Alternative exon definition events control the choice between nuclear retention and cytoplasmic export of U11/U12-65K mRNA

Cellular homeostasis of the minor spliceosome is regulated by a negative feed-back loop that targets U11-48K and U11/U12-65K mRNAs encoding essential components of the U12-type intron-specific U11/U12 di-snRNP. This involves interaction of the U11 snRNP with an evolutionarily conserved splicing enhancer giving rise to unproductive mRNA isoforms. In the case of U11/U12-65K, this mechanism controls the length of the 3' untranslated region (3'UTR). We show that this process is dynamically regulated in developing neurons and some other cell types, and involves a binary switch between translation-competent mRNAs with a short 3'UTR to non-productive isoforms with a long 3'UTR that are retained in the nucleus or/and spliced to the downstream amylase locus. Importantly, the choice between these alternatives is determined by alternative terminal exon definition events regulated by conserved U12-and U2-type 50 splice sites as well as sequence signals used for pre-mRNA cleavage and polyadenylation. We additionally show that U11 snRNP binding to the U11/U12-65K mRNA species with a long 3'UTR is required for their nuclear retention. Together, our studies uncover an intricate molecular circuitry regulating the abundance of a key spliceosomal protein and shed new light on the mechanisms limiting the export of non-productively spliced mRNAs from the nucleus to the cytoplasm.Peer reviewe

The DNA/RNA-Dependent RNA Polymerase QDE-1 Generates Aberrant RNA and dsRNA for RNAi in a Process Requiring Replication Protein A and a DNA Helicase

The Neurospora RNA-dependent RNA polymerase QDE-1 is an RNA polymerase that can use both RNA and DNA as templates, suggesting a new mechanism for small RNA production

Appraisal of the effectiveness of a controlled course of the biological prophylaxis of envirommetally induced diseases in children dwellers of the Asbest city

We carried out on children attending two kindergartens in the Asbestos city a one month course of the biological prophylaxis based on a bio-protective complex (comprising glutamate, a polyvitamin-polymineral preparation “Vitrum Kids", and methionine) which had been successfully teased in animal experiments. It was demonstrated that administration of this complex significantly improved the general health status of the children, influenced beneficially the lipoperoxidation and humoral immune indices as well as cytogenetic index in cells of buccal mucosa and DNA fragmentation in blood leucocytes. The last two effects may be considered as an evidence for a probable cancer preventive action of the tested biological prophylaxis.В двух ДОУ города Асбеста проведен месячный контролируемый курс биопрофилактики на основе комплекса биопротекторов с различными механизмами действия (глутамат, поливитаминно-минеральный комплекс «Витрум-Кидс», метионин), ранее успешно испытанного в эксперименте на животных в условиях воздействия хризотил-асбеста. Прием этого комплекса существенно улучшил общее состояние здоровья детей, благоприятно повлиял на уровень перекисного окисления и на показатели гуморального иммунитета, а также снизил цитогенетический показатель, оцененный в клетках слизистой рта, и фрагментацию ДНК в лейкоцитах крови. Последние два эффекта могут расцениваться как свидетельствующие о вероятном противоканцерогенном действии испытанной биологической профилактики

MicroRNA-122 Modulates the Rhythmic Expression Profile of the Circadian Deadenylase Nocturnin in Mouse Liver

Nocturnin is a circadian clock-regulated deadenylase thought to control mRNA expression post-transcriptionally through poly(A) tail removal. The expression of Nocturnin is robustly rhythmic in liver at both the mRNA and protein levels, and mice lacking Nocturnin are resistant to diet-induced obesity and hepatic steatosis. Here we report that Nocturnin expression is regulated by microRNA-122 (miR-122), a liver specific miRNA. We found that the 3′-untranslated region (3′-UTR) of Nocturnin mRNA harbors one putative recognition site for miR-122, and this site is conserved among mammals. Using a luciferase reporter construct with wild-type or mutant Nocturnin 3′-UTR sequence, we demonstrated that overexpression of miR-122 can down-regulate luciferase activity levels and that this effect is dependent on the presence of the putative miR-122 recognition site. Additionally, the use of an antisense oligonucleotide to knock down miR-122 in vivo resulted in significant up-regulation of both Nocturnin mRNA and protein expression in mouse liver during the night, resulting in Nocturnin rhythms with increased amplitude. Together, these data demonstrate that the normal rhythmic profile of Nocturnin expression in liver is shaped in part by miR-122. Previous studies have implicated Nocturnin and miR-122 as important post-transcriptional regulators of both lipid metabolism and circadian clock controlled gene expression in the liver. Therefore, the demonstration that miR-122 plays a role in regulating Nocturnin expression suggests that this may be an important intersection between hepatic metabolic and circadian control

Plate tectonics of virus shell assembly and reorganization in phage φ8, a distant relative of mammalian reoviruses

The hallmark of a virus is its capsid, which harbors the viral genome and is formed from protein subunits, which assemble following precise geometric rules. dsRNA viruses use an unusual protein multiplicity (120 copies) to form their closed capsids. We have determined the atomic structure of the capsid protein (P1) from the dsRNA cystovirus Φ8. In the crystal P1 forms pentamers, very similar in shape to facets of empty procapsids, suggesting an unexpected assembly pathway that proceeds via a pentameric intermediate. Unlike the elongated proteins used by dsRNA mammalian reoviruses, P1 has a compact trapezoid-like shape and a distinct arrangement in the shell, with two near-identical conformers in nonequivalent structural environments. Nevertheless, structural similarity with the analogous protein from the mammalian viruses suggests a common ancestor. The unusual shape of the molecule may facilitate dramatic capsid expansion during phage maturation, allowing P1 to switch interaction interfaces to provide capsid plasticity

Editing of hnRNP K protein mRNA in colorectal adenocarcinoma and surrounding mucosa

The heterogeneous nuclear ribonucleoprotein K (hnRNP K) protein is an RNA-binding protein involved in many processes that compose gene expression. K protein is upregulated in the malignant processes and has been shown to modulate the expression of genes involved in mitogenic responses and tumorigenesis. To explore the possibility that there are alternative isoforms of K protein expressed in colon cancer, we amplified and sequenced K protein mRNA that was isolated from colorectal cancers as well as from normal tissues surrounding the tumours. Sequencing revealed a single G-to-A base substitution at position 274 that was found in tumours and surrounding mucosa, but not in individuals that had no colorectal tumour. This substitution most likely reflects an RNA editing event because it was not found in the corresponding genomic DNAs. Sequencing of RNA from normal colonic mucosa of patients with prior resection of colorectal cancer revealed only the wild-type K protein transcript, indicating that G274A isoform is tumour related. To our knowledge, this is the first example of an RNA editing event in cancer and its surrounding tissue, a finding that may offer a new diagnostic and treatment marker

The dynamics and efficacy of antiviral RNA silencing: A model study

<p>Abstract</p> <p>Background</p> <p>Mathematical modeling is important to provide insight in the complicated pathway of RNA silencing. RNA silencing is an RNA based mechanism that is widely used by eukaryotes to fight viruses, and to control gene expression.</p> <p>Results</p> <p>We here present the first mathematical model that combines viral growth with RNA silencing. The model involves a plus-strand RNA virus that replicates through a double-strand RNA intermediate. The model of the RNA silencing pathway consists of cleavage of viral RNA into siRNA by Dicer, target cleavage of viral RNA via the RISC complex, and a secondary response. We found that, depending on the strength of the silencing response, different viral growth patterns can occur. Silencing can decrease viral growth, cause oscillations, or clear the virus completely. Our model can explain various observed phenomena, even when they seem contradictory at first: the diverse responses to the removal of RNA dependent RNA polymerase; different viral growth curves; and the great diversity in observed siRNA ratios.</p> <p>Conclusion</p> <p>The model presented here is an important step in the understanding of the natural functioning of RNA silencing in viral infections.</p

Expression of Human nPTB Is Limited by Extreme Suboptimal Codon Content

Background: The frequency of synonymous codon usage varies widely between organisms. Suboptimal codon content limits expression of viral, experimental or therapeutic heterologous proteins due to limiting cognate tRNAs. Codon content is therefore often adjusted to match codon bias of the host organism. Codon content also varies between genes within individual mammalian species. However, little attention has been paid to the consequences of codon content upon translation of host proteins. Methodology/Principal Findings: In comparing the splicing repressor activities of transfected human PTB and its two tissue-restricted paralogs–nPTB and ROD1–we found that the three proteins were expressed at widely varying levels. nPTB was expressed at 1–3 % the level of PTB despite similar levels of mRNA expression and 74 % amino acid identity. The low nPTB expression was due to the high proportion of codons with A or U at the third codon position, which are suboptimal in human mRNAs. Optimization of the nPTB codon content, akin to the ‘‘humanization’ ’ of foreign ORFs, allowed efficient translation in vivo and in vitro to levels comparable with PTB. We were then able to demonstrate that all three proteins act as splicing repressors. Conclusions/Significance: Our results provide a striking illustration of the importance of mRNA codon content in determining levels of protein expression, even within cells of the natural host species

High Glucose Suppresses Human Islet Insulin Biosynthesis by Inducing miR-133a Leading to Decreased Polypyrimidine Tract Binding Protein-Expression

BACKGROUND: Prolonged periods of high glucose exposure results in human islet dysfunction in vitro. The underlying mechanisms behind this effect of high glucose are, however, unknown. The polypyrimidine tract binding protein (PTB) is required for stabilization of insulin mRNA and the PTB mRNA 3'-UTR contains binding sites for the microRNA molecules miR-133a, miR-124a and miR-146. The aim of this study was therefore to investigate whether high glucose increased the levels of these three miRNAs in association with lower PTB levels and lower insulin biosynthesis rates. METHODOLOGY/PRINCIPAL FINDINGS: Human islets were cultured for 24 hours in the presence of low (5.6 mM) or high glucose (20 mM). Islets were also exposed to sodium palmitate or the proinflammatory cytokines IL-1beta and IFN-gamma, since saturated free fatty acids and cytokines also cause islet dysfunction. RNA was then isolated for real-time RT-PCR analysis of miR-133a, miR-124a, miR-146, insulin mRNA and PTB mRNA contents. Insulin biosynthesis rates were determined by radioactive labeling and immunoprecipitation. Synthetic miR-133a precursor and inhibitor were delivered to dispersed islet cells by lipofection, and PTB was analyzed by immunoblotting following culture at low or high glucose. Culture in high glucose resulted in increased islet contents of miR-133a and reduced contents of miR-146. Cytokines increased the contents of miR-146. The insulin and PTB mRNA contents were unaffected by high glucose. However, both PTB protein levels and insulin biosynthesis rates were decreased in response to high glucose. The miR-133a inhibitor prevented the high glucose-induced decrease in PTB and insulin biosynthesis, and the miR-133a precursor decreased PTB levels and insulin biosynthesis similarly to high glucose. CONCLUSION: Prolonged high-glucose exposure down-regulates PTB levels and insulin biosynthesis rates in human islets by increasing miR-133a levels. We propose that this mechanism contributes to hyperglycemia-induced beta-cell dysfunction

Regulation of Retention of FosB Intron 4 by PTB

One effect of stressors such as chronic drug administration is that sequence within the terminal exon of the transcription factor FosB is recognized as intronic and removed by alternative splicing. This results in an open-reading-frame shift that produces a translation stop codon and ultimately a truncated protein, termed ΔFosB. In vitro splicing assays with control and mutated transcripts generated from a fosB mini-gene construct indicated a CU-rich sequence at the 3′ end of intron 4 (I4) plays an important role in regulating fosB pre-mRNA splicing due to its binding of polypyrimidine tract binding protein (PTB). PTB binding to this sequence is dependent upon phosphorylation by protein kinase A and is blocked if the CU-rich sequence is mutated to a U-rich region. When this mutated fosB minigene is expressed in HeLa cells, the splicing efficiency of its product is increased compared to wild type. Moreover, transient transfection of PTB-1 in HeLa cells decreased the splicing efficiency of a wild type fosB minigene transcript. Depletion of PTB from nuclear extracts facilitated U2AF65 binding to wild type sequence in vitro, suggesting these proteins function in a dynamic equilibrium to modulate fosB pre-mRNA alternative splicing. These results demonstrate for the first time that phosphorylated PTB promotes intron retention and thereby silences the splicing of fosB I4