RACK1 mRNA translation is regulated via a rapamycin-sensitive pathway and coordinated with ribosomal protein synthesis

AbstractRACK1 has been shown to interact with several proteins, this suggesting that it may play a central role in cell growth regulation. Some recent articles have described RACK1 as a component of the small ribosomal subunit. To investigate the relationship between RACK1 and ribosome, we analyzed RACK1 mRNA structure and regulation. Translational regulation was studied in HeLa cells subjected to serum or amino acid deprivation and stimulation. The results show that RACK1 mRNA has a 5′ terminal oligopyrimidine sequence and that its translation is dependent on the availability of serum and amino acids in exactly the same way as any other vertebrate ribosomal protein mRNA

Farmaci oppioidi e Cannabis nella terapia del dolore

[English]:“Opioid and Cannabis in Pain Control” is the result of studies performed by the Pharmacy Department and the “Centro Interdipartimentale di Ricerca in Farmacoeconomia e Farmacoutilizzazione” (CIRFF) of the University of Naples, “Federico II”. This book is aimed to those who work in a pharmacy and who, scholars, teachers or students, are interested in delve into the issue. The text analyzes different topics with an interdisciplinary approach. A large part is devoted to the chemical and pharmacological aspects related to this topic. Subsequently, the text focuses the theme, still very debated, of using opioids and Cannabis in therapy through an exhaustive analysis of the entire existing legislation: from the first laws promulgated by the Kingdom of Italy until the last ministerial circulars by Italian republic. Finally yet importantly, an important part of the book focuses on medical and therapeutic interpretation with regard to the effects on pain control, where opioids and Cannabis are not only a fruitful frontier of research, but also a consolidated and effective tool to counteract some types of pain / [Italiano]: “Farmaci oppioidi e Cannabis nella terapia del dolore” rappresenta il frutto di alcuni studi, condotti per almeno tre lustri nel Dipartimento di Farmacia e nel Centro Interdipartimentale di Ricerca in Farmacoeconomia e Farmacoutilizzazione (CIRFF) della Federico II, e si rivolge sia a chi presta servizio ogni giorno in una farmacia, sia a chi, studioso, docente o studente, è interessato ad approfondire l’argomento. Il testo, utilizzando un approccio interdisciplinare, si muove su piani euristici differenti. Naturalmente, ampio spazio è stato dedicato alla parte farmaceutica, analizzando tutti gli aspetti chimici e farmacologici connessi a questo tema. Un secondo punto di rilievo riguarda la problematica normativa legata alla dibattuta questione dell’utilizzo in terapia degli oppioidi e della Cannabis. In tal senso, si è cercato di offrire una prospettiva chiara ed esauriente del complesso quadro legislativo vigente: a partire dalle prime leggi promulgate dal Regno d’Italia, fino ad arrivare alle ultime circolari ministeriali in materia, è stata rivista ed esaminata l’intera normativa sulle sostanze stupefacenti, spiegandone anche i passaggi più delicati e controversi. Infine, soprattutto per ciò che concerne le ricadute sulla terapia del dolore, una parte significativa del libro si è concentrata sull’interpretazione medica e terapeutica, dove i farmaci oppioidi e la Cannabis costituiscono non solo una feconda frontiera di ricerca, ma anche un consolidato ed efficace strumento per contrastare alcune tipologie di dolore

Impairing the production of ribosomal RNA activates mammalian target of rapamycin complex 1 signalling and downstream translation factors

Ribosome biogenesis is a key process for maintaining protein synthetic capacity in dividing or growing cells, and requires coordinated production of ribosomal proteins and ribosomal RNA (rRNA), including the processing of the latter. Signalling through mammalian target of rapamycin complex 1 (mTORC1) activates all these processes. Here, we show that, in human cells, impaired rRNA processing, caused by expressing an interfering mutant of BOP1 or by knocking down components of the PeBoW complex elicits activation of mTORC1 signalling. This leads to enhanced phosphorylation of its substrates S6K1 and 4E-BP1, and stimulation of proteins involved in translation initiation and elongation. In particular, we observe both inactivation and downregulation of the eukaryotic elongation factor 2 kinase, which normally inhibits translation elongation. The latter effect involves decreased expression of the eEF2K mRNA. The mRNAs for ribosomal proteins, whose translation is positively regulated by mTORC1 signalling, also remain associated with ribosomes. Therefore, our data demonstrate that disrupting rRNA production activates mTORC1 signalling to enhance the efficiency of the translational machinery, likely to help compensate for impaired ribosome production

A post-transcriptional regulatory landscape of aging in the female mouse hippocampus

Aging is associated with substantial physiological changes and constitutes a major risk factor for neurological disorders including dementia. Alterations in gene expression upon aging have been extensively studied; however, an in-depth characterization of post-transcriptional regulatory events remains elusive. Here, we profiled the age-related changes of the transcriptome and translatome in the female mouse hippocampus by RNA sequencing of total RNA and polysome preparations at four ages (3-, 6-, 12-, 20-month-old); and we implemented a variety of bioinformatics approaches to unravel alterations in transcript abundance, alternative splicing, and polyadenylation site selection. We observed mostly well-coordinated transcriptome and translatome expression signatures across age including upregulation of transcripts related to immune system processes and neuroinflammation, though transcripts encoding ribonucleoproteins or associated with mitochondrial functions, calcium signaling and the cell-cycle displayed substantial discordant profiles, suggesting translational control associated with age-related deficits in hippocampal-dependent behavior. By contrast, alternative splicing was less preserved, increased with age and was associated with distinct functionally-related transcripts encoding proteins acting at synapses/dendrites, RNA-binding proteins; thereby predicting regulatory roles for RBM3 and CIRBP. Only minor changes in polyadenylation site selection were identified, indicating pivotal 3′-end selection in young adults compared to older groups. Overall, our study provides a comprehensive resource of age-associated post-transcriptional regulatory events in the mouse hippocampus, enabling further examination of the molecular features underlying age-associated neurological diseases

Norovirus infection results in eIF2α independent host translation shut-off and remodels the G3BP1 interactome evading stress granule formation.

Viral infections impose major stress on the host cell. In response, stress pathways can rapidly deploy defence mechanisms by shutting off the protein synthesis machinery and triggering the accumulation of mRNAs into stress granules to limit the use of energy and nutrients. Because this threatens viral gene expression, viruses need to evade these pathways to propagate. Human norovirus is responsible for gastroenteritis outbreaks worldwide. Here we examined how norovirus interacts with the eIF2α signaling axis controlling translation and stress granules. While norovirus infection represses host cell translation, our mechanistic analyses revealed that eIF2α signaling mediated by the stress kinase GCN2 is uncoupled from translational stalling. Moreover, infection results in a redistribution of the RNA-binding protein G3BP1 to replication complexes and remodelling of its interacting partners, allowing the avoidance from canonical stress granules. These results define novel strategies by which norovirus undergo efficient replication whilst avoiding the host stress response and manipulating the G3BP1 interactome

Il farmaco: ricerca, sviluppo e applicazione in terapia

[Italiano]:Il farmaco: ricerca, sviluppo e applicazione in terapia si propone l’obiettivo di offrire una panoramica sul processo di Ricerca e Sviluppo che un farmaco compie a partire dal momento in cui viene progettato fino alla sua pratica utilizzazione. Quando una molecola è ritenuta potenzialmente adatta per creare un medicinale, si attiva un lungo percorso che ha come traguardo la realizzazione di un nuovo mezzo terapeutico e la sua approvazione per l’immissione in commercio. Un percorso scandito dalla rigorosa osservanza di regolamenti e leggi che si sono evoluti nel tempo di pari passo con il progresso scientifico e tecnologico, ma spesso anche a seguito di reazioni avverse o eventi dannosi irreversibili che hanno innescato processi di revisione delle norme e dei protocolli sperimentali. Questo libro parte con una densa ricognizione sulla storia della farmacologia occidentale, al fine di agevolare la comprensione del coacervo di vicende e circostanze che nel tempo hanno fatto da sfondo a tutte quelle dinamiche attraverso cui il processo di Ricerca e Sviluppo si è gradualmente affermato e consolidato. Notevole attenzione è stata poi dedicata ad alcuni risvolti divenuti oramai cruciali all’interno dell’articolato universo normativo in cui il farmaco è collocato, quali le terapie avanzate e i nuovi approcci per la ricerca clinica. Inoltre, gli autori si sono concentrati sulla prescrizione dei cosiddetti off-label e sulle tematiche di farmacoutilizzazione e farmacovigilanza che, nel giro di pochi decenni, sono assurte a sfere di conoscenza sempre più significative e influenti nelle prospettive presenti e future, non solo delle scienze farmaceutiche ma dell’intera società. Lo sforzo compiuto per redigere questo volume trova la sua ragion d’essere proprio nel voler mettere a disposizione dei lettori uno sguardo d’insieme sul farmaco e sulle complesse sfide che ancora lo attendono./ [English]:“The drug: research, development and application in therapy” is an in-depth study on the Research and Development process that a drug performs from the moment it is designed up to its practical use. When a molecule is considered suitable for a medicine, a long process is activated which has as its goal the creation of a new therapeutic tool and its approval for marketing. A path marked by the strict observance of regulations and laws that have evolved over time in step with scientific and technological progress. A path that however has often been determined also by tragic events following damaging adverse reactions that have triggered processes of revision of the norms and experimental protocols. This book starts with a summary on the history of Western pharmacology, written to allow the reader to understand the circumstances that have been the background to those dynamics through which the Research and Development process has gradually consolidated. An important part of the book is dedicated to some aspects that are crucial in the normative universe in which the drug is placed, such as the advanced therapies and new approaches for clinical research. The authors also focused on the prescriptions of off-label drugs and on the issues of pharmacoutilization and pharmacovigilance, two disciplines that, in a few years, have become increasingly influential in the present and future perspectives, not only of the pharmaceutical sciences but of the entire society

Ruolo della chinasi PIM1 in risposta allo stress ribosomale

PIM1 is an oncogenic serine/threonine kinase mainly expressed in hematopoietic cells. Expression of PIM1 is regulated by a variety of growth factors and cytokines from transcriptional to post-translational level. Together with the other two members of the family (PIM2 and PIM3), PIM1 plays a role in hematopoietic cell growth and survival. By studying the molecular mechanisms of Diamond-Blackfan Anemia (DBA) we have found out that PIM1 interacts with the ribosomal protein RPS19 and cosediments with ribosomes. RPS19 gene is mutated in roughly one fourth of DBA patients, causing a defect in hematopoiesis due to an excess of apoptosis of erythroid precursors. To investigate the possible implication of PIM1 in DBA pathogenesis we have here analyzed the expression of this kinase in cultured cell model systems. These include a stably transfected cell line, inducible for PIM1 overexpression (PIM-REx) and an hematopoietic cell line (K562C), inducible for RPS19 downregulation. We have found that the depletion of RPS19 causes a drastic destabilization of PIM1 partly dependent on proteasome. The rapid degradation of PIM1 could be responsible for some defects observed in cells from DBA patients

Ruolo della chinasi PIM1 in risposta allo stress ribosomale

PIM1 is an oncogenic serine/threonine kinase mainly expressed in hematopoietic cells. Expression of PIM1 is regulated by a variety of growth factors and cytokines from transcriptional to post-translational level. Together with the other two members of the family (PIM2 and PIM3), PIM1 plays a role in hematopoietic cell growth and survival. By studying the molecular mechanisms of Diamond-Blackfan Anemia (DBA) we have found out that PIM1 interacts with the ribosomal protein RPS19 and cosediments with ribosomes. RPS19 gene is mutated in roughly one fourth of DBA patients, causing a defect in hematopoiesis due to an excess of apoptosis of erythroid precursors. To investigate the possible implication of PIM1 in DBA pathogenesis we have here analyzed the expression of this kinase in cultured cell model systems. These include a stably transfected cell line, inducible for PIM1 overexpression (PIM-REx) and an hematopoietic cell line (K562C), inducible for RPS19 downregulation. We have found that the depletion of RPS19 causes a drastic destabilization of PIM1 partly dependent on proteasome. The rapid degradation of PIM1 could be responsible for some defects observed in cells from DBA patients

A versatile tandem RNA isolation procedure to capture in vivo formed mRNA-protein complexes

We describe a tandem RNA isolation procedure (TRIP) that enables purification of in vivo formed messenger ribonucleoprotein (mRNP) complexes. The procedure relies on the purification of polyadenylated mRNAs with oligo(dT) beads from cellular extracts, followed by the capture of specific mRNAs with 3'-biotinylated 2'-O-methylated antisense RNA oligonucleotides, which are recovered with streptavidin beads. TRIP was applied to isolate in vivo crosslinked mRNP complexes from yeast, nematodes and human cells for subsequent analysis of RNAs and bound proteins. The method provides a basis for adaptation to other types of polyadenylated RNAs, enabling the comprehensive identification of bound proteins/RNAs, and the investigation of dynamic rearrangement of mRNPs imposed by cellular or environmental cues