Intercistronic Region Research Articles

Exploring the transcription start sites and other genomic features facilitates the accurate identification and annotation of small RNAs across multiple stress conditions in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTB) is a pathogen that is known for its ability to persist in harsh environments and cause chronic infections. Understanding the regulatory networks of MTB is crucial for developing effective treatments. Small regulatory RNAs (sRNAs) play important roles in gene expression regulation in all kingdoms of life, and their classification based solely on genomic location can be imprecise due to the computational-based prediction of protein-coding genes in bacteria, which often neglects segments of mRNA such as 5'UTRs, 3'UTRs, and intercistronic regions of operons. To address this issue, our study simultaneously discovered genomic features such as TSSs, UTRs, and operons together with sRNAs in the M. tuberculosis H37Rv strain (ATCC 27294) across multiple stress conditions. Our analysis identified 1,376 sRNA candidates and 8,173 TSSs in MTB, providing valuable insights into its complex regulatory landscape. TSS mapping enabled us to classify these sRNAs into more specific categories, including promoter-associated sRNAs, 5'UTR-derived sRNAs, 3'UTR-derived sRNAs, true intergenic sRNAs, and antisense sRNAs. Three of these sRNA candidates were experimentally validated using 3'-RACE-PCR: predictedRNA_0240, predictedRNA_0325, and predictedRNA_0578. Future characterization and validation are necessary to fully elucidate the functions and roles of these sRNAs in MTB. Our study is the first to simultaneously unravel TSSs and sRNAs in MTB and demonstrate that the identification of other genomic features, such as TSSs, UTRs, and operons, allows for more accurate and specific classification of sRNAs.

Differential Binding of Carbapenems with the AdeABC Efflux Pump and Modulation of the Expression of AdeB Linked to Novel Mutations within Two-Component System AdeRS in Carbapenem-Resistant Acinetobacter baumannii.

ABSTRACTResistance-nodulation-division-type efflux system AdeABC plays an important role in carbapenem resistance among Acinetobacter baumannii. However, a knowledge gap is observed regarding the role of its regulator AdeRS in carbapenem-resistant A. baumannii (CRAB). This study effectively combines microbiological analysis with an in-silico structural approach to understand the contribution of AdeRS among CRAB (n = 38). Additionally, molecular docking was performed for the first time to study the interaction of FDA-approved carbapenems and pump inhibitor PAβN with the open and closed structure of AdeB at the three binding sites (periplasmic, proximal, distal). It was observed that open conformation of AdeB facilitates the binding of carbapenems and PAβN at entrance and proximal sites compared to the closed conformation. PAβN was found to block carbapenem interacting residues in AdeB, establishing its role as a competitive inhibitor of AdeB substrates. Overexpression of AdeABC was detected by q-RT-PCR among 29% of CRABs, and several mutations within AdeS (GLY186VAL, SER188PHE, GLU121LYS, VAL255ILE) and AdeR (VAL120ILE, ALA136VAL) were detected by sequencing. The sequence and structure-based study of AdeRS was performed to analyze the probable effect of these mutations on regulation of the two-component system (TCS), especially, utilizing its three-dimensional structure. AdeS mutations inhibited the transfer of a phosphate group to AdeR, preventing the binding of AdeR to the intercistronic region, leading to overexpression of AdeABC. The elucidation of the role of mutations in AdeRS improves our understanding of TCS-based regulation. Identification of the key residues of AdeB interacting with carbapenems and PAβN may help in future designing of novel inhibitors.IMPORTANCE AdeABC is an important efflux pump in A. baumannii that plays a role in resistance toward different antibiotics including the “last resort” antibiotic, carbapenem. This pump is regulated by a two-component system, AdeRS. To understand the binding of carbapenems with AdeABC and pump inhibition by PAβN, we analyzed for the first time the possible atomic level interactions of carbapenems and PAβN with AdeB. In the current study, AdeRS-associated novel mutations in clinical A. baumannii are reported for the first time, and a sequence-structure based in-silico approach was used to interpret their role in AdeABC overexpression, leading to carbapenem resistance. None of the previous studies had undertaken both these aspects simultaneously. This study analyzes the open and closed conformation of AdeB, their binding with carbapenems, and key residues involved in it. This helps in visualizing the plausible atomic level causes of pump inhibition driving the discovery of novel inhibitors.

A rice mTERF protein V14 sustains photosynthesis establishment and temperature acclimation in early seedling leaves

BackgroundPlant mitochondrial transcription termination factor (mTERF) family members play important roles in development and stress tolerance through regulation of organellar gene expression. However, their molecular functions have yet to be clearly defined.ResultsHere an mTERF gene V14 was identified by fine mapping using a conditional albino mutant v14 that displayed albinism only in the first two true leaves, which was confirmed by transgenic complementation tests. Subcellular localization and real-time PCR analyses indicated that V14 encodes a chloroplastic protein ubiquitously expressed in leaves while spiking in the second true leaf. Chloroplastic gene expression profiling in the pale leaves of v14 through real-time PCR and Northern blotting analyses showed abnormal accumulation of the unprocessed transcripts covering the rpoB-rpoC1 and/or rpoC1-rpoC2 intercistronic regions accompanied by reduced abundance of the mature rpoC1 and rpoC2 transcripts, which encode two core subunits of the plastid-encoded plastid RNA polymerase (PEP). Subsequent immunoblotting analyses confirmed the reduced accumulation of RpoC1 and RpoC2. A light-inducible photosynthetic gene psbD was also found down-regulated at both the mRNA and protein levels. Interestingly, such stage-specific aberrant posttranscriptional regulation and psbD expression can be reversed by high temperatures (30 ~ 35 °C), although V14 expression lacks thermo-sensitivity. Meanwhile, three V14 homologous genes were found heat-inducible with similar temporal expression patterns, implicating their possible functional redundancy to V14.ConclusionsThese data revealed a critical role of V14 in chloroplast development, which impacts, in a stage-specific and thermo-sensitive way, the appropriate processing of rpoB-rpoC1-rpoC2 precursors and the expression of certain photosynthetic proteins. Our findings thus expand the knowledge of the molecular functions of rice mTERFs and suggest the contributions of plant mTERFs to photosynthesis establishment and temperature acclimation.

Exploring the genetic determinants underlying the differential production of an inducible chromosomal cephalosporinase - BlaB in Yersinia enterocolitica biotypes 1A, 1B, 2 and 4

Yersinia enterocolitica is an enteric bacterium which can cause severe gastroenteritis. Beta-lactams are the most widely used antibiotics against Y. enterocolitica. Y. enterocolitica produces two chromosomal β-lactamases, BlaA and BlaB. BlaB is an Ambler Class C inducible broad spectrum cephlaosporinase which showed differential enzyme activity in different biotypes of Y. enterocolitica. The expression of blaB is mainly regulated by ampR- the transcriptional regulator and, ampD - which helps in peptidoglycan recycling. The aim of this study was to identify and characterize genetic determinants underlying differential enzyme activity of BlaB in Y. enterocolitica biotypes 1 A, IB, 2 and 4. Thus, ampR, blaB and ampD were PCR-amplified and modeled in silico. The intercistronic region containing promoters of ampR and blaB was also investigated. Our results indicated that blaB was more inducible in biotypes 2 and 4, than in biotypes 1 A and 1B. Superimposition of in silico modeled proteins suggested that variations in amino acid sequences of AmpR, BlaB and AmpD were not responsible for hyper-production of BlaB in biotypes 2 and 4. Analysis of promoter regions of ampR and blaB revealed variations at −30, −37 and −58 positions from blaB transcription start site. Studies on relative expression levels of blaB in different biotypes by qRT-PCR indicated that nucleotide variations at these positions might contribute to a higher enzyme activity of BlaB in biotypes 2 and 4. However, this is a preliminary study and further studies including more strains of each biotype are required to strengthen our findings. Nevertheless, to the best of our knowledge, this is the first study which has investigated the genetic determinants underlying differential inducible production of BlaB in different biotypes of Y. enterocolitica.

Architecture of divergent flagellar promoters controlled by CtrA in Rhodobacter sphaeroides

BackgroundRhodobacter sphaeroides has two sets of flagellar genes, fla1 and fla2, that are responsible for the synthesis of two different flagellar structures. The expression of the fla2 genes is under control of CtrA. In several α-proteobacteria CtrA is also required for the expression of the flagellar genes, but the architecture of CtrA-dependent promoters has only been studied in detail in Caulobacter crescentus. In many cases the expression of fla genes originates from divergent promoters located a few base pairs apart, suggesting a particular arrangement of the cis-acting sites.ResultsHere we characterized several control regions of the R. sphaeroides fla2 genes and analyzed in detail two regions containing the divergent promoters flgB2p-fliI2p, and fliL2p-fliF2p. Binding sites for CtrA of these promoters were identified in silico and tested by site directed mutagenesis. We conclude that each one of these promoter regions has a particular arrangement, either a single CtrA binding site for activation of fliL2p and fliF2p, or two independent sites for activation of flgB2p and fliI2p. ChIP experiments confirmed that CtrA binds to the control region containing the flgB2 and fliI2 promoters, supporting the notion that CtrA directly controls the expression of the fla2 genes. The flgB and fliI genes are syntenic and show a short intercistronic region in closely related bacterial species. We analyzed these regions and found that the arrangement of the CtrA binding sites varies considerably.ConclusionsThe results in this work reveal the arrangement of the fla2 divergent promoters showing that CtrA promotes transcriptional activation using more than a single architecture.

Intercistronic expression elements (IEE) from the chloroplast of Chlamydomonas reinhardtii can be used for the expression of foreign genes in synthetic operons.

Two intercistronic regions were identified as functional intercistronic expression elements (IEE) for the simultaneous expression of aphA-6 and gfp in a synthetic operon in the chloroplast of C. reinhardtii. Chlamydomonas reinhardtii, a biflagellate photosynthetic microalga, has been widely used in basic and applied science. Already three decades ago, Chlamydomonas had its chloroplast genome transformed and to this day constitutes the only alga routinely used in transplastomic technology. Despite the fact that over a 100 foreign genes have been expressed from the chloroplast genome, little has been done to address the challenge of expressing multiple genes in the form of operons, a development that is needed and crucial to push forward metabolic engineering and synthetic biology in this organism. Here, we studied five intercistronic regions and investigated if they can be used as intercistronic expression elements (IEE) in synthetic operons to drive the expression of foreign genes in the chloroplast of C. reinhardtii. The intercistronic regions were those from the psbB-psbT, psbN-psbH, psaC-petL, petL-trnN and tscA-chlN chloroplast operons, and the foreign genes were the aminoglycoside 3'-phosphotransferase (aphA-6), which confers resistance to kanamycin, and the green fluorescent protein gene (gfp). While all the intercistronic regions yielded lines that were resistant to kanamycin, only two (obtained with intercistronic regions from psbN-psbH and tscA-chlN) were identified as functional IEEs, yielding lines in which the second cistron (gfp) was translated and generated GFP. The IEEs we have identified could be useful for the stacking of genes for metabolic engineering or synthetic biology circuits in the chloroplast of C. reinhardtii.

Unexpected functional versatility of the pentatricopeptide repeat proteins PGR3, PPR5 and PPR10.

Pentatricopeptide repeat (PPR) proteins are a large family of helical repeat proteins that bind RNA in mitochondria and chloroplasts. Sites of PPR action have been inferred primarily from genetic data, which have led to the view that most PPR proteins act at a very small number of sites in vivo. Here, we report new functions for three chloroplast PPR proteins that had already been studied in depth. Maize PPR5, previously shown to promote trnG splicing, is also required for rpl16 splicing. Maize PPR10, previously shown to bind the atpI-atpH and psaJ-rpl33 intercistronic regions, also stabilizes a 3′-end downstream from psaI. Arabidopsis PGR3, shown previously to bind upstream of petL, also binds the rpl14-rps8 intercistronic region where it stabilizes a 3′-end and stimulates rps8 translation. These functions of PGR3 are conserved in maize. The discovery of new functions for three proteins that were already among the best characterized members of the PPR family implies that functional repertoires of PPR proteins are more complex than have been appreciated. The diversity of sequences bound by PPR10 and PGR3 in vivo highlights challenges of predicting binding sites of native PPR proteins based on the amino acid code for nucleotide recognition by PPR motifs.

Mechanistic insight into how multidrug resistant Acinetobacter baumannii response regulator AdeR recognizes an intercistronic region.

AdeR–AdeS is a two-component regulatory system, which controls expression of the adeABC efflux pump involved in Acinetobacter baumannii multidrug resistance. AdeR is a response regulator consisting of an N-terminal receiver domain and a C-terminal DNA-binding-domain. AdeR binds to a direct-repeat DNA in the intercistronic region between adeR and adeABC. We demonstrate a markedly high affinity binding between unphosphorylated AdeR and DNA with a dissociation constant of 20 nM. In addition, we provide a 2.75 Å crystal structure of AdeR DNA-binding-domain complexed with the intercistronic DNA. This structure shows that the α3 and β hairpin formed by β5–β6 interacts with the major and minor groove of the DNA, which in turn leads to the introduction of a bend. The AdeR receiver domain structure revealed a dimerization motif mediated by a gearwheel-like structure involving the D108F109-R122 motif through cation π stack interaction. The structure of AdeR receiver domain bound with magnesium indicated a conserved Glu19Asp20-Asp63 magnesium-binding motif, and revealed that the potential phosphorylation site Asp63OD1 forms a hydrogen bond with Lys112. We thus dissected the mechanism of how AdeR recognizes the intercistronic DNA, which leads to a diverse mode of response regulation. Unlocking the AdeRS mechanism provides ways to circumvent A. baumannii antibiotic resistance.

XRN2 Autoregulation and Control of Polycistronic Gene Expresssion in Caenorhabditis elegans.

XRN2 is a conserved 5’→3’ exoribonuclease that complexes with proteins that contain XRN2-binding domains (XTBDs). In Caenorhabditis elegans (C. elegans), the XTBD-protein PAXT-1 stabilizes XRN2 to retain its activity. XRN2 activity is also promoted by 3'(2'),5'-bisphosphate nucleotidase 1 (BPNT1) through hydrolysis of an endogenous XRN inhibitor 3’-phosphoadenosine-5'-phosphate (PAP). Here, we find through unbiased screening that loss of bpnt-1 function suppresses lethality caused by paxt-1 deletion. This unexpected finding is explained by XRN2 autoregulation, which occurs through repression of a cryptic promoter activity and destabilization of the xrn-2 transcript. De-repression appears to be triggered such that more robust XRN2 perturbation, by elimination of both PAXT-1 and BPNT1, is less detrimental to worm viability than absence of PAXT-1 alone. Indeed, we find that two distinct XRN2 repression mechanisms are alleviated at different thresholds of XRN2 inactivation. Like more than 15% of C. elegans genes, xrn-2 occurs in an operon, and we identify additional operons under its control, consistent with a broader function of XRN2 in polycistronic gene regulation. Regulation occurs through intercistronic regions that link genes in an operon, but a part of the mechanisms may allow XRN2 to operate on monocistronic genes in organisms lacking operons.

A novel set of vectors for Fur-controlled protein expression under iron deprivation in Escherichia coli

BackgroundIn the presence of sufficient iron, the Escherichia coli protein Fur (Ferric Uptake Regulator) represses genes controlled by the Fur box, a consensus sequence near or within promoters of target genes. De-repression of Fur-controlled genes occurs upon iron deprivation. In the E. coli chromosome, there is a bidirectional intercistronic promoter region with two non-overlapping Fur boxes. This region controls Fur-regulated expression of entCEBAH in the clockwise direction and fepB in the anticlockwise direction.ResultsWe cloned the E. coli bidirectional fepB/entC promoter region into low-copy-number plasmid backbones (pACYC184 and pBR322) along with downstream sequences encoding epitope tags and a multiple cloning site (MCS) compatible with the bacterial adenylate cyclase two-hybrid (BACTH) system. The vector pFCF1 allows for iron-controlled expression of FLAG-tagged proteins, whereas the pFBH1 vector allows for iron-controlled expression of HA-tagged proteins. We showed that E. coli knockout strains transformed with pFCF1-entA, pFCF1-entE and pFBH1-entB express corresponding proteins with appropriate epitope tags when grown under iron restriction. Furthermore, transformants exhibited positive chrome azurol S (CAS) assay signals under iron deprivation, indicating that the transformants were functional for siderophore biosynthesis. Western blotting and growth studies in rich and iron-depleted media demonstrated that protein expression from these plasmids was under iron control. Finally, we produced the vector pFCF2, a pFCF1 derivative in which a kanamycin resistance (KanR) gene was engineered in the direction opposite of the MCS. The entA ORF was then subcloned into the pFCF2 MCS. Bidirectional protein expression in an iron-deprived pFCF2-entA transformant was confirmed using antibiotic selection, CAS assays and growth studies.ConclusionsThe vectors pFCF1, pFCF2, and pFBH1 have been shown to use the fepB/entC promoter region to control bidirectional in trans expression of epitope-tagged proteins in iron-depleted transformants. In the presence of intracellular iron, protein expression from these constructs was abrogated due to Fur repression. The compatibility of the pFCF1 and pFBH1 backbones allows for iron-controlled expression of multiple epitope-tagged proteins from a single co-transformant.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0298-1) contains supplementary material, which is available to authorized users.

Insights into the Regulation of mRNA Processing of Polycistronic Transcripts Mediated by DRBD4/PTB2, a Trypanosome Homolog of the Polypyrimidine Tract-Binding Protein.

Trypanosomes regulate gene expression mostly by posttranscriptional mechanisms, including control of mRNA turnover and translation efficiency. This regulation is carried out via certain elements located at the 3'-untranslated regions of mRNAs, which are recognized by RNA-binding proteins. In trypanosomes, trans-splicing is of central importance to control mRNA maturation. We have previously shown that TcDRBD4/PTB2, a trypanosome homolog of the human polypyrimidine tract-binding protein splicing regulator, interacts with the intergenic region of one specific dicistronic transcript, referred to as TcUBP (and encoding for TcUBP1 and TcUBP2, two closely kinetoplastid-specific proteins). In this work, a survey of TcUBP RNA processing revealed certain TcDRBD4/PTB2-regulatory elements within its intercistronic region, which are likely to influence the trans-splicing rate of monocistronic-derived transcripts. Furthermore, TcDRBD4/PTB2 overexpression in epimastigote cells notably decreased both UBP1 and UBP2 protein expression. This type of posttranscriptional gene regulatory mechanism could be extended to other transcripts as well, as we identified several other RNA precursor molecules that specifically bind to TcDRBD4/PTB2. Altogether, these findings support a model in which TcDRBD4/PTB2-containing ribonucleoprotein complexes can prevent trans-splicing. This could represent another stage of gene expression regulation mediated by the masking of trans-splicing/polyadenylation signals.

Virus-induced gene silencing (VIGS) in Lilium leichtlinii using the Cucumber mosaic virus vector.

Lilies (Lilium) are among the most important floriculture crops, and to accelerate research regarding lily genetics, the development of reverse-genetics tools is necessary. However, Agrobacterium-mediated transformation in Lilium is time-consuming, since the plants require several years to progress from acclimation to flowering. Thus, virus-induced gene silencing (VIGS) is an attractive method for assaying gene function. In the present study, we modified a lily-derived strain of Cucumber mosaic virus (CMV-HL) as a VIGS vector and evaluated its effectiveness for inducing gene silencing in Lilium leichtlinii by introducing L. leichtlinii phytoene desaturase (LlPDS) gene fragments into an intercistronic region between the 3a and 3b genes of the CMV-HL RNA3 genome. At 30 days after inoculation (dpi) with LlPDS-containing CMV-HL, photo-bleaching was observed in the upper leaves of L. leichtlinii, and at 57 dpi, we observed that the natural orange color in flower tepals had faded. Reduced LlPDS expression and the detection of small interfering LlPDS RNA indicated that the color changes were the result of LlPDS gene silencing. In addition, the leaves also exhibited a mild photo-bleaching phenotype in the following year. Therefore, our results indicate that CMV-HL spreads systemically in the leaves and flowers of Lilium during the first year of infection, as well as in new shoots during the following year, and that the vector system can be successfully applied to induce short-term endogenous gene silencing in lilies.

Operons are a conserved feature of nematode genomes.

The organization of genes into operons, clusters of genes that are co-transcribed to produce polycistronic pre-mRNAs, is a trait found in a wide range of eukaryotic groups, including multiple animal phyla. Operons are present in the class Chromadorea, one of the two main nematode classes, but their distribution in the other class, the Enoplea, is not known. We have surveyed the genomes of Trichinella spiralis, Trichuris muris, and Romanomermis culicivorax and identified the first putative operons in members of the Enoplea. Consistent with the mechanism of polycistronic RNA resolution in other nematodes, the mRNAs produced by genes downstream of the first gene in the T. spiralis and T. muris operons are trans-spliced to spliced leader RNAs, and we are able to detect polycistronic RNAs derived from these operons. Importantly, a putative intercistronic region from one of these potential enoplean operons confers polycistronic processing activity when expressed as part of a chimeric operon in Caenorhabditis elegans. We find that T. spiralis genes located in operons have an increased likelihood of having operonic C. elegans homologs. However, operon structure in terms of synteny and gene content is not tightly conserved between the two taxa, consistent with models of operon evolution. We have nevertheless identified putative operons conserved between Enoplea and Chromadorea. Our data suggest that operons and “spliced leader” (SL) trans-splicing predate the radiation of the nematode phylum, an inference which is supported by the phylogenetic profile of proteins known to be involved in nematode SL trans-splicing.

Regulation of Expression of abcA and Its Response to Environmental Conditions

The ATP-dependent transporter gene abcA in Staphylococcus aureus confers resistance to hydrophobic β-lactams. In strain ISP794, abcA is regulated by the transcriptional regulators MgrA and NorG and shares a 420-nucleotide intercistronic region with the divergently transcribed pbp4 gene, which encodes the transpeptidase Pbp4. Exposure of exponentially growing cells to iron-limited media, oxidative stress, and acidic pH (5.5) for 0.5 to 2 h had no effect on abcA expression. In contrast, nutrient limitation produced a significant increase in abcA transcripts. We identified three additional regulators (SarA, SarZ, and Rot) that bind to the overlapping promoter region of abcA and pbp4 in strain MW2 and investigated their role in the regulation of abcA expression. Expression of abcA is decreased by 10.0-fold in vivo in a subcutaneous abscess model. In vitro, abcA expression depends on rot and sarZ regulators. Moenomycin A exposure of strain MW2 produced an increase in abcA transcripts. Relative to MW2, the MIC of moenomycin was decreased 8-fold for MW2ΔabcA and increased 10-fold for the MW2 abcA overexpresser, suggesting that moenomycin is a substrate of AbcA.

Mapping of internal ribosome entry sites (IRES).

The dicistronic luciferase reporter gene system is the most common method to isolate and characterize internal ribosome entry sites (IRES). It is based on the expression of a dicistronic RNA comprising two independent reporter genes in 3' and 5'cistrons, and the putative IRES inserted into intercistronic region. The most convenient aspect of using Renilla and firefly luciferase genes is that both gene products can be detected in a single assay using Dual-Luciferase(®) Reporter Assay System from Promega. The Renilla luciferase coding sequence is often inserted into the 5'cistron and serves as internal control. It is translated cap dependently, as it is close to the cap structure at the 5' end. The 3'cistron located far downstream to the cap structure can only be translated by a cap-independent mechanism when the intercistronic sequence is capable of ribosome binding and re-initiation of translation. Expression level of the 3'cistron is usually normalized to the expression of 5'cistron to estimate the relative IRES activity of intercistronic sequences.

The AfaR small RNA controls expression of the AfaD-VIII invasin in pathogenic Escherichia coli strains

Pathogenic Escherichia coli strains carrying the afa-8 gene cluster are frequently associated with extra-intestinal infections in humans and animals. The afa-8 A to E genes determine the formation of an afimbrial adhesive sheath consisting of the AfaD-VIII invasin and the AfaE-VIII adhesin at the bacterial cell surface. This structure is thought to be required for host colonization. We characterized a new gene encoding the small RNA AfaR, which is transcribed in cis from the complementary strand of the 3′ untranslated region of the afaD messenger RNA, within the afaD–afaE intercistronic region. AfaR is a trans-acting Hfq-dependent antisense small RNA that binds the 5′ untranslated region of the afaD messenger RNA, initiating several ribonuclease E-dependent cleavages, thereby downregulating production of the AfaD-VIII invasin. AfaR transcription is dependent on σE, a member of the stress response family of extracytoplasmic alternative sigma factors. We found that the AfaR-dependent regulatory pathway was controlled by temperature, allowing the production of the AfaD-VIII invasin at temperatures above 37°C. Our findings suggest that the entry of afa-8-positive pathogenic E. coli strains into epithelial cells is tightly regulated by the AfaR small RNA.

Complex RNA metabolism in the chloroplast: an update on the psbB operon

Expression of most plastid genes involves multiple post-transcriptional processing events, such as splicing, editing, and intercistronic processing. The latter involves the formation of mono-, di-, and multicistronic transcripts, which can further be regulated by differential stability and expression. The plastid pentacistronic psbB transcription unit has been well characterized in vascular plants. It encodes the subunits CP47 (psbB), T (psbT), and H (psbH) of photosystem II as well as cytochrome b 6 (petB) and subunit IV (petD) of the cytochrome b 6 f complex. Each of the petB and petD genes contains a group II intron, which is spliced during post-transcriptional modification. The small subunit of photosystem II, PsbN, is encoded in the intercistronic region between psbH and psbT but is transcribed in the opposite direction. Expression of the psbB gene cluster necessitates different processing events along with numerous newly evolved specificity factors conferring stability to many of the processed RNA transcripts, and thus exemplarily shows the complexity of RNA metabolism in the chloroplast.

Translation Initiation on mRNAs Bound by Nuclear Cap-binding Protein Complex CBP80/20 Requires Interaction between CBP80/20-dependent Translation Initiation Factor and Eukaryotic Translation Initiation Factor 3g

In the cytoplasm of mammalian cells, either cap-binding proteins 80 and 20 (CBP80/20) or eukaryotic translation initiation factor (eIF) 4E can direct the initiation of translation. Although the recruitment of ribosomes to mRNAs during eIF4E-dependent translation (ET) is well characterized, the molecular mechanism for CBP80/20-dependent translation (CT) remains obscure. Here, we show that CBP80/20-dependent translation initiation factor (CTIF), which has been shown to be preferentially involved in CT but not ET, specifically interacts with eIF3g, a component of the eIF3 complex involved in ribosome recruitment. By interacting with eIF3g, CTIF serves as an adaptor protein to bridge the CBP80/20 and the eIF3 complex, leading to efficient ribosome recruitment during CT. Accordingly, down-regulation of CTIF using a small interfering RNA causes a redistribution of CBP80 from polysome fractions to subpolysome fractions, without significant consequence to eIF4E distribution. In addition, down-regulation of eIF3g inhibits the efficiency of nonsense-mediated mRNA decay, which is tightly coupled to CT but not to ET. Moreover, the artificial tethering of CTIF to an intercistronic region of dicistronic mRNA results in translation of the downstream cistron in an eIF3-dependent manner. These findings support the idea that CT mechanistically differs from ET.

Protein-mediated protection as the predominant mechanism for defining processed mRNA termini in land plant chloroplasts.

Most chloroplast mRNAs are processed from larger precursors. Several mechanisms have been proposed to mediate these processing events, including site-specific cleavage and the stalling of exonucleases by RNA structures. A protein barrier mechanism was proposed based on analysis of the pentatricopeptide repeat (PPR) protein PPR10: PPR10 binds two intercistronic regions and impedes 5′- and 3′-exonucleases, resulting in processed RNAs with PPR10 bound at the 5′- or 3′-end. In this study, we provide evidence that protein barriers are the predominant means for defining processed mRNA termini in chloroplasts. First, we map additional RNA termini whose arrangement suggests biogenesis via a PPR10-like mechanism. Second, we show that the PPR protein HCF152 binds to the immediate 5′- or 3′-termini of transcripts that require HCF152 for their accumulation, providing evidence that HCF152 defines RNA termini by blocking exonucleases. Finally, we build on the observation that the PPR10 and HCF152 binding sites accumulate as small chloroplast RNAs to infer binding sites of other PPR proteins. We show that most processed mRNA termini are represented by small RNAs whose sequences are highly conserved. We suggest that each such small RNA is the footprint of a PPR-like protein that protects the adjacent RNA from degradation.

A Novel Borna Disease Virus Vector System That Stably Expresses Foreign Proteins from an Intercistronic Noncoding Region

Borna disease virus (BDV), a nonsegmented, negative-strand RNA virus, infects a wide variety of mammalian species and readily establishes a long-lasting, persistent infection in brain cells. Therefore, this virus could be a promising candidate as a novel RNA virus vector enabling stable gene expression in the central nervous system (CNS). Previous studies demonstrated that the 5' untranslated region of the genome is the only site for insertion and expression of a foreign gene. In this study, we established a novel BDV vector in which an additional transcription cassette has been inserted into an intercistronic noncoding region between the viral phosphoprotein (P) and matrix (M) genes. The recombinant BDV (rBDV) carrying green fluorescent protein (GFP) between the P and M genes, rBDV P/M-GFP, expressed GFP efficiently in cultured cells and rodent brains for a long period of time without attenuation. Furthermore, we generated a nonpropagating rBDV, ΔGLLP/M, which lacks the envelope glycoprotein (G) and a splicing intron within the polymerase gene (L), by the transcomplementation system with either transient or stable expression of the G gene. Interestingly, rBDV ΔGLLP/M established a persistent infection in cultured cells with stable expression of GFP in the absence of the expression of G. Using persistently infected rBDV ΔGLLP/M-infected cells, we determined the amino acid region in the cytoplasmic tail (CT) of BDV G important for the release of infectious rBDV particles and also demonstrated that the CT region may be critical for the generation of pseudotyped rBDV having vesicular stomatitis virus G protein. Our results revealed that the newly established BDV vector constitutes an alternative tool not only for stable expression of foreign genes in the CNS but also for understanding the mechanism of the release of enveloped virions.