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The mechanisms involved in the increase in potassium excretion during the first week after uninephrectomy (UNX) were investigated. Data from three sets of experiments showed an increase in arterial plasma K concentrations. Rats infused with intragastric KCl showed an acute increase in arterial plasma K concentrations, which increased K excretion after UNX. In contrast, infusion with amiloride increased sodium excretion but decreased arterial plasma K concentration per kidney.

A study of circadium rhythms and the duration of adaptive potassium and sodium excretion in rats subjected to uninephrectomy (UNX) reveals that excretion circadian cycles were associated with UNX effect on plasma K and distal secretary mechanism alteration. Circadian control of excretion is non-dependent on UNX. Plasma K concentration controls distal K secretion.

Alternative precursor messenger RNA splicing is instrumental in expanding the proteome of higher eukaryotes, and changes in 3' splice site (3'ss) usage contribute to human disease. We demonstrate by small interfering RNA-mediated knockdowns, followed by RNA sequencing, that many proteins first recruited to human C* spliceosomes, which catalyze step 2 of splicing, regulate alternative splicing, including the selection of alternatively spliced NAGNAG 3'ss. Cryo-electron microscopy and protein cross-linking reveal the molecular architecture of these proteins in C* spliceosomes, providing mechanistic and structural insights into how they influence 3'ss usage. They further elucidate the path of the 3' region of the intron, allowing a structure-based model for how the C* spliceosome potentially scans for the proximal 3'ss. By combining biochemical and structural approaches with genome-wide functional analyses, our studies reveal widespread regulation of alternative 3'ss usage after step 1 of splicing and the likely mechanisms whereby C* proteins influence NAGNAG 3'ss choices.

The aspects of fire retardation in some phosphorus-modified polymethyl methacrylate (PMMA) and polystyrene (PSt) polymers are reported in the present paper. Both additive and reactive strategies were employed to obtain the desired level of loading of the phosphorus-bearing compound/moiety (2 wt.% of P in each case). Test samples were obtained using bulk polymerization. The modifying compounds contained the P-atom in various chemical environments, as well as in an oxidation state of either III or V. With a view to gain an understanding of the chemical constitution of the gaseous products formed from the thermal decomposition of liquid additives/reactives, these materials were subjected to GC/MS analysis, whereas the decomposition of solid additives was detailed using the pyrolysis-GC/MS technique. Other investigations included the use of: Inductively-coupled Plasma/Optical Emission Spectroscopy (ICP/OES), solid-state NMR and FT-IR spectroscopy. In the case of PMMA-based systems, it was found that the modifying phosphonate ester function, upon thermal cracking, produced 'phosphorus' acid species which initiated the charring process. In the case of solid additives, it is more likely that the resultant phosphorus- and/or oxygenated phosphorus-containing volatiles acted as flame inhibitors in the gaseous phase. With the PSt-based systems, a probable process involving the phosphorylation of the phenyl groups leading to crosslinking and char formation is feasible.

Post-transcriptional 3' end addition of nucleotides is important in a variety of RNA decay pathways. We have examined the 3' end addition of nucleotides during the decay of the Hsp70 mRNA and a corresponding reporter RNA in Drosophila S2 cells by conventional sequencing of cDNAs obtained after mRNA circularization and by deep sequencing of dedicated libraries enriched for 3' decay intermediates along the length of the mRNA. Approximately 5%-10% of 3' decay intermediates carried nonencoded oligo(A) tails with a mean length of 2-3 nucleotides. RNAi experiments showed that the oligoadenylated RNA fragments were intermediates of exosomal decay and the noncanonical poly(A) polymerase Trf4-1 was mainly responsible for A addition. A hot spot of A addition corresponded to an intermediate of 3' decay that accumulated upon inhibition of decapping, and knockdown of Trf4-1 increased the abundance of this intermediate, suggesting that oligoadenylation facilitates 3' decay. Oligoadenylated 3' decay intermediates were found in the cytoplasmic fraction in association with ribosomes, and fluorescence microscopy revealed a cytoplasmic localization of Trf4-1. Thus, oligoadenylation enhances exosomal mRNA degradation in the cytoplasm., (© 2016 Harnisch et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Small nucleolar and small Cajal body RNAs (snoRNAs and scaRNAs) of the H/ACA box and C/D box type are generated by exonucleolytic shortening of longer precursors. Removal of the last few nucleotides at the 3' end is known to be a distinct step. We report that, in human cells, knock-down of the poly(A) specific ribonuclease (PARN), previously implicated only in mRNA metabolism, causes the accumulation of oligoadenylated processing intermediates of H/ACA box but not C/D box RNAs. In agreement with a role of PARN in snoRNA and scaRNA processing, the enzyme is concentrated in nucleoli and Cajal bodies. Oligo(A) tails are attached to a short stub of intron sequence remaining beyond the mature 3' end of the snoRNAs. The noncanonical poly(A) polymerase PAPD5 is responsible for addition of the oligo(A) tails. We suggest that deadenylation is coupled to clean 3' end trimming, which might serve to enhance snoRNA stability.

Shortening of the poly(A) tail is the first and often rate-limiting step in mRNA degradation. Three poly(A)-specific 3' exonucleases have been described that can carry out this reaction: PAN, composed of two subunits; PARN, a homodimer; and the CCR4-NOT complex, a heterooligomer that contains two catalytic subunits and may have additional functions in the cell. Current evidence indicates that all three enzymes use a two-metal ion mechanism to release nucleoside monophosphates in a hydrolytic reaction. The CCR4-NOT is the main deadenylase in all organisms examined, and mutations affecting the complex can be lethal. The contribution of PAN, apparently an initial deadenylation preceding the activity of CCR4-NOT, is less important, whereas the activity of PARN seems to be restricted to specific substrates or circumstances, for example, stress conditions. Rapid deadenylation and decay of specific mRNAs can be caused by recruitment of both PAN and the CCR4-NOT complex. This function can be carried out by RNA-binding proteins, for example, members of the PUF family. Alternatively, miRNAs can recruit the deadenylase complexes with the help of their associated GW182 proteins., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Erythroid precursor cells lose the capacity for mRNA synthesis due to exclusion of the nucleus during maturation. Therefore, the stability and translation of mRNAs that code for specific proteins, which function in late stages of maturation when reticulocytes become erythrocytes, are controlled tightly. Reticulocyte 15-lipoxygenase (r15-LOX) initiates the breakdown of mitochondria in mature reticulocytes. Through the temporal restriction of mRNA translation, the synthesis of r15-LOX is prevented in premature cells. The enzyme is synthesized only in mature reticulocytes, although r15-LOX mRNA is already present in erythroid precursor cells. Translation of r15-LOX mRNA is inhibited by hnRNP K and hnRNP E1, which bind to the differentiation control element (DICE) in its 3' untranslated region (3'UTR). The hnRNP K/E1-DICE complex interferes with the joining of the 60S ribosomal subunit to the 40S subunit at the AUG. We took advantage of the inducible human erythroid K562 cell system that fully recapitulates this process to identify so far unknown factors, which are critical for DICE-dependent translational regulation. Applying RNA chromatography with the DICE as bait combined with hnRNP K immunoprecipitation, we specifically purified the DEAD-box RNA helicase 6 (DDX6) that interacts with hnRNP K and hnRNP E1 in a DICE-dependent manner. Employing RNA interference and fluorescence in situ hybridization, we show that DDX6 colocalizes with endogenous human (h)r15-LOX mRNA to P-body-like RNP granules, from which 60S ribosomal subunits are excluded. Our data suggest that in premature erythroid cells translational silencing of hr15-LOX mRNA is maintained by DDX6 mediated storage in these RNP granules.

Erythroid precursor cells undergo nuclear extrusion and degradation of mitochondria when they mature to erythrocytes. It has been suggested before that the reticulocyte 15-lipoxygenase (r15-LOX) plays an important role in initiating the breakdown of mitochondria in rabbit reticulocytes. The expression of rabbit r15-LOX is regulated by the heterogeneous nuclear ribonucleoproteins (hnRNP) K and E1 at the translational level. However, this mechanism has never been confirmed in human erythropoiesis. Based on K562 cells we have set up an inducible human erythroid cell system. We show that, during induction, K562 cells exhibit changes in morphology and protein expression that are characteristic for terminal erythroid maturation: nuclear exclusion, expression of endogenous human r15-LOX regulated by hnRNP K and hnRNP E1, and loss of mitochondria. Importantly, induction of terminal erythroid maturation in primary human CD34(+) cells recapitulated the results obtained in K562 cells. Employing the physiologically relevant K562 cell system we uncovered a new mechanism of interdependent post-transcriptional regulation of gene expression. The timely expression of the tyrosine kinase c-Src, which phosphorylates hnRNP K in later stages, is controlled by hnRNP K in early stages of erythroid maturation. hnRNP K binds to the 3'-untranslated region of the c-Src mRNA and inhibits its translation by blocking 80 S ribosome formation. In premature erythroid cells, small interfering RNA-mediated knockdown of hnRNP K, but not of hnRNP E1, leads to the de-repression of c-Src synthesis.

hnRNP K and hnRNP E1/E2 are RNA-binding proteins comprised of three hnRNP K-homology (KH) domains. These proteins are involved in the translational control and stabilization of mRNAs in erythroid cells. hnRNP E1 and hnRNP K regulate the translation of reticulocyte 15-lipoxygenase (r15-LOX) mRNA. Both proteins bind specifically to the differentiation control element (DICE) in the 3' untranslated region (3'UTR) of the r15-LOX mRNA. It has been shown that hnRNP K is a substrate of the tyrosine kinase c-Src and that tyrosine phosphorylation by c-Src inhibits the binding of hnRNP K to the DICE. Here, we investigate which of the three KH domains of hnRNP E1 and hnRNP K mediate the DICE interaction. Using RNA-binding assays, we demonstrate DICE-binding of the KH domains 1 and 3 of hnRNP E1, and KH domain 3 of hnRNP K. Furthermore, with RNA-binding assays, NMR experiments and in vitro translation studies, we show that tyrosine 458 in KH domain 3 of hnRNP K is important for the DICE interaction and we provide evidence that it is a target of c-Src.