A silencing safeguard: links between RNA silencing and mRNA processing in Arabidopsis

RNA silencing is a genome defense mechanism used by many eukaryotic organisms to fight viruses and to control transposable elements. Work by Gregory et al. on Arabidopsis thaliana (in this issue of Developmental Cell) revealed a mechanism whereby the plant protects its endogenous messenger RNAs from undergoing RNA silencing and uncovered an unexpected role of the cap-binding protein ABH1 in miRNA biogenesis.     

Xenogeneic silencing relies on temperature-dependent phosphorylation of the host H-NS protein in Shewanella

Lateral gene transfer (LGT) plays a key role in shaping the genome evolution and environmental adaptation of bacteria. Xenogeneic silencing is crucial to ensure the safe acquisition of LGT genes into host pre-existing regulatory networks. We previously found that the host nucleoid structuring protein (H-NS) silences prophage CP4So at warm temperatures yet enables this prophage to excise at cold temperatures in Shewanella oneidensis. However, whether H-NS silences other genes and how bacteria modulate H-NS to regulate the expression of genes have not been fully elucidated. In this study, we discovered that the H-NS silences many LGT genes and the xenogeneic silencing of H-NS relies on a temperature-dependent phosphorylation at warm temperatures in S. oneidensis. Specifically, phosphorylation of H-NS at Ser42 is critical for silencing the cold-inducible genes including the excisionase of CP4So prophage, a cold shock protein, and a stress-related chemosensory system. By contrast, nonphosphorylated H-NS derepresses the promoter activity of these genes/operons to enable their expression at cold temperatures. Taken together, our results reveal that the posttranslational modification of H-NS can function as a regulatory switch to control LGT gene expression in host genomes to enable the host bacterium to react and thrive when environmental temperature changes.     

Translation reinitiation relies on the interaction between eIF3a/TIF32 and progressively folded cis-acting mRNA elements preceding short uORFs

Reinitiation is a gene-specific translational control mechanism characterized by the ability of some short upstream uORFs to retain post-termination 40S subunits on mRNA. Its efficiency depends on surrounding cis-acting sequences, uORF elongation rates, various initiation factors, and the intercistronic distance. To unravel effects of cis-acting sequences, we investigated previously unconsidered structural properties of one such a cis-enhancer in the mRNA leader of GCN4 using yeast genetics and biochemistry. This leader contains four uORFs but only uORF1, flanked by two transferrable 5' and 3' cis-acting sequences, and allows efficient reinitiation. Recently we showed that the 5' cis-acting sequences stimulate reinitiation by interacting with the N-terminal domain (NTD) of the eIF3a/TIF32 subunit of the initiation factor eIF3 to stabilize post-termination 40S subunits on uORF1 to resume scanning downstream. Here we identify four discernible reinitiation-promoting elements (RPEs) within the 5' sequences making up the 5' enhancer. Genetic epistasis experiments revealed that two of these RPEs operate in the eIF3a/TIF32-dependent manner. Likewise, two separate regions in the eIF3a/TIF32-NTD were identified that stimulate reinitiation in concert with the 5' enhancer. Computational modeling supported by experimental data suggests that, in order to act, the 5' enhancer must progressively fold into a specific secondary structure while the ribosome scans through it prior uORF1 translation. Finally, we demonstrate that the 5' enhancer's stimulatory activity is strictly dependent on and thus follows the 3' enhancer's activity. These findings allow us to propose for the first time a model of events required for efficient post-termination resumption of scanning. Strikingly, structurally similar RPE was predicted and identified also in the 5' leader of reinitiation-permissive uORF of yeast YAP1. The fact that it likewise operates in the eIF3a/TIF32-dependent manner strongly suggests that at least in yeasts the underlying mechanism of reinitiation on short uORFs is conserved.     

The distribution of functional bacteriophage T4 mRNA on polysomes.

Functional bacteriophage T4 deoxynucleotide kinase and α-glucosyl transferase mRNAs can be isolated from polysomes extracted from cells 8 min after infection. At least 55% of the 8-min deoxynucleotide kinase mRNA is associated with polysomes and is released from the cell membrane by deoxyribonuclease (DNase) treatment (soluble mRNA). Approximately 20% of the kinase mRNA remains tightly bound to membrane after DNase treatment (membrane mRNA) and 25% of the kinase mRNA is routinely lost during fractionation. The membrane-bound kinase mRNA is about three times as stable in vitro as the soluble kinase mRNA. Soluble kinase mRNA (14.5S) is found associated with as few as one ribosome and as many as 22 ribosomes; however, 14.5S α-glucosyl transferase mRNA is found predominantly in six ribosome polysomes. The size of the α-glucosyl transferase mRNA is heterogenous, ranging between 14.5 and 20S. The larger α-glucosyl transferase mRNAs are never found on small polysomes but appear only in polysomes containing at least nine ribosomes (18S α-glucosyl transferase mRNA). Maximum-size α-glucosyl transferase mRNA (approximately 20S) appears on polysomes containing at least 14 ribosomes. The relationships between decay of T4 mRNA and polysome size and the location of ribosome loading sites on the 20S α-glucosyl transferase message are also discussed.     

Mitochondrial translocation of APE1 relies on the MIA pathway

APE1 is a multifunctional protein with a fundamental role in repairing nuclear and mitochondrial DNA lesions caused by oxidative and alkylating agents. Unfortunately, comprehensions of the mechanisms regulating APE1 intracellular trafficking are still fragmentary and contrasting. Recent data demonstrate that APE1 interacts with the mitochondrial import and assembly protein Mia40 suggesting the involvement of a redox-assisted mechanism, dependent on the disulfide transfer system, to be responsible of APE1 trafficking into the mitochondria. The MIA pathway is an import machinery that uses a redox system for cysteine enriched proteins to drive them in this compartment. It is composed by two main proteins: Mia40 is the oxidoreductase that catalyzes the formation of the disulfide bonds in the substrate, while ALR reoxidizes Mia40 after the import. In this study, we demonstrated that: (i) APE1 and Mia40 interact through disulfide bond formation; and (ii) Mia40 expression levels directly affect APE1''s mitochondrial translocation and, consequently, play a role in the maintenance of mitochondrial DNA integrity. In summary, our data strongly support the hypothesis of a redox-assisted mechanism, dependent on Mia40, in controlling APE1 translocation into the mitochondrial inner membrane space and thus highlight the role of this protein transport pathway in the maintenance of mitochondrial DNA stability and cell survival.     

Correlation between mRNA levels and functional role of alpha1-adrenoceptor subtypes in arteries: evidence of alpha1L as a functional isoform of the alpha1A-adrenoceptor

The mRNA levels for the three alpha1-adrenoceptor subtypes, alpha1A, alpha1B, and alpha1D, were quantified by real-time RT-PCR in arteries from Wistar rats. The alpha1D-adrenoceptor was prominent in both aorta (79.0%) and mesenteric artery (68.7%), alpha1A predominated in tail (61.7%) and small mesenteric artery (73.3%), and both alpha1A- and alpha1D-subtypes were expressed at similar levels in iliac artery. The mRNA levels of the alpha1B-subtype were a minority in all vessels (1.7-11.1%). Concentration-response curves of contraction in response to phenylephrine or relaxation in response to alpha1-adrenoceptor antagonists on maximal sustained contraction induced by phenylephrine were constructed from control vessels and vessels pretreated with 100 micromol/l chloroethylclonidine (CEC) for 30 min. The significant decrease in the phenylephrine potency observed after CEC treatment together with the inhibitory potency displayed by 8-{2-[4-(2-methoxyphenyl)-1-piperazinyl]-8-azaspiro (4,5) decane-7-dionedihydrochloride} (BMY-7378, an alpha1D-adrenoceptor antagonist) confirm the relevant role of alpha1D-adrenoceptors in aorta and iliac and proximal mesenteric arteries. The potency of 5-methylurapidil (an alpha1A-adrenoceptor antagonist) and the changes in the potency of both BMY-7378 and 5-methylurapidil after CEC treatment provided evidence of a mixed population of alpha1A- and alpha1D-adrenoceptors in iliac and distal mesenteric arteries. The low potency of prazosin (pIC50 < 9) as well as the high 5-methylurapidil potency in tail and small mesenteric arteries suggest the main role of alpha1A/alpha1L-adrenoceptors with minor participation of the alpha1D-subtype. The mRNA levels and CEC treatment corroborated this pattern and confirmed that the alpha1L-adrenoceptor could be a functional isoform of the alpha1A-subtype.     

The functional cooperation of MAP1A heavy chain and light chain 2 in the binding of microtubules

Microtubule-associated protein 1A (MAP1A) is a high-molecular-weight protein that is comprised of a heavy chain and a light chain (LC2) and is widely distributed along the microtubules in both mature neurons and glial cells. To illustrate the interaction among the MAP1A heavy chain, light chain, and microtubule, we prepared DNA constructs with Myc-, EGFP-, or DsRed-tags for full-length MAP1A DNA expressing whole MAP1A protein, two domains of MAP1A heavy chain, and light chain. Distribution patterns of various MAP1A domains as well as their interactions with microtubules were monitored in a non-neuronal COS7 and a neuronal Neuro2A cells. Our data revealed that a complete MAP1A protein, which contains both heavy chain and LC2, could be colocalized with microtubule networks not only in Neuro2A cells but also in transfected COS7 cells. Filamentous structures failed to be visualized along microtubules in COS7 cells transfected with MAP1A heavy chain or LC2 alone. Whereas, after introducing MAP1A heavy chain with LC2 into COS7 cells, both heavy chain and LC2 could be colocalized with microtubules. From our functional analysis, both MAP1A and its LC2 could protect microtubules against the challenge of nacodazol. Data collected from yeast two-hybrid assays of various MAP1A domains confirmed that the interaction of LC2 and NH2-terminal of MAP1A heavy chain is important for microtubule binding. From our analysis of MAP1A functional domains, we suggest that interactions between MAP1A heavy chain and LC2 are critical for the binding of microtubules.