this kinetic approach to analyze mRNA biogenesis in living cells are discussed

The Hsp90 chaperone controls the biogenesis of L7Ae RNPs through conserved machinery

RNA-binding proteins of the L7Ae family are at the heart of many essential ribonucleoproteins (RNPs), including box C/D and H/ACA small nucleolar RNPs, U4 small nuclear RNP, telomerase, and messenger RNPs coding for selenoproteins. In this study, we show that Nufip and its yeast homologue Rsa1 are key components of the machinery that assembles these RNPs. We observed that Rsa1 and Nufip bind several L7Ae proteins and tether them to other core proteins in the immature particles. Surprisingly, Rsa1 and Nufip also link assembling RNPs with the AAA + adenosine triphosphatases hRvb1 and hRvb2 and with the Hsp90 chaperone through two conserved adaptors, Tah1/hSpagh and Pih1. Inhibition of Hsp90 in human cells prevents the accumulation of U3, U4, and telomerase RNAs and decreases the levels of newly synthesized hNop58, hNHP2, 15.5K, and SBP2. Thus, Hsp90 may control the folding of these proteins during the formation of new RNPs. This suggests that Hsp90 functions as a master regulator of cell proliferation by allowing simultaneous control of cell signaling and cell growth

α-MSH regulates intergenic splicing of MC1R and TUBB3 in human melanocytes

Alternative splicing enables higher eukaryotes to increase their repertoire of proteins derived from a restricted number of genes. However, the possibility that functional diversity may also be augmented by splicing between adjacent genes has been largely neglected. Here, we show that the human melanocortin 1 receptor (MC1R) gene, a critical component of the facultative skin pigmentation system, has a highly complex and inefficient poly(A) site which is instrumental in allowing intergenic splicing between this locus and its immediate downstream neighbour tubulin-β-III (TUBB3). These transcripts, which produce two distinct protein isoforms localizing to the plasma membrane and the endoplasmic reticulum, seem to be restricted to humans as no detectable chimeric mRNA could be found in MC1R expressing mouse melanocytes. Significantly, treatment with the MC1R agonist α-MSH or activation of the stress response kinase p38-MAPK, both key molecules associated with ultraviolet radiation dermal insult and subsequent skin tanning, result in a shift in expression from MC1R in favour of chimeric MC1R-TUBB3 isoforms in cultured melanocytes. We propose that these chimeric proteins serve to equip melanocytes with novel cellular phenotypes required as part of the pigmentation response

The influence of physical exercise on the generation of TGF-β1, PDGF-AA, and VEGF-A in adipose tissue

Adipose tissue is an important organ that produces and secretes hormones and cytokines, including TGF-β1, PDGF-AA, and VEGF-A. The goal of the present study was to investigate the influence of a single session of acute exercise, as well as the prolonged endurance training on the production of TGF-β1, PDGF-AA, and VEGF-A in the subcutaneous white adipose tissue in rats. Rats were randomly divided into two groups: untrained (UT, n = 30) and trained rats (T, subjected to 6-week endurance training with increasing load, n = 29). Both groups were subjected to an acute exercise session with the same work load. The rats were killed before (UTpre, Tpre), immediately after (UT0h, T0h), or 3 h (UT3h, T3h) after exercise and adipose tissue samples collected. Growth factor mRNA was evaluated using RT-PCR; the protein levels were measured before and after training (UTpre and Tpre) using the immunoenzymatic method. TGF-β1 and PDGF-AA mRNA levels were decreased in the UT3h rats compared to the UTpre rats (P = 0.0001 and P = 0.03, respectively), but the VEGF-A mRNA level remained unchanged in the UT0h and UT3h rats compared to UTpre rats. TGF-β1, PDGF-AA and VEGF-A mRNA levels were decreased in the T3h rats compared to Tpre (P = 0.0002, P = 0.02, and P = 0.03, respectively). TGF-β1, PDGF-AA and VEGF-A mRNA levels significantly increased in the Tpre rats compared to UTpre (all P = 0.0002). However, the protein levels remained constant. In conclusion, prolonged physical exercise increases growth factor mRNA in adipose tissue but not protein levels

Expression of Conjoined Genes: Another Mechanism for Gene Regulation in Eukaryotes

From the ENCODE project, it is realized that almost every base of the entire human genome is transcribed. One class of transcripts resulting from this arises from the conjoined gene, which is formed by combining the exons of two or more distinct (parent) genes lying on the same strand of a chromosome. Only a very limited number of such genes are known, and the definition and terminologies used for them are highly variable in the public databases. In this work, we have computationally identified and manually curated 751 conjoined genes (CGs) in the human genome that are supported by at least one mRNA or EST sequence available in the NCBI database. 353 representative CGs, of which 291 (82%) could be confirmed, were subjected to experimental validation using RT-PCR and sequencing methods. We speculate that these genes are arising out of novel functional requirements and are not merely artifacts of transcription, since more than 70% of them are conserved in other vertebrate genomes. The unique splicing patterns exhibited by CGs reveal their possible roles in protein evolution or gene regulation. Novel CGs, for which no transcript is available, could be identified in 80% of randomly selected potential CG forming regions, indicating that their formation is a routine process. Formation of CGs is not only limited to human, as we have also identified 270 CGs in mouse and 227 in drosophila using our approach. Additionally, we propose a novel mechanism for the formation of CGs. Finally, we developed a database, ConjoinG, which contains detailed information about all the CGs (800 in total) identified in the human genome. In summary, our findings reveal new insights about the functionality of CGs in terms of another possible mechanism for gene regulation and genomic evolution and the mechanism leading to their formation

Proteomic and 3D structure analyses highlight the C/D box snoRNP assembly mechanism and its control

International audienceIn vitro, assembly of box C/D small nucleolar ribonucleoproteins (snoRNPs) involves the sequential recruitment of core proteins to snoRNAs. In vivo, however, assembly factors are required (NUFIP, BCD1, and the FISP90-R2TP complex), and it is unknown whether a similar sequential scheme applies. In this paper, we describe systematic quantitative stable isotope labeling by amino acids in cell culture proteomic experiments and the crystal structure of the core protein Snu 13p/15.5K bound to a fragment of the assembly factor Rsa1p/NUFIP. This revealed several unexpected features: (a) the existence of a protein-only pre-snoRNP complex containing five assembly factors and two core proteins, 15.5K and Nop58; (b) the characterization of ZNHIT3, which is present in the protein-only complex but gets released upon binding to C/D snoRNAs; (c) the dynamics of the R2TP complex, which,appears a to load/unload RuvBL AAA(+) adenosine triphosphatase from pre-snoRNPs; and (d) a potential mechanism for preventing premature activation of snoRNP catalytic activity. These data provide a framework for understanding the assembly of box C/D snoRNPs

Reversal of Cocaine-Conditioned Place Preference through Methyl Supplementation in Mice: Altering Global DNA Methylation in the Prefrontal Cortex

Analysis of global methylation in cells has revealed correlations between overall DNA methylation status and some biological states. Recent studies suggest that epigenetic regulation through DNA methylation could be responsible for neuroadaptations induced by addictive drugs. However, there is no investigation to determine global DNA methylation status following repeated exposure to addictive drugs. Using mice conditioned place preference (CPP) procedure, we measured global DNA methylation level in the nucleus accumbens (NAc) and the prefrontal cortex (PFC) associated with drug rewarding effects. We found that cocaine-, but not morphine- or food-CPP training decreased global DNA methylation in the PFC. Chronic treatment with methionine, a methyl donor, for 25 consecutive days prior to and during CPP training inhibited the establishment of cocaine, but not morphine or food CPP. We also found that both mRNA and protein level of DNMT (DNA methytransferase) 3b in the PFC were downregulated following the establishment of cocaine CPP, and the downregulation could be reversed by repeated administration of methionine. Our study indicates a crucial role of global PFC DNA hypomethylation in the rewarding effects of cocaine. Reversal of global DNA hypomethylation could significantly attenuate the rewarding effects induced by cocaine. Our results suggest that methionine may have become a potential therapeutic target to treat cocaine addiction

The Secrets of a Functional Synapse – From a Computational and Experimental Viewpoint

BACKGROUND: Neuronal communication is tightly regulated in time and in space. The neuronal transmission takes place in the nerve terminal, at a specialized structure called the synapse. Following neuronal activation, an electrical signal triggers neurotransmitter (NT) release at the active zone. The process starts by the signal reaching the synapse followed by a fusion of the synaptic vesicle and diffusion of the released NT in the synaptic cleft; the NT then binds to the appropriate receptor, and as a result, a potential change at the target cell membrane is induced. The entire process lasts for only a fraction of a millisecond. An essential property of the synapse is its capacity to undergo biochemical and morphological changes, a phenomenon that is referred to as synaptic plasticity. RESULTS: In this survey, we consider the mammalian brain synapse as our model. We take a cell biological and a molecular perspective to present fundamental properties of the synapse:(i) the accurate and efficient delivery of organelles and material to and from the synapse; (ii) the coordination of gene expression that underlies a particular NT phenotype; (iii) the induction of local protein expression in a subset of stimulated synapses. We describe the computational facet and the formulation of the problem for each of these topics. CONCLUSION: Predicting the behavior of a synapse under changing conditions must incorporate genomics and proteomics information with new approaches in computational biology

Polysomal mRNA Association and Gene Expression in <i>Trypanosoma brucei</i>

Background: The contrasting physiological environments of Trypanosoma brucei procyclic (insect vector) and bloodstream (mammalian host) forms necessitates deployment of different molecular processes and, therefore, changes in protein expression. Transcriptional regulation is unusual in T. brucei because the arrangement of genes is polycistronic; however, genes which are transcribed together are subsequently cleaved into separate mRNAs by trans-splicing. Following pre-mRNA processing, the regulation of mature mRNA stability is a tightly controlled cellular process. While many stage-specific transcripts have been identified, previous studies using RNA-seq suggest that changes in overall transcript level do not necessarily reflect the abundance of the corresponding protein. Methods: To better understand the regulation of gene expression in T. brucei, we performed a bioinformatic analysis of RNA-seq on total, sub-polysomal, and polysomal mRNA samples. We further cross-referenced our dataset with a previously published proteomics dataset to identify new protein coding sequences. Results: Our analyses showed that several long non-coding RNAs are more abundant in the sub-polysome samples, which possibly implicates them in regulating cellular differentiation in T. brucei. We also improved the annotation of the T.brucei genome by identifying new putative protein coding transcripts that were confirmed by mass spectrometry data. Conclusions: Several long non-coding RNAs are more abundant in the sub-polysome cellular fractions and might pay a role in the regulation of gene expression. We hope that these data will be of wide general interest, as well as being of specific value to researchers studying gene regulation expression and life stage transitions in T. brucei