Co-transcriptional degradation of aberrant pre-mRNA by Xrn2

Eukaryotic protein-coding genes are transcribed as pre-mRNAs that are matured by capping, splicing and cleavage and polyadenylation. Although human pre-mRNAs can be long and complex, containing multiple introns and many alternative processing sites, they are usually processed co-transcriptionally. Mistakes during nuclear mRNA maturation could lead to potentially harmful transcripts that are important to eliminate. However, the processes of human pre-mRNA degradation are not well characterised in the human nucleus. We have studied how aberrantly processed pre-mRNAs are degraded and find a role for the 5′→3′ exonuclease, Xrn2. Xrn2 associates with and co-transcriptionally degrades nascent β-globin transcripts, mutated to inhibit splicing or 3′ end processing. Importantly, we provide evidence that many endogenous pre-mRNAs are also co-transcriptionally degraded by Xrn2 when their processing is inhibited by Spliceostatin A. Our data therefore establish a previously unknown function for Xrn2 and an important further aspect of pre-mRNA metabolism that occurs co-transcriptionally

The PMC2NT domain of the catalytic exosome subunit Rrp6p provides the interface for binding with its cofactor Rrp47p, a nucleic acid-binding protein

The exosome complex is a key component of the cellular RNA surveillance machinery and is required for normal 3′ end processing of many stable RNAs. Exosome activity requires additional factors such as the Ski or TRAMP complexes to activate the complex or facilitate substrate binding. Rrp47p promotes the catalytic activity of the exosome component Rrp6p, but its precise function is unknown. Here we show that recombinant Rrp47p is expressed as an apparently hexameric complex that specifically binds structured nucleic acids. Furthermore, pull-down assays demonstrated that Rrp47p interacts directly with the N-terminal region of Rrp6p that contains the functionally uncharacterized PMC2NT domain. Strains expressing a mutant form of Rrp6p lacking the N-terminal region failed to accumulate Rrp47p at normal levels, exhibited a slow growth phenotype characteristic of rrp47-Δ mutants and showed RNA processing defects consistent with loss of Rrp47p function. These findings suggest Rrp47p promotes Rrp6p activity by facilitating binding via the PMC2NT domain to structural elements within RNA. Notably, characterized Rrp6p substrates such as the 5.8S+30 species are predicted to contain helices at their 3′ termini, while others such as intergenic or antisense cryptic unstable transcripts could potentially form extensive double-stranded molecules with overlapping mRNAs

Evidence for core exosome independent function of the nuclear exoribonuclease Rrp6p

The RNA exosome processes and degrades RNAs in archaeal and eukaryotic cells. Exosomes from yeast and humans contain two active exoribonuclease components, Rrp6p and Dis3p/Rrp44p. Rrp6p is concentrated in the nucleus and the dependence of its function on the nine-subunit core exosome and Dis3p remains unclear. We found that cells lacking Rrp6p accumulate poly(A)+ rRNA degradation intermediates distinct from those found in cells depleted of Dis3p, or the core exosome component Rrp43p. Depletion of Dis3p in the absence of Rrp6p causes a synergistic increase in the levels of degradation substrates common to the core exosome and Rrp6p, but has no effect on Rrp6p-specific substrates. Rrp6p lacking a portion of its C-terminal domain no longer co-purifies with the core exosome, but continues to carry out RNA 3′-end processing of 5.8S rRNA and snoRNAs, as well as the degradation of certain truncated Rrp6-specific rRNA intermediates. However, disruption of Rrp6p–core exosome interaction results in the inability of the cell to efficiently degrade certain poly(A)+ rRNA processing products that require the combined activities of Dis3p and Rrp6p. These findings indicate that Rrp6p may carry out some of its critical functions without physical association with the core exosome

Role of ghrelin in food reward: impact of ghrelin on sucrose self-administration and mesolimbic dopamine and acetylcholine receptor gene expression

The decision to eat is strongly influenced by non-homeostatic factors such as food palatability. Indeed, the rewarding and motivational value of food can override homeostatic signals, leading to increased consumption and hence, obesity. Ghrelin, a gut-derived orexigenic hormone, has a prominent role in homeostatic feeding. Recently, however, it has emerged as a potent modulator of the mesolimbic dopaminergic reward pathway, suggesting a role for ghrelin in food reward. Here, we sought to determine whether ghrelin and its receptors are important for reinforcing motivation for natural sugar reward by examining the role of ghrelin receptor (GHS-R1A) stimulation and blockade for sucrose progressive ratio operant conditioning, a procedure used to measure motivational drive to obtain a reward. Peripherally and centrally administered ghrelin significantly increased operant responding and therefore, incentive motivation for sucrose. Utilizing the GHS-R1A antagonist JMV2959, we demonstrated that blockade of GHS-R1A signaling significantly decreased operant responding for sucrose. We further investigated ghrelin's effects on key mesolimbic reward nodes, the ventral tegmental area (VTA) and nucleus accumbens (NAcc), by evaluating the effects of chronic central ghrelin treatment on the expression of genes encoding major reward neurotransmitter receptors, namely dopamine and acetylcholine. Ghrelin treatment was associated with an increased dopamine receptor D5 and acetylcholine receptor nAChRβ2 gene expression in the VTA and decreased expression of D1, D3, D5 and nAChRα3 in the NAcc. Our data indicate that ghrelin plays an important role in motivation and reinforcement for sucrose and impacts on the expression of dopamine and acetylcholine encoding genes in the mesolimbic reward circuitry. These findings suggest that ghrelin antagonists have therapeutic potential for the treatment of obesity and to suppress the overconsumption of sweet food

Substrate specificity of the TRAMP nuclear surveillance complexes

During nuclear surveillance in yeast and human cells, the RNA exosome functions together with the TRAMP complexes. Here, the authors defined the protein composition of the TRAMP complexes and identified specific RNA binding sites for the different TRAMP components

The CCR4-NOT Complex Physically and Functionally Interacts with TRAMP and the Nuclear Exosome

BACKGROUND: Ccr4-Not is a highly conserved multi-protein complex consisting in yeast of 9 subunits, including Not5 and the major yeast deadenylase Ccr4. It has been connected functionally in the nucleus to transcription by RNA polymerase II and in the cytoplasm to mRNA degradation. However, there has been no evidence so far that this complex is important for RNA degradation in the nucleus. METHODOLOGY/PRINCIPAL FINDINGS: In this work we point to a new role for the Ccr4-Not complex in nuclear RNA metabolism. We determine the importance of the Ccr4-Not complex for the levels of non-coding nuclear RNAs, such as mis-processed and polyadenylated snoRNAs, whose turnover depends upon the nuclear exosome and TRAMP. Consistently, mutation of both the Ccr4-Not complex and the nuclear exosome results in synthetic slow growth phenotypes. We demonstrate physical interactions between the Ccr4-Not complex and the exosome. First, Not5 co-purifies with the exosome. Second, several exosome subunits co-purify with the Ccr4-Not complex. Third, the Ccr4-Not complex is important for the integrity of large exosome-containing complexes. Finally, we reveal a connection between the Ccr4-Not complex and TRAMP through the association of the Mtr4 helicase with the Ccr4-Not complex and the importance of specific subunits of Ccr4-Not for the association of Mtr4 with the nuclear exosome subunit Rrp6. CONCLUSIONS/SIGNIFICANCE: We propose a model in which the Ccr4-Not complex may provide a platform contributing to dynamic interactions between the nuclear exosome and its co-factor TRAMP. Our findings connect for the first time the different players involved in nuclear and cytoplasmic RNA degradation

Distinct Roles of Non-Canonical Poly(A) Polymerases in RNA Metabolism

Trf4p and Trf5p are non-canonical poly(A) polymerases and are part of the heteromeric protein complexes TRAMP4 and TRAMP5 that promote the degradation of aberrant and short-lived RNA substrates by interacting with the nuclear exosome. To assess the level of functional redundancy between the paralogous Trf4 and Trf5 proteins and to investigate the role of the Trf4-dependent polyadenylation in vivo, we used DNA microarrays to compare gene expression of the wild-type yeast strain of S. cerevisiae with either that of trf4Δ or trf5Δ mutant strains or the trf4Δ mutant expressing the polyadenylation-defective Trf4(DADA) protein. We found little overlap between the sets of transcripts with altered expression in the trf4Δ or the trf5Δ mutants, suggesting that Trf4p and Trf5p target distinct groups of RNAs for degradation. Surprisingly, most RNAs the expression of which was altered by the trf4 deletion were restored to wild-type levels by overexpression of TRF4(DADA), showing that the polyadenylation activity of Trf4p is dispensable in vivo. Apart from previously reported Trf4p and Trf5p target RNAs, this analysis along with in vivo cross-linking and RNA immunopurification-chip experiments revealed that both the TRAMP4 and the TRAMP5 complexes stimulate the degradation of spliced-out introns via a mechanism that is independent of the polyadenylation activity of Trf4p. In addition, we show that disruption of trf4 causes severe shortening of telomeres suggesting that TRF4 functions in the maintenance of telomere length. Finally, our study demonstrates that TRF4, the exosome, and TRF5 participate in antisense RNA–mediated regulation of genes involved in phosphate metabolism. In conclusion, our results suggest that paralogous TRAMP complexes have distinct RNA selectivities with functional implications in RNA surveillance as well as other RNA–related processes. This indicates widespread and integrative functions of TRAMP complexes for the coordination of different gene expression regulatory processes

Ghrelin Modulates the fMRI BOLD Response of Homeostatic and Hedonic Brain Centers Regulating Energy Balance in the Rat

The orexigenic gut-brain peptide, ghrelin and its G-protein coupled receptor, the growth hormone secretagogue receptor 1a (GHS-R1A) are pivotal regulators of hypothalamic feeding centers and reward processing neuronal circuits of the brain. These systems operate in a cooperative manner and receive a wide array of neuronal hormone/transmitter messages and metabolic signals. Functional magnetic resonance imaging was employed in the current study to map BOLD responses to ghrelin in different brain regions with special reference on homeostatic and hedonic regulatory centers of energy balance. Experimental groups involved male, ovariectomized female and ovariectomized estradiol-replaced rats. Putative modulation of ghrelin signaling by endocannabinoids was also studied. Ghrelin-evoked effects were calculated as mean of the BOLD responses 30 minutes after administration. In the male rat, ghrelin evoked a slowly decreasing BOLD response in all studied regions of interest (ROI) within the limbic system. This effect was antagonized by pretreatment with GHS-R1A antagonist JMV2959. The comparison of ghrelin effects in the presence or absence of JMV2959 in individual ROIs revealed significant changes in the prefrontal cortex, nucleus accumbens of the telencephalon, and also within hypothalamic centers like the lateral hypothalamus, ventromedial nucleus, paraventricular nucleus and suprachiasmatic nucleus. In the female rat, the ghrelin effects were almost identical to those observed in males. Ovariectomy and chronic estradiol replacement had no effect on the BOLD response. Inhibition of the endocannabinoid signaling by rimonabant significantly attenuated the response of the nucleus accumbens and septum. In summary, ghrelin can modulate hypothalamic and mesolimbic structures controlling energy balance in both sexes. The endocannabinoid signaling system contributes to the manifestation of ghrelin’s BOLD effect in a region specific manner. In females, the estradiol milieu does not influence the BOLD response to ghrelin

Hedonic and incentive signals for body weight control

Here we review the emerging neurobiological understanding of the role of the brain’s reward system in the regulation of body weight in health and in disease. Common obesity is characterized by the over-consumption of palatable/rewarding foods, reflecting an imbalance in the relative importance of hedonic versus homeostatic signals. The popular ‘incentive salience theory’ of food reward recognises not only a hedonic/pleasure component (‘liking’) but also an incentive motivation component (‘wanting’ or ‘reward-seeking’). Central to the neurobiology of the reward mechanism is the mesoaccumbal dopamine system that confers incentive motivation not only for natural rewards such as food but also by artificial rewards (eg. addictive drugs). Indeed, this mesoaccumbal dopamine system receives and integrates information about the incentive (rewarding) value of foods with information about metabolic status. Problematic over-eating likely reflects a changing balance in the control exerted by hypothalamic versus reward circuits and/or it could reflect an allostatic shift in the hedonic set point for food reward. Certainly, for obesity to prevail, metabolic satiety signals such as leptin and insulin fail to regain control of appetitive brain networks, including those involved in food reward. On the other hand, metabolic control could reflect increased signalling by the stomach-derived orexigenic hormone, ghrelin. We have shown that ghrelin activates the mesoaccumbal dopamine system and that central ghrelin signalling is required for reward from both chemical drugs (eg alcohol) and also from palatable food. Future therapies for problematic over-eating and obesity may include drugs that interfere with incentive motivation, such as ghrelin antagonists

Current and Future Drug Targets in Weight Management

Obesity will continue to be one of the leading causes of chronic disease unless the ongoing rise in the prevalence of this condition is reversed. Accumulating morbidity figures and a shortage of effective drugs have generated substantial research activity with several molecular targets being investigated. However, pharmacological modulation of body weight is extremely complex, since it is essentially a battle against one of the strongest human instincts and highly efficient mechanisms of energy uptake and storage. This review provides an overview of the different molecular strategies intended to lower body weight or adipose tissue mass. Weight-loss drugs in development include molecules intended to reduce the absorption of lipids from the GI tract, various ways to limit food intake, and compounds that increase energy expenditure or reduce adipose tissue size. A number of new preparations, including combinations of the existing drugs topiramate plus phentermine, bupropion plus naltrexone, and the selective 5-HT2C agonist lorcaserin have recently been filed for approval. Behind these leading candidates are several other potentially promising compounds and combinations currently undergoing phase II and III testing. Some interesting targets further on the horizon are also discussed