Intragenic Transcription Research Articles

RNAseq analysis of \u03b1-proteobacterium Gluconobacter oxydans 621H

BackgroundThe acetic acid bacterium Gluconobacter oxydans 621H is characterized by its exceptional ability to incompletely oxidize a great variety of carbohydrates in the periplasm. The metabolism of this α-proteobacterium has been characterized to some extent, yet little is known about its transcriptomes and related data. In this study, we applied two different RNAseq approaches. Primary transcriptomes enriched for 5′-ends of transcripts were sequenced to detect transcription start sites, which allow subsequent analysis of promoter motifs, ribosome binding sites, and 5´-UTRs. Whole transcriptomes were sequenced to identify expressed genes and operon structures.ResultsSequencing of primary transcriptomes of G. oxydans revealed 2449 TSSs, which were classified according to their genomic context followed by identification of promoter and ribosome binding site motifs, analysis of 5´-UTRs including validation of predicted cis-regulatory elements and correction of start codons. 1144 (41%) of all genes were found to be expressed monocistronically, whereas 1634 genes were organized in 571 operons. Together, TSSs and whole transcriptome data were also used to identify novel intergenic (18), intragenic (328), and antisense transcripts (313).ConclusionsThis study provides deep insights into the transcriptional landscapes of G. oxydans. The comprehensive transcriptome data, which we made publicly available, facilitate further analysis of promoters and other regulatory elements. This will support future approaches for rational strain development and targeted gene expression in G. oxydans. The corrections of start codons further improve the high quality genome reference and support future proteome analysis.

Analysis of primary microRNA loci from nascent transcriptomes reveals regulatory domains governed by chromatin architecture.

Changes in mature microRNA (miRNA) levels that occur downstream of signaling cascades play an important role during human development and disease. However, the regulation of primary microRNA (pri-miRNA) genes remains to be dissected in detail. To address this, we followed a data-driven approach and developed a transcript identification, validation and quantification pipeline for characterizing the regulatory domains of pri-miRNAs. Integration of 92 nascent transcriptomes and multilevel data from cells arising from ecto-, endo- and mesoderm lineages reveals cell type-specific expression patterns, allows fine-resolution mapping of transcription start sites (TSS) and identification of candidate regulatory regions. We show that inter- and intragenic pri-miRNA transcripts span vast genomic regions and active TSS locations differ across cell types, exemplified by the mir-29a∼29b-1, mir-100∼let-7a-2∼125b-1 and miR-221∼222 clusters. Considering the presence of multiple TSS as an important regulatory feature at miRNA loci, we developed a strategy to quantify differential TSS usage. We demonstrate that the TSS activities associate with cell type-specific super-enhancers, differential stimulus responsiveness and higher-order chromatin structure. These results pave the way for building detailed regulatory maps of miRNA loci.

Intragenic Enhancers Attenuate Host Gene Expression

Eukaryotic gene transcription is regulated at many steps, including RNA polymerase II (Pol II) recruitment, transcription initiation, promoter-proximal Pol II pause release, and transcription termination; however, mechanisms regulating transcription during productive elongation remain poorly understood. Enhancers, which activate gene transcription, themselves undergo Pol II-mediated transcription, but our understanding of enhancer transcription and enhancer RNAs (eRNAs) remains incomplete. Here we show that transcription at intragenic enhancers interferes with and attenuates host gene transcription during productive elongation. While the extent of attenuation correlates positively with nascent eRNA expression, the act of intragenic enhancer transcription alone, but not eRNAs, explains the attenuation. Through CRISPR/Cas9-mediated deletions, we demonstrate a physiological role for intragenic enhancer-mediated transcription attenuation in cell fate determination. We propose that intragenic enhancers not only enhance transcription of one or more genes from a distance but also fine-tune transcription of their host gene through transcription interference, facilitating differential utilization of the same regulatory element for disparate functions.

C-terminal BRE overexpression in 11q23-rearranged and t(8;16) acute myeloid leukemia is caused by intragenic transcription initiation.

Overexpression of the BRE (brain and reproductive organ-expressed) gene defines a distinct pediatric and adult acute myeloid leukemia (AML) subgroup. Here we identify a promoter enriched for active chromatin marks in BRE intron 4 causing strong biallelic expression of a previously unknown C-terminal BRE transcript. This transcript starts with BRE intron 4 sequences spliced to exon 5 and downstream sequences, and if translated might code for an N terminally truncated BRE protein. Remarkably, the new BRE transcript was highly expressed in over 50% of 11q23/KMT2A (lysine methyl transferase 2A)-rearranged and t(8;16)/KAT6A-CREBBP cases, while it was virtually absent from other AML subsets and normal tissues. In gene reporter assays, the leukemia-specific fusion protein KMT2A-MLLT3 transactivated the intragenic BRE promoter. Further epigenome analyses revealed 97 additional intragenic promoter marks frequently bound by KMT2A in AML with C-terminal BRE expression. The corresponding genes may be part of a context-dependent KMT2A-MLLT3-driven oncogenic program, because they were higher expressed in this AML subtype compared with other groups. C-terminal BRE might be an important contributor to this program because in a case with relapsed AML, we observed an ins(11;2) fusing CHORDC1 to BRE at the region where intragenic transcription starts in KMT2A-rearranged and KAT6A-CREBBP AML.

Analysis of primary microRNA loci from nascent transcriptomes reveals regulatory domains governed by chromatin architecture.

Changes in mature microRNA (miRNA) levels that occur downstream of signaling cascades play an important role during human development and disease. However, the regulation of primary microRNA (pri-miRNA) genes remains to be dissected in detail. To address this, we followed a data-driven approach and developed a transcript identification, validation and quantification pipeline for characterizing the regulatory domains of pri-miRNAs. Integration of 92 nascent transcriptomes and multilevel data from cells arising from ecto-, endo- and mesoderm lineages reveals cell type-specific expression patterns, allows fine-resolution mapping of transcription start sites (TSS) and identification of candidate regulatory regions. We show that inter- and intragenic pri-miRNA transcripts span vast genomic regions and active TSS locations differ across cell types, exemplified by the mir-29a∼29b-1, mir-100∼let-7a-2∼125b-1 and miR-221∼222 clusters. Considering the presence of multiple TSS as an important regulatory feature at miRNA loci, we developed a strategy to quantify differential TSS usage. We demonstrate that the TSS activities associate with cell type-specific super-enhancers, differential stimulus responsiveness and higher-order chromatin structure. These results pave the way for building detailed regulatory maps of miRNA loci.

Termination factor Rho: From the control of pervasive transcription to cell fate determination in Bacillus subtilis

In eukaryotes, RNA species originating from pervasive transcription are regulators of various cellular processes, from the expression of individual genes to the control of cellular development and oncogenesis. In prokaryotes, the function of pervasive transcription and its output on cell physiology is still unknown. Most bacteria possess termination factor Rho, which represses pervasive, mostly antisense, transcription. Here, we investigate the biological significance of Rho-controlled transcription in the Gram-positive model bacterium Bacillus subtilis. Rho inactivation strongly affected gene expression in B. subtilis, as assessed by transcriptome and proteome analysis of a rho–null mutant during exponential growth in rich medium. Subsequent physiological analyses demonstrated that a considerable part of Rho-controlled transcription is connected to balanced regulation of three mutually exclusive differentiation programs: cell motility, biofilm formation, and sporulation. In the absence of Rho, several up-regulated sense and antisense transcripts affect key structural and regulatory elements of these differentiation programs, thereby suppressing motility and biofilm formation and stimulating sporulation. We dissected how Rho is involved in the activity of the cell fate decision-making network, centered on the master regulator Spo0A. We also revealed a novel regulatory mechanism of Spo0A activation through Rho-dependent intragenic transcription termination of the protein kinase kinB gene. Altogether, our findings indicate that distinct Rho-controlled transcripts are functional and constitute a previously unknown built-in module for the control of cell differentiation in B. subtilis. In a broader context, our results highlight the recruitment of the termination factor Rho, for which the conserved biological role is probably to repress pervasive transcription, in highly integrated, bacterium-specific, regulatory networks.

Genome-wide determination of transcription start sites reveals new insights into promoter structures in the actinomycete Corynebacterium glutamicum

The genome-wide identification of transcription start sites, enabled by high-throughput sequencing of a cDNA library enriched for native 5′ transcript ends, is ideally suited for the analysis of promoters. Here, the transcriptome of Corynebacterium glutamicum, a non-pathogenic soil bacterium from the actinomycetes branch that is used in industry for the production of amino acids, was analysed by transcriptome sequencing of the 5′-ends of native transcripts. Total RNA samples were harvested from the exponential phase of growth, therefore the study mainly addressed promoters recognized by the main house-keeping sigma factor σA. The identification of 2454 transcription start sites (TSS) allowed the detailed analysis of most promoters recognized by σA and furthermore enabled us to form different promoter groups according to their location relative to protein-coding regions. These groups included leaderless transcripts (546 promoters), short-leadered (<500 bases) transcripts (917), and long-leadered (>500 bases) transcripts (173) as well as intragenic (557) and antisense transcripts (261). All promoters and the individual groups were searched for information, e.g. conserved residues and promoter motifs, and general design features as well as group-specific preferences were identified. A purine was found highly favored as TSS, whereas the −1 position was dominated by pyrimidines. The spacer between TSS and ‐10 region were consistently 6–7 bases and the −10 promoter motif was generally visible, whereas a recognizable −35 region was only occurring in a smaller fraction of promoters (7.5%) and enriched for leadered and antisense transcripts but depleted for leaderless transcripts. Promoters showing an extended −10 region were especially frequent in case of non-canonical −10 motifs (45.5%). Two bases downstream of the −10 core region, a G was conserved, exceeding 40% abundance in most groups. This fraction reached 74.6% for a group of putative σB-dependent promoters, thus giving a hint to a specific property of these promoters. In addition, the high number of promoters analysed allowed finding of subtle signals only showing up significantly with this large set. This included the observation of a periodically changing A+T-content with maxima spaced by a full turn of the DNA helix. This periodic structure includes the A+T-rich UP-element of bacterial promoters known before but was found to extend up to −100, indicating hitherto unknown constraints influencing promoter architecture and possibly also promoter function.

Bidirectional terminators in Saccharomyces cerevisiae prevent cryptic transcription from invading neighboring genes

Transcription can be quite disruptive for chromatin so cells have evolved mechanisms to preserve chromatin integrity during transcription, thereby preventing the emergence of cryptic transcripts from spurious promoter sequences. How these transcripts are regulated and processed remains poorly characterized. Notably, very little is known about the termination of cryptic transcripts. Here, we used RNA-Seq to identify and characterize cryptic transcripts in Spt6 mutant cells (spt6-1004) in Saccharomyces cerevisiae. We found polyadenylated cryptic transcripts running both sense and antisense relative to genes in this mutant. Cryptic promoters were enriched for TATA boxes, suggesting that the underlying DNA sequence defines the location of cryptic promoters. While intragenic sense cryptic transcripts terminate at the terminator of the genes that host them, we found that antisense cryptic transcripts preferentially terminate near the 3΄-end of the upstream gene. This finding led us to demonstrate that most terminators in yeast are bidirectional, leading to termination and polyadenylation of transcripts coming from both directions. We propose that S. cerevisiae has evolved this mechanism in order to prevent/attenuate spurious transcription from invading neighbouring genes, a feature that is particularly critical for organisms with small compact genomes.

DNA Methylation in Cancer Tissues

Cancer linked DNA hypo-methylation and hyper-methylation are present throughout the human genome. The hyper-methylation facilitates cancer progress by repressing the tumor suppressor gene. Hypo-methylation contribution towards cancer has not yet been clear. Recent studies of tissue specific methylation have suggested that DNA hypo-methylation aid tumor formation by many pathways. Loss of DNA methylation associated with cancer may alter transcription. In addition, DNA hypo-methylation might affect promoter usage production of intra-genic non-coding RNA transcripts, co-transcriptional splicing and initiation and elongation of transcription. Studies of hemi methylation of DNA in cancerous cells as well as normal tissues suggest that active de-methylation can explain cancer associated DNA hypo-methylation. New studies that genomic 5-hydroxymethylcytosine is intermediate in DNA de-methylation exhibits cancer associated losses. It suggests that both decreased hydroxyl-methylation and methylation of DNA play important role in carcinogenesis.

In vivo expression technology and 5' end mapping of the Borrelia burgdorferi transcriptome identify novel RNAs expressed during mammalian infection.

Borrelia burgdorferi, the bacterial pathogen responsible for Lyme disease, modulates its gene expression profile in response to the environments encountered throughout its tick-mammal infectious cycle. To begin to characterize the B. burgdorferi transcriptome during murine infection, we previously employed an in vivo expression technology-based approach (BbIVET). This identified 233 putative promoters, many of which mapped to un-annotated regions of the complex, segmented genome. Herein, we globally identify the 5′ end transcriptome of B. burgdorferi grown in culture as a means to validate non-ORF associated promoters discovered through BbIVET. We demonstrate that 119 BbIVET promoters are associated with transcription start sites (TSSs) and validate novel RNA transcripts using Northern blots and luciferase promoter fusions. Strikingly, 49% of BbIVET promoters were not found to associate with TSSs. This finding suggests that these sequences may be primarily active in the mammalian host. Furthermore, characterization of the 6042 B. burgdorferi TSSs reveals a variety of RNAs including numerous antisense and intragenic transcripts, leaderless RNAs, long untranslated regions and a unique nucleotide frequency for initiating intragenic transcription. Collectively, this is the first comprehensive map of TSSs in B. burgdorferi and characterization of previously un-annotated RNA transcripts expressed by the spirochete during murine infection.

Relationships Between RNA Polymerase II Activity and Spt Elongation Factors to Spt- Phenotype and Growth in Saccharomyces cerevisiae.

The interplay between adjacent transcription units can result in transcription-dependent alterations in chromatin structure or recruitment of factors that determine transcription outcomes, including the generation of intragenic or other cryptic transcripts derived from cryptic promoters. Mutations in a number of genes in Saccharomyces cerevisiae confer both cryptic intragenic transcription and the Suppressor of Ty (Spt-) phenotype for the lys2-128∂ allele of the LYS2 gene. Mutants that suppress lys2-128∂ allow transcription from a normally inactive Ty1 ∂ promoter, conferring a LYS+ phenotype. The arrangement of transcription units at lys2-128∂ is reminiscent of genes containing cryptic promoters within their open reading frames. We set out to examine the relationship between RNA Polymerase II (Pol II) activity, functions of Spt elongation factors, and cryptic transcription because of the previous observation that increased-activity Pol II alleles confer an Spt- phenotype. We identify both cooperating and antagonistic genetic interactions between Pol II alleles and alleles of elongation factors SPT4, SPT5, and SPT6. We find that cryptic transcription at FLO8 and STE11 is distinct from that at lys2-128∂, though all show sensitivity to reduction in Pol II activity, especially the expression of lys2-128∂ found in Spt- mutants. We determine that the lys2-128∂ Spt- phenotypes for spt6-1004 and increased activity rpo21/rpb1 alleles each require transcription from the LYS2 promoter. Furthermore, we identify the Ty1 transcription start site (TSS) within the ∂ element as the position of Spt- transcription in tested Spt- mutants.

Riboregulation of bacterial and archaeal transposition.

The coexistence of transposons with their hosts depends largely on transposition levels being tightly regulated to limit the mutagenic burden associated with frequent transposition. For 'DNA-based' (class II) bacterial transposons there is growing evidence that regulation through small noncoding RNAs and/or the RNA-binding protein Hfq are prominent mechanisms of defense against transposition. Recent transcriptomics analyses have identified many new cases of antisense RNAs (asRNA) that potentially could regulate the expression of transposon-encoded genes giving the impression that asRNA regulation of DNA-based transposons is much more frequent than previously thought. Hfq is a highly conserved bacterial protein that plays a central role in posttranscriptional gene regulation and stress response pathways in many bacteria. Three different mechanisms for Hfq-directed control of bacterial transposons have been identified to date highlighting the versatility of this protein as a regulator of bacterial transposons. There is also evidence emerging that some DNA-based transposons encode RNAs that could regulate expression of host genes. In the case of IS200, which appears to have lost its ability to transpose, contributing a regulatory RNA to its host could account for the persistence of this mobile element in a wide range of bacterial species. It remains to be seen how prevalent these transposon-encoded RNA regulators are, but given the relatively large amount of intragenic transcription in bacterial genomes, it would not be surprising if new examples are forthcoming. WIREs RNA 2016, 7:382-398. doi: 10.1002/wrna.1341 For further resources related to this article, please visit the WIREs website.

Damage-inducible intragenic demethylation of the human TP53 tumor suppressor gene is associated with transcription from an alternative intronic promoter.

Wild‐type TP53 exons 5–8 contain CpG dinucleotides that are prone to methylation‐dependent mutation during carcinogenesis, but the regulatory effects of methylation affecting these CpG sites are unclear. To clarify this, we first assessed site‐specific TP53 CpG methylation in normal and transformed cells. Both DNA damage and cell ageing were associated with site‐specific CpG demethylation in exon 5 accompanied by induction of a truncated TP53 isoform regulated by an adjacent intronic promoter (P2). We then synthesized novel synonymous TP53 alleles with divergent CpG content but stable encodement of the wild‐type polypeptide. Expression of CpG‐enriched TP53 constructs selectively reduced production of the full‐length transcript (P1), consistent with a causal relationship between intragenic demethylation and transcription. 450K methylation comparison of normal (TP53‐wildtype) and cancerous (TP53‐mutant) human cells and tissues revealed focal cancer‐associated declines in CpG methylation near the P1 transcription start site, accompanied by rises near the alternate exon 5 start site. These data confirm that site‐specific changes of intragenic TP53 CpG methylation are extrinsically inducible, and suggest that human cancer progression is mediated in part by dysregulation of damage‐inducible intragenic CpG demethylation that alters TP53 P1/P2 isoform expression. © 2015 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc.

Abo1, a conserved bromodomain AAA-ATPase, maintains global nucleosome occupancy and organisation.

Maintenance of the correct level and organisation of nucleosomes is crucial for genome function. Here, we uncover a role for a conserved bromodomain AAA-ATPase, Abo1, in the maintenance of nucleosome architecture in fission yeast. Cells lacking abo1(+) experience both a reduction and mis-positioning of nucleosomes at transcribed sequences in addition to increased intragenic transcription, phenotypes that are hallmarks of defective chromatin re-establishment behind RNA polymerase II. Abo1 is recruited to gene sequences and associates with histone H3 and the histone chaperone FACT. Furthermore, the distribution of Abo1 on chromatin is disturbed by impaired FACT function. The role of Abo1 extends to some promoters and also to silent heterochromatin. Abo1 is recruited to pericentromeric heterochromatin independently of the HP1 ortholog, Swi6, where it enforces proper nucleosome occupancy. Consequently, loss of Abo1 alleviates silencing and causes elevated chromosome mis-segregation. We suggest that Abo1 provides a histone chaperone function that maintains nucleosome architecture genome-wide.

H-NS suppresses pilE intragenic transcription and antigenic variation in Neisseria gonorrhoeae.

Initially, pilE transcription in Neisseria gonorrhoeae appeared to be complicated, yet it was eventually simplified into a model where integration host factor activates a single -35/ -10 promoter. However, with the advent of high-throughput RNA sequencing, numerous small pil-specific RNAs (sense as well as antisense) have been identified at the pilE locus as well as at various pilS loci. Using a combination of in vitro transcription, site-directed mutagenesis, Northern analysis and quantitative reverse transcriptase PCR (qRT-PCR) analysis, we have identified three additional non-canonical promoter elements within the pilE gene; two are located within the midgene region (one sense and one antisense), with the third, an antisense promoter, located immediately downstream of the pilE ORF. Using strand-specific qRT-PCR analysis, an inverse correlation exists between the level of antisense expression and the amount of sense message. By their nature, promoter sequences tend to be AT-rich. In Escherichia coli, the small DNA-binding protein H-NS binds to AT-rich sequences and inhibits intragenic transcription. In N. gonorrhoeae hns mutants, pilE antisense transcription was increased twofold, with a concomitant decrease in sense transcript levels. However, most noticeably in these mutants, the absence of H-NS protein caused pilE/pilS recombination to increase dramatically when compared with WT values. Consequently, H-NS protein suppresses pilE intragenic transcription as well as antigenic variation through the pilE/pilS recombination system.

A systematic mutational analysis of a histone H3 residue in budding yeast provides insights into chromatin dynamics.

In previous work using the Saccharomyces cerevisiae model system, a mutant version of histone H3—H3-L61W—was found to confer a variety of abnormal growth phenotypes and defects in specific aspects of the transcription process, including a pronounced alteration in the distribution pattern of the transcription elongation factor Spt16 across transcribed genes and promotion of cryptic transcription initiation within the FLO8 gene. To gain insights into the contribution of the H3-L61 residue to chromatin function, we have generated yeast strains expressing versions of histone H3 harboring all possible natural amino acid substitutions at position 61 (H3-L61X mutants) and tested them in a series of assays. We found that whereas 16 of the 19 H3-L61X mutants support viability when expressed as the sole source of histone H3 in cells, all 19 confer abnormal phenotypes ranging from very mild to severe, a finding that might in part explain the high degree of conservation of the H3-L61 residue among eukaryotes. An examination of the strength of the defects conferred by each H3-L61X mutant and the nature of the corresponding substituted residue provides insights into structural features of the nucleosome required for proper Spt16−gene interactions and for prevention of cryptic transcription initiation events. Finally, we provide evidence that the defects imparted by H3-L61X mutants on Spt16−gene interactions and on repression of intragenic transcription initiation are mechanistically related to each other.

Transcriptome analysis of thermophilic methylotrophic Bacillus methanolicus MGA3 using RNA-sequencing provides detailed insights into its previously uncharted transcriptional landscape.

BackgroundBacillus methanolicus MGA3 is a thermophilic, facultative ribulose monophosphate (RuMP) cycle methylotroph. Together with its ability to produce high yields of amino acids, the relevance of this microorganism as a promising candidate for biotechnological applications is evident. The B. methanolicus MGA3 genome consists of a 3,337,035 nucleotides (nt) circular chromosome, the 19,174 nt plasmid pBM19 and the 68,999 nt plasmid pBM69. 3,218 protein-coding regions were annotated on the chromosome, 22 on pBM19 and 82 on pBM69. In the present study, the RNA-seq approach was used to comprehensively investigate the transcriptome of B. methanolicus MGA3 in order to improve the genome annotation, identify novel transcripts, analyze conserved sequence motifs involved in gene expression and reveal operon structures. For this aim, two different cDNA library preparation methods were applied: one which allows characterization of the whole transcriptome and another which includes enrichment of primary transcript 5′-ends.ResultsAnalysis of the primary transcriptome data enabled the detection of 2,167 putative transcription start sites (TSSs) which were categorized into 1,642 TSSs located in the upstream region (5′-UTR) of known protein-coding genes and 525 TSSs of novel antisense, intragenic, or intergenic transcripts. Firstly, 14 wrongly annotated translation start sites (TLSs) were corrected based on primary transcriptome data. Further investigation of the identified 5′-UTRs resulted in the detailed characterization of their length distribution and the detection of 75 hitherto unknown cis-regulatory RNA elements. Moreover, the exact TSSs positions were utilized to define conserved sequence motifs for translation start sites, ribosome binding sites and promoters in B. methanolicus MGA3. Based on the whole transcriptome data set, novel transcripts, operon structures and mRNA abundances were determined. The analysis of the operon structures revealed that almost half of the genes are transcribed monocistronically (940), whereas 1,164 genes are organized in 381 operons. Several of the genes related to methylotrophy had highly abundant transcripts.ConclusionThe extensive insights into the transcriptional landscape of B. methanolicus MGA3, gained in this study, represent a valuable foundation for further comparative quantitative transcriptome analyses and possibly also for the development of molecular biology tools which at present are very limited for this organism.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1239-4) contains supplementary material, which is available to authorized users.

IgE recognition of chimeric isoforms of the honeybee (Apis mellifera) venom allergen Api m 10 evaluated by protein array technology

Api m 10 has recently been established as novel major allergen that is recognized by more than 60% of honeybee venom (HBV) allergic patients. Previous studies suggest Api m 10 protein heterogeneity which may have implications for diagnosis and immunotherapy of HBV allergy. In the present study, RT-PCR revealed the expression of at least nine additional Api m 10 transcript isoforms by the venom glands. Two distinct mechanisms are responsible for the generation of these isoforms: while the previously known variant 2 is produced by an alternative splicing event, novel identified isoforms are intragenic chimeric transcripts. To the best of our knowledge, this is the first report of the identification of chimeric transcripts generated by the honeybee. By a retrospective proteomic analysis we found evidence for the presence of several of these isoforms in the venom proteome. Additionally, we analyzed IgE reactivity to different isoforms by protein array technology using sera from HBV allergic patients, which revealed that IgE recognition of Api m 10 is both isoform- and patient-specific. While it was previously demonstrated that the majority of HBV allergic patients display IgE reactivity to variant 2, our study also shows that some patients lacking IgE antibodies for variant 2 display IgE reactivity to two of the novel identified Api m 10 variants, i.e. variants 3 and 4.

The influence of Argonaute proteins on alternative RNA splicing.

Alternative splicing of precursor RNAs is an important process in multicellular species because it impacts several aspects of gene expression: from the increase of protein repertoire to the level of expression. A large body of evidences demonstrates that factors regulating chromatin and transcription impact the outcomes of alternative splicing. Argonaute (AGO) proteins were known to play key roles in the regulation of gene expression at the post-transcriptional level. More recently, their role in the nucleus of human somatic cells has emerged. Here, we will discuss some of the nuclear functions of AGO, with special emphasis on alternative splicing. The AGO-mediated modulation of alternative splicing is based on several properties of these proteins: their binding to transcripts on chromatin and their interactions with many proteins, especially histone tail-modifying enzymes, HP1γ and splicing factors. AGO proteins may favor a decrease in the RNA-polymerase II kinetics at actively transcribed genes leading to the modulation of alternative splicing decisions. They could also influence alternative splicing through their interaction with core components of the splicing machinery and several splicing factors. We will discuss the modes of AGO recruitment on chromatin at active genes. We suggest that long intragenic antisense transcripts (lincRNA) might be an important feature of genes containing splicing events regulated by AGO.

Arabidopsis MRG domain proteins bridge two histone modifications to elevate expression of flowering genes.

Trimethylation of lysine 36 of histone H3 (H3K36me3) is found to be associated with various transcription events. In Arabidopsis, the H3K36me3 level peaks in the first half of coding regions, which is in contrast to the 3′-end enrichment in animals. The MRG15 family proteins function as ‘reader’ proteins by binding to H3K36me3 to control alternative splicing or prevent spurious intragenic transcription in animals. Here, we demonstrate that two closely related Arabidopsis homologues (MRG1 and MRG2) are localised to the euchromatin and redundantly ensure the increased transcriptional levels of two flowering time genes with opposing functions, FLOWERING LOCUS C and FLOWERING LOCUS T (FT). MRG2 directly binds to the FT locus and elevates the expression in an H3K36me3-dependent manner. MRG1/2 binds to H3K36me3 with their chromodomain and interact with the histone H4-specific acetyltransferases (HAM1 and HAM2) to achieve a high expression level through active histone acetylation at the promoter and 5′ regions of target loci. Together, this study presents a mechanistic link between H3K36me3 and histone H4 acetylation. Our data also indicate that the biological functions of MRG1/2 have diversified from their animal homologues during evolution, yet they still maintain their conserved H3K36me3-binding molecular function.