Single amino acid changes in the predicted RNase H domain of Escherichia coli RNase G lead to complementation of RNase E deletion mutants

The endoribonuclease RNase E of Escherichia coli is an essential enzyme that plays a major role in all aspects of RNA metabolism. In contrast, its paralog, RNase G, seems to have more limited functions. It is involved in the maturation of the 5′ terminus of 16S rRNA, the processing of a few tRNAs, and the initiation of decay of a limited number of mRNAs but is not required for cell viability and cannot substitute for RNase E under normal physiological conditions. Here we show that neither the native nor N-terminal extended form of RNase G can restore the growth defect associated with either the rne-1 or rneΔ1018 alleles even when expressed at very high protein levels. In contrast, two distinct spontaneously derived single amino acid substitutions within the predicted RNase H domain of RNase G, generating the rng-219 and rng-248 alleles, result in complementation of the growth defect associated with various RNase E mutants, suggesting that this region of the two proteins may help distinguish their in vivo biological activities. Analysis of rneΔ1018/rng-219 and rneΔ1018/rng-248 double mutants has provided interesting insights into the distinct roles of RNase E and RNase G in mRNA decay and tRNA processing.     

Differential Subcellular Distributions and Trafficking Functions of hnRNP A2/B1 Spliceoforms

Trafficking of mRNA molecules from the nucleus to distal processes in neural cells is mediated by heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 trans‐acting factors. Although hnRNP A2/B1 is alternatively spliced to generate four isoforms, most functional studies have not distinguished between these isoforms. Here, we show, using isoform‐specific antibodies and isoform‐specific green fluorescent protein (GFP)‐fusion expression constructs, that A2b is the predominant cytoplasmic isoform in neural cells, suggesting that it may play a key role in mRNA trafficking. The differential subcellular distribution patterns of the individual isoforms are determined by the presence or absence of alternative exons that also affect their dynamic behavior in different cellular compartments, as measured by fluorescence correlation spectroscopy. Expression of A2b is also differentially regulated with age, species and cellular development. Furthermore, coinjection of isoform‐specific antibodies and labeled RNA into live oligodendrocytes shows that the assembly of RNA granules is impaired by blockade of A2b function. These findings suggest that neural cells modulate mRNA trafficking by regulating alternative splicing of hnRNP A2/B1 and controlling expression levels of A2b, which may be the predominant mediator of cytoplasmic‐trafficking functions. These findings highlight the importance of considering isoform‐specific functions for alternatively spliced proteins.     

Dismantling of Arabidopsis thaliana mesophyll cell chloroplasts during natural leaf senescence

One of the earliest events in the process of leaf senescence is dismantling of chloroplasts. Mesophyll cell chloroplasts from rosette leaves were studied in Arabidopsis thaliana undergoing natural senescence. The number of chloroplasts decreased by only 17% in fully yellow leaves, and chloroplasts were found to undergo progressive photosynthetic and ultrastructural changes as senescence proceeded. In ultrastructural studies, an intact tonoplast could not be visualized, thus, a 35S-GFP::δ-TIP line with a GFP-labeled tonoplast was used to demonstrate that chloroplasts remain outside of the tonoplast even at late stages of senescence. Chloroplast DNA was measured by real-time PCR at four different chloroplast loci, and a fourfold decrease in chloroplast DNA per chloroplast was noted in yellow senescent leaves when compared to green leaves from plants of the same age. Although chloroplast DNA did decrease, the chloroplast/nuclear gene copy ratio was still 31:1 in yellow leaves. Interestingly, mRNA levels for the four loci differed: psbA and ndhB mRNAs remained abundant late into senescence, while rpoC1 and rbcL mRNAs decreased in parallel to chloroplast DNA. Together, these data demonstrate that, during senescence, chloroplasts remain outside of the vacuole as distinct organelles while the thylakoid membranes are dismantled internally. As thylakoids were dismantled, Rubisco large subunit, Lhcb1, and chloroplast DNA levels declined, but variable levels of mRNA persisted.     

Noncognate Mycobacterium tuberculosis Toxin-Antitoxins Can Physically and Functionally Interact

The Mycobacterium tuberculosis genome harbors a striking number (>40) of toxin-antitoxin systems. Among them are at least seven MazF orthologs, designated MazF-mt1 through MazF-mt7, four of which have been demonstrated to function as mRNA interferases that selectively target mRNA for cleavage at distinct consensus sequences. As is characteristic of all toxin-antitoxin systems, each of the mazF-mt toxin genes is organized in an operon downstream of putative antitoxin genes. However, only one of the seven putative upstream antitoxins (designated MazE-mt1 through MazE-mt7) has significant sequence similarity to Escherichia coli MazE, the cognate antitoxin for E. coli MazF. Interestingly, the M. tuberculosis genome contains two independent operons encoding E. coli MazE orthologs, but they are not paired with mazF-mt-like genes. Instead, the genes encoding these two MazE orthologs are each paired with proteins containing a PIN domain, indicating that they may be members of the very large VapBC toxin-antitoxin family. We tested a spectrum of pair-wise combinations of cognate and noncognate Mtb toxin-antitoxins using in vivo toxicity and rescue experiments along with in vitro interaction experiments. Surprisingly, we uncovered several examples of noncognate toxin-antitoxin association, even among different families (e.g. MazF toxins and VapB antitoxins). These results challenge the “one toxin for one antitoxin” dogma and suggest that M. tuberculosis may enlist a sophisticated toxin-antitoxin network to alter its physiology in response to environmental cues.     

A Synonymous Single Nucleotide Polymorphism in ΔF508 CFTR Alters the Secondary Structure of the mRNA and the Expression of the Mutant Protein

Recent advances in our understanding of translational dynamics indicate that codon usage and mRNA secondary structure influence translation and protein folding. The most frequent cause of cystic fibrosis (CF) is the deletion of three nucleotides (CTT) from the cystic fibrosis transmembrane conductance regulator (CFTR) gene that includes the last cytosine (C) of isoleucine 507 (Ile507ATC) and the two thymidines (T) of phenylalanine 508 (Phe508TTT) codons. The consequences of the deletion are the loss of phenylalanine at the 508 position of the CFTR protein (ΔF508), a synonymous codon change for isoleucine 507 (Ile507ATT), and protein misfolding. Here we demonstrate that the ΔF508 mutation alters the secondary structure of the CFTR mRNA. Molecular modeling predicts and RNase assays support the presence of two enlarged single stranded loops in the ΔF508 CFTR mRNA in the vicinity of the mutation. The consequence of ΔF508 CFTR mRNA “misfolding” is decreased translational rate. A synonymous single nucleotide variant of the ΔF508 CFTR (Ile507ATC), that could exist naturally if Phe-508 was encoded by TTC, has wild type-like mRNA structure, and enhanced expression levels when compared with native ΔF508 CFTR. Because CFTR folding is predominantly cotranslational, changes in translational dynamics may promote ΔF508 CFTR misfolding. Therefore, we propose that mRNA “misfolding” contributes to ΔF508 CFTR protein misfolding and consequently to the severity of the human ΔF508 phenotype. Our studies suggest that in addition to modifier genes, SNPs may also contribute to the differences observed in the symptoms of various ΔF508 homozygous CF patients.     

Recognition of Adenosine Residues by the Active Site of Poly(A)-specific Ribonuclease

Poly(A)-specific ribonuclease (PARN) is a mammalian 3′-exoribonuclease that degrades poly(A) with high specificity. To reveal mechanisms by which poly(A) is recognized by the active site of PARN, we have performed a kinetic analysis using a large repertoire of trinucleotide substrates. Our analysis demonstrated that PARN harbors specificity for adenosine recognition in its active site and that the nucleotides surrounding the scissile bond are critical for adenosine recognition. We propose that two binding pockets, which interact with the nucleotides surrounding the scissile bond, play a pivotal role in providing specificity for the recognition of adenosine residues by the active site of PARN. In addition, we show that PARN, besides poly(A), also quite efficiently degrades poly(U), ∼10-fold less efficiently than poly(A). The poly(U)-degrading property of PARN could be of biological significance as oligo(U) tails recently have been proposed to play a role in RNA stabilization and destabilization.     

Cloning,characterization and tissue distribution of a pi-class glutathione S-transferase from clam (Venerupis philippinarum): Response to benzo[α]pyrene exposure

Glutathione S-transferases (GSTs) are phase II enzymes involved in major detoxification reactions of xenobiotics in many organisms. In this study, a full-length cDNA of GST-pi was cloned from the gill of Venerupis philippinarum by rapid amplification of cDNA ends (RACE) method for the first time. The full-length cDNA of V. philippinarum GST-pi (denoted as VpGSTp) was 1142 bp, with a 5′ untranslated region (UTR) of 87 bp, a 3′ UTR of 438 bp, and an open reading frame (ORF) of 618 bp encoding a protein of 205 amino acid residues with an estimated molecular mass of 23.9 kDa and an predicted isoelectric point (pI) of 7.9. The comparison of the deduced amino acid sequences with GSTs from other species showed that the enzyme belongs to the pi-class, and the amino acids defining the binding sites of glutathione (G-site) and for xenobiotic substrates (H-site) were highly conserved. Tissue distribution analysis of the VpGSTp mRNA revealed that the GST-pi expression level was observed higher in gill, adductor muscle, mantle and foot while lower in digestive gland. Using quantitative real-time PCR, the dose- and time-related effects of benzo[α]pyrene (B[α]P) on VpGSTp mRNA expression were investigated in gills and digestive gland. The results showed that a time-dependant increase in the expression of VpGSTp was induced by B[α]P and appeared a good linear relationship with B[α]P concentrations. All these results suggested that GST-pi in bivalve had an antioxidant role and VpGSTp expression may be a useful biomarker candidate for monitoring environmental contaminants such as PAHs.