Polyadenylated Transcripts Research Articles

Extensive Rearrangement of the Arabidopsis Mitochondrial Genome Elicits Cellular Conditions for Thermotolerance

Three nuclear genes involved in plant mitochondrial recombination surveillance have been previously identified. Simultaneous disruption of two of these genes, MutS Homolog1 (MSH1) and RECA3, results in extensive rearrangement of the mitochondrial genome and dramatic changes in plant growth. We have capitalized on these changes in mitochondrial genome organization to understand the role mitochondria play in plant cellular and developmental processes. Transcript profiling of the double mutants grown under normal conditions revealed differential regulation of numerous nuclear genes involved in stress responses together with increased levels of polyadenylated mitochondrial transcripts. We show that extensive rearrangement of the mitochondrial genome in Arabidopsis (Arabidopsis thaliana) directly elicits physiological stress responses in plants, with msh1 recA3 double mutants exhibiting enhanced thermotolerance. Likewise, we show that mitochondrial transcriptional changes are associated with genome recombination, so that differential gene modulation is accomplished, at least in part, through altered gene copy number.

Polyadenylation and degradation of incomplete RNA polymerase I transcripts in mammalian cells

Most transcripts in growing cells are ribosomal RNA precursors (pre-rRNA). Here, we show that in mammals, aberrant pre-rRNA transcripts generated by RNA polymerase I (Pol I) are polyadenylated and accumulate markedly after treatment with low concentrations of actinomycin D (ActD), which blocks the synthesis of full-length rRNA. The poly(A) polymerase-associated domain-containing protein 5 is required for polyadenylation, whereas the exosome is partly responsible for the degradation of the short aberrant transcripts. Thus, polyadenylation functions in the quality control of Pol I transcription in metazoan cells. The impact of excessive aberrant RNAs on the degradation machinery is an unrecognized mechanism that might contribute to biological properties of ActD.

Large-scale transcriptome data reveals transcriptional activity of fission yeast LTR retrotransposons

BackgroundRetrotransposons are transposable elements that proliferate within eukaryotic genomes through a process involving reverse transcription. The numbers of retrotransposons within genomes and differences between closely related species may yield insight into the evolutionary history of the elements. Less is known about the ongoing dynamics of retrotransposons, as analysis of genome sequences will only reveal insertions of retrotransposons that are fixed - or near fixation - in the population or strain from which genetic material has been extracted for sequencing. One pre-requisite for retrotransposition is transcription of the elements. Given their intrinsic sequence redundancy, transcriptome-level analyses of transposable elements are scarce. We have used recently published transcriptome data from the fission yeast Schizosaccharomyces pombe to assess the ability to detect and describe transcriptional activity from Long Terminal Repeat (LTR) retrotransposons. LTR retrotransposons are normally flanked by two LTR sequences. However, the majority of LTR sequences in S. pombe exist as solitary LTRs, i.e. as single terminal repeat sequences not flanking a retrotransposon. Transcriptional activity was analysed for both full-length LTR retrotransposons and solitary LTRs.ResultsTwo independent sets of transcriptome data reveal the presence of full-length, polyadenylated transcripts from LTR retrotransposons in S. pombe during growth phase in rich medium. The redundancy of retrotransposon sequences makes it difficult to assess which elements are transcriptionally active, but data strongly indicates that only a subset of the LTR retrotransposons contribute significantly to the detected transcription. A considerable level of reverse strand transcription is also detected. Equal levels of transcriptional activity are observed from both strands of solitary LTR sequences. Transcriptome data collected during meiosis suggests that transcription of solitary LTRs is correlated with the transcription of nearby protein-coding genes.ConclusionsPresumably, the host organism negatively regulates proliferation of LTR retrotransposons. The finding of considerable transcriptional activity of retrotransposons suggests that part of this regulation is likely to take place at a post-transcriptional level. Alternatively, the transcriptional activity may signify a hitherto unrecognized activity level of retrotransposon proliferation. Our findings underline the usefulness of transcriptome data in elucidating dynamics in retrotransposon transcription.

3 ANALYSIS OF HOX EXPRESSION IN BOVINE OOCYTES AND EARLY EMBRYOS

HOX genes encode transcription factors known to play a major role in patterning the main body axis of vertebrate embryos from the gastrulation stage onward. A few studies have provided evidence that some HOX genes might be expressed before implantation in mammalian embryos. Translation of maternally inherited transcripts is regulated by modifications of the poly(A) tail length until embryonic genome activation (EGA), occurring during the 4th cell cycle in the bovine. The objective of this work was to establish the expression pattern of various HOX genes and to study the polyadenylation of their transcripts during oocyte maturation and early embryonic development. Pools of 20 bovine oocytes before and after in vitro maturation and 20 in vitro-produced embryos at different stages of development up to the blastocyst stage were collected. Three to 12 pools were used for each stage. RNA was extracted and reverse transcribed (RT) using random hexamers. Quantitative real-time PCR (qPCR) was performed to establish expression profiles of 4 HOX genes: HOXD1, HOXA3, HOXB9, and HOXC9. Two distinct patterns of expression were observed. First, relative amounts of HOXD1, HOXA3, and HOXC9 were lower in morulae and blastocysts than in oocytes. On the other hand, relative expression of HOXB9 increased between the 5 to 8 cell stage and the morula stage (Mann-Whitney, P < 0.05). Those expression patterns were not modified when embryos were cultured in presence of α-amanitin, a RNA polymerase II inhibitor, indicating the maternal origin of the transcripts until EGA. Total amount of mRNAs, estimated by RT-qPCR with random hexamers, was stable for all studied genes during oocyte maturation. The relative amount of polyadenylated GAPDH mRNAs, estimated by RT-qPCR with poly(dT), decreased greatly in mature oocytes compared with immature oocytes indicating massive deadenylation of those transcripts. The relative amount of polyadenylated HOXC9 transcripts decreased slightly but significantly during oocyte maturation (Mann-Whitney, P < 0.05).The relative amount of polyadenylatedm RNAs corresponding to HOXD1, HOXA3, and HOXB9 was stable during oocyte maturation. This indicates that those transcripts escape the default deadenylation pathways followed by housekeeping genes. This experiment has been repeated 3 to 4 times. In conclusion, we confirmed the presence of HOXD1, HOXA3, HOXB9, and HOXC9 transcripts in bovine oocytes and early-stage embryos. Their role during oocyte maturation and the first stages of embryonic development will be investigated through loss of function studies. This work is funded by the Fonds National de la Recherche Scientifique (Belgium).

347 TRANSCRIPTIONAL REGULATION DURING IN VITRO BOVINE OOCYTE MATURATION: FACT OR ARTIFACT?

Understanding the molecular dynamics of oocyte maturation may assist in improvements to maturation protocols and outcomes of in vitro embryo production. There are conflicting results on the occurrence of transcriptional changes during meiotic maturation. A previous microarray study from our group (Carter et al. 2007 Reprod. Fertil. Dev. 19 248) indicated upregulation of a number of transcripts during oocyte maturation in vitro. The aim of this study was to attempt to ascertain whether such changes are real or artifacts of methods used for analysis. COCs were aspirated from follicles of bovine ovaries collected at the local abattoir. Immature COC were immediately denuded and half of them were snap frozen in pools of 12 oocytes in liquid nitrogen. The remaining oocytes were matured in vitro for 24 h at 39°C under an atmosphere of 5% CO2 in air with maximum humidity in TCM-199 supplemented with 10% (v/v) fetal calf serum and 10 ng mL-1 epidermal growth factor. They were subsequently denuded and snap frozen in pools of 12 as above. Samples were stored at -80°C until analysis. Six different pools per stage were collected from four separate collections. Prior to RNA isolation, samples were spiked with luciferase mRNA and total RNA was isolated using RNeasy Micro Kit (Qiagen, Hilden, Germany). The RNA was then divided into two equal parts for priming either with Oligo (dT) or random primers during cDNA synthesis.The expression of eight genes (LUM, PLAT, SERPINE1, STC1, PLAU, ANXA1, TGFB2, and MAOA), previously reportedtobe up regulated (P < 0.05) during bovine oocyte maturation and luciferase was examined. Quantitative real-time PCR was used to compare transcript abundance and data were analyzed using the relative standard curve method. The expression level of these genes was normalized to the average luciferase levels and means were compared by the student t-test. With the exception of MAOA, the expression of all transcripts up regulated (P < 0.05) in the previous microarray study showed similar trend in this analysis. However, the relative expression levels varied with the type of primer used during cDNA synthesis. Oligo (dT) primed samples had higher relative expression ratios compared to samples with random primers, indicating the preferential amplification of polyadenylated transcripts by oligo primers in matured oocytes. Despite the variations in the ratio, the expression patterns remained the same, indicating the existence of transcription during the in vitro bovine oocyte maturation. Future studies will involve in vivo-matured oocytes and identify whether the observed transcription in the in vitro samples is a normal phenomenon or a consequence of suboptimal in vitro culture environment. Supported by Science Foundation Ireland (07/SRC/B1156).

Regulation of White Cell Development.

Abstract SCI-9During hematopoiesis, determination of lineage and maturation to functional leukocytes depend upon cytokine-mediated changes in the transcriptional programs of progenitor and precursor cells. The classic binary branching tree of hematopoiesis now appears to be a more subtle series of gradual changes in differentiation probabilities, with competitive promotion and inhibition of lineage pathways by regulatory transcription factors such as (among others) PU.1, C/EBPα, GFI-1, EGR1/2, and NAB2 for the myeloid lineages and RUNX1, Notch-1, E2A, GATA-3, EBF, and PAX5 for lymphoid cell development. In addition, the recent discovery of regulatory non-coding RNAs (ncRNAs) has revealed another, important layer of control of hematopoiesis. The best studied members of this diverse group are the microRNAs, which often down-regulate multiple target transcripts. miRNAs involved in the regulation of myeloid development and function include miR-155, miR-223, and miR-17-19 cluster members. In addition, miR-9, miR-146a, miR-155, and miR-181a regulate the responses of immunocytes of the innate and acquired immune systems. Most recently, increasing numbers of long ncRNAs have been identified and found to regulate expression of other genes, both in cis and in trans. EGO (eosinophil granule ontogeny), a 500 base pair spliced, polyadenylated transcript regulates eosinophil granule protein gene expression. HOTAIRM1 (Hox antisense intergenic RNA, myeloid-1), a ∼500 base pair spliced polyadenylated ncRNA, affects neutrophil expression of both contiguous and distant HoxA cluster genes, as well as transcripts for CD18 integrin. Thus the control of white cell development depends not simply on a small number of key transcription factors, but rather on a complex network of interacting protein and ncRNA regulators of the transcriptional and translational programs of cell differentiation and function. DisclosuresNo relevant conflicts of interest to declare.

Characterization of polyadenylated cryIA(b) transcripts in maize MON810 commercial varieties

The Zea mays L. event MON810 is one of the major commercialized genetically modified crops. The inserted expression cassette has a 3' truncation partially affecting the cryIA(b) coding sequence, resulting in the lack of the NOS terminator, with transcription of the transgene reported to read-through 3'-past the truncation site. Here, we demonstrate that the cryIA(b) transgene gives rise to a variety of polyadenylated transcripts of different sizes that extend to around 1 kbp downstream the truncation site. A Stop codon at position +7 downstream the truncation site indicates the production of a transgenic protein with two additional amino acids; which is compatible with the reported size of the CryIA(b) protein in MON810. There is no evidence of the existence of other translated products. Several main 3' transcription termination regions were detected close to the truncation site and in the transgene 3' flanking sequence. Next to these main termination sites, we identified some sequence motifs that could potentially act as 3'-end-processing elements and drive termination of the transgene transcripts. The MON810 transgene has been introduced into different commercial varieties through breeding programs. Here, we demonstrate that there are no significant differences among the levels of transgene mRNA accumulation, major transcript sizes and 3' termini profiles comparing a number of MON810 commercial varieties grown under similar environmental conditions. Commercial varieties of this event appear to be stable in terms of transgene expression.

Fail‐safe termination elements: a common feature of the eukaryotic genome?

A recent scan of the human genome (1) identified approximately 11 million hairpins. Some have been linked to known sequences, such as viruses, transposable elements, and, more recently, regulating or microRNAs, but the significance, if any, of most sequences that can be predicted to form hairpins remains unknown. Here we show that hairpins that are cleaved by RNase III- like nucleases can induce termination, even with normally polyadenylated transcripts, and that a cleaved hairpin downstream of a normal termination signal can induce fail-safe termination. Because such cleavage sites appear common to intergenic regions, the results raise the possibility that similar fail-safe termination elements are widely distributed in the eukaryotic genome to prevent read-through transcription from disrupting downstream promoter elements or opposing transcription.-Nabavi, S., Nazar, R. N. Fail-safe termination elements: a common feature of the eukaryotic genome?

Hyaluronan-CD44 Interaction with Protein Kinase Cϵ Promotes Oncogenic Signaling by the Stem Cell Marker Nanog and the Production of MicroRNA-21, Leading to Down-regulation of the Tumor Suppressor Protein PDCD4, Anti-apoptosis, and Chemotherapy Resistance in Breast Tumor Cells

Multidrug resistance and disease relapse is a challenging clinical problem in the treatment of breast cancer. In this study, we investigated the hyaluronan (HA)-induced interaction between CD44 (a primary HA receptor) and protein kinase Cepsilon (PKCepsilon), which regulates a number of human breast tumor cell functions. Our results indicate that HA binding to CD44 promotes PKCepsilon activation, which, in turn, increases the phosphorylation of the stem cell marker, Nanog, in the breast tumor cell line MCF-7. Phosphorylated Nanog is then translocated from the cytosol to the nucleus and becomes associated with RNase III DROSHA and the RNA helicase p68. This process leads to microRNA-21 (miR-21) production and a tumor suppressor protein (e.g. PDCD4 (program cell death 4)) reduction. All of these events contribute to up-regulation of inhibitors of apoptosis proteins (IAPs) and MDR1 (multidrug-resistant protein), resulting in anti-apoptosis and chemotherapy resistance. Transfection of MCF-7 cells with PKCepsilon or Nanog-specific small interfering RNAs effectively blocks HA-mediated PKCepsilon-Nanog signaling events, abrogates miR-21 production, and increases PDCD4 expression/eIF4A binding. Subsequently, this PKCepsilon-Nanog signaling inhibition causes IAP/MDR1 down-regulation, apoptosis, and chemosensitivity. To further evaluate the role of miR-21 in oncogenesis and chemoresistance, MCF-7 cells were also transfected with a specific anti-miR-21 inhibitor in order to silence miR-21 expression and inhibit its target functions. Our results indicate that anti-miR-21 inhibitor not only enhances PDCD4 expression/eIF4A binding but also blocks HA-CD44-mediated tumor cell behaviors. Thus, this newly discovered HA-CD44 signaling pathway should provide important drug targets for sensitizing tumor cell apoptosis and overcoming chemotherapy resistance in breast cancer cells.

Production and Processing of siRNA Precursor Transcripts from the Highly Repetitive Maize Genome

Mutations affecting the maintenance of heritable epigenetic states in maize identify multiple RNA–directed DNA methylation (RdDM) factors including RMR1, a novel member of a plant-specific clade of Snf2-related proteins. Here we show that RMR1 is necessary for the accumulation of a majority of 24 nt small RNAs, including those derived from Long-Terminal Repeat (LTR) retrotransposons, the most common repetitive feature in the maize genome. A genetic analysis of DNA transposon repression indicates that RMR1 acts upstream of the RNA–dependent RNA polymerase, RDR2 (MOP1). Surprisingly, we show that non-polyadenylated transcripts from a sampling of LTR retrotransposons are lost in both rmr1 and rdr2 mutants. In contrast, plants deficient for RNA Polymerase IV (Pol IV) function show an increase in polyadenylated LTR RNA transcripts. These findings support a model in which Pol IV functions independently of the small RNA accumulation facilitated by RMR1 and RDR2 and support that a loss of Pol IV leads to RNA Polymerase II–based transcription. Additionally, the lack of changes in general genome homeostasis in rmr1 mutants, despite the global loss of 24 nt small RNAs, challenges the perceived roles of siRNAs in maintaining functional heterochromatin in the genomes of outcrossing grass species.

Quantification of the yeast transcriptome by single-molecule sequencing

We present single-molecule sequencing digital gene expression (smsDGE), a high-throughput, amplification-free method for accurate quantification of the full range of cellular polyadenylated RNA transcripts using a Helicos Genetic Analysis system. smsDGE involves a reverse-transcription and polyA-tailing sample preparation procedure followed by sequencing that generates a single read per transcript. We applied smsDGE to the transcriptome of Saccharomyces cerevisiae strain DBY746, using 6 of the available 50 channels in a single sequencing run, yielding on average 12 million aligned reads per channel. Using spiked-in RNA, accurate quantitative measurements were obtained over four orders of magnitude. High correlation was demonstrated across independent flow-cell channels, instrument runs and sample preparations. Transcript counting in smsDGE is highly efficient due to the representation of each transcript molecule by a single read. This efficiency, coupled with the high throughput enabled by the single-molecule sequencing platform, provides an alternative method for expression profiling.

Regulation of bovine oocyte‐specific transcripts during in vitro oocyte maturation and after maternal–embryonic transition analyzed using a transcriptomic approach

Oocyte/embryo genomics in mammals faces specific challenges due to limited biological material, to the comparison of models with different total RNA contents, and to expression of a specific set of genes often absent from commercially available microarrays. Here, we report experimental validation of a RNA amplification protocol for bovine oocytes and blastocysts. Using real-time PCR, we have confirmed that the profile of both abundant and scarce polyadenylated transcripts was conserved after RNA amplification. Next, amplified probes generated from immature oocytes, in vitro matured oocytes, and in vitro produced hatched blastocysts were hybridized onto a macroarray that included oocyte-specific genes. Following an original approach, we have compared two normalization procedures, based on the median signal or an exogenous standard. We have evidenced the expected difference in sets of differential genes depending on the normalization procedure. Using a 1.5-fold threshold, no transcript was found to be upregulated when data were normalized to an exogenous standard, which reflects the absence of transcription during in vitro oocyte maturation. In blastocysts, the majority of oocyte-preferentially expressed genes were not activated, as previously observed in mouse. Finally, microarray data were validated by real-time PCR on a random subset of genes. Our study sheds new light on and complements previous transcriptomic analyses of bovine oocyte to embryo transition using commercial platforms.

A retrospective model of oocyte competence: global mRNA and housekeeping transcripts are not associated with in vitro developmental outcome

SummaryOocyte developmental competence depends on maternal stores that support development throughout a transcriptionally silent period during early embryogenesis. Previous attempts to investigate transcripts associated with oocyte competence have relied on prospective models, which are mostly based on morphological criteria. Using a retrospective model, we quantitatively compared mRNA among oocytes with different embryo development competence. A cytoplasm biopsy was removed from in vitro matured oocytes to perform comparative analysis of amounts of global polyadenylated (polyA) mRNA and housekeeping gene transcripts. After parthenogenetic activation of biopsied oocytes, presumptive zygotes were cultured individually in vitro and oocytes were classified according to embryo development: (i) blocked before the 8-cell stage; (ii) blocked between the 8-cell and morulae stages; or (iii) developed to the blastocyst stage. Sham-manipulated controls confirmed that biopsies did not alter development outcome. Total polyA mRNA amounts correlate with oocyte diameter but not with the ability to develop to the 8-cell and blastocyst stages. The last was also confirmed by relative quantification of GAPDH, H2A and Hprt1 transcripts. In conclusion, we describe a novel retrospective model to identify putative markers of development competence in single oocytes and demonstrate that global mRNA amounts at the metaphase II stage do not correlate with embryo development in vitro.

Regulation of NAB2 mRNA 3′-end formation requires the core exosome and the Trf4p component of the TRAMP complex

The nuclear exosome functions in a variety of pathways catalyzing formation of mature RNA 3'-ends or the destruction of aberrant RNA transcripts. The RNA 3'-end formation activity of the exosome appeared restricted to small noncoding RNAs. However, the nuclear exosome controls the level of the mRNA encoding the poly(A)-binding protein Nab2p in a manner requiring an A(26) sequence in the mRNA 3' untranslated regions (UTR), and the activities of Nab2p and the exosome-associated exoribonuclease Rrp6p. Here we show that the A(26) sequence inhibits normal 3'-end processing of NAB2 mRNA in vivo and in vitro, and makes formation of the mature 3'-end dependent on trimming of the transcript by the core exosome and the Trf4p component of the TRAMP complex from a downstream site. The detection of mature, polyadenylated transcripts ending at, or within, the A(26) sequence indicates that exosome trimming sometimes gives way to polyadenylation of the mRNA. Alternatively, Rrp6p and the TRAMP-associated Mtr4p degrade these transcripts thereby limiting the amount of Nab2p in the cell. These findings suggest that NAB2 mRNA 3'-end formation requires the exosome and TRAMP complex, and that competition between polyadenylation and Rrp6p-dependent degradation controls the level of this mRNA.

Alternative polyadenylation variants of the RNA binding protein, HuR: abundance, role of AU-rich elements and auto-Regulation.

The RNA-binding protein, HuR, is involved in the stabilization of AU-rich element-containing mRNAs with products that are involved in cell-cycle progression, cell differentiation and inflammation. We show that there are multiple polyadenylation variants of HuR mRNA that differ in their abundance, using both bioinformatics and experimental approaches. A polyadenylation variant with distal poly(A) signal is a rare transcript that harbors functional AU-rich elements (ARE) in the 3′UTR. A minimal 60-nt region, but not a mutant form, fused to reporter-3′UTR constructs was able to downregulate the reporter activity. The most predominant and alternatively polyadenylated mature transcript does not contain the ARE. HuR itself binds HuR mRNA, and upregulated the activity of reporter from constructs fused with ARE-isoform and the HuR ARE. Wild-type tristetraprolin (TTP), but not the zinc finger mutant TTP, competes for HuR binding and upregulation of HuR mRNA. The study shows that the HuR gene codes for several polyadenylation variants differentially regulated by AU-rich elements, and demonstrates an auto-regulatory role of HuR.

Fail‐safe Termination Elements: A Common Feature of the Eukaryotic Genome?

A recent scan of the human genome identified approximately 11 million hairpins. Some have been linked to known sequences such as viruses, transposable elements and, more recently, regulating or microRNAs but the significance, if any, of most sequences which can be predicted to form hairpins remains unknown. Here we show that hairpins which are cleaved by RNase III‐like nucleases can induce termination, even with normally polyadenylated transcripts and that a cleaved hairpin, downstream of a normal termination signal can induce fail‐safe termination. The results raise the possibility that similar fail‐safe termination elements are widely distributed in the eukaryotic genome to prevent read‐through transcription from disrupting downstream promoter elements. Supported by the Natural Sciences and Engineering Research Council of Canada.

Transcriptomic profiling of Ichthyophthirius multifiliis reveals polyadenylation of the large subunit ribosomal RNA

Polyadenylation of eukaryotic transcripts is usually restricted to mRNA, providing transcripts with stability from degradation by nucleases. Conversely, an RNA degradation pathway can be signaled through poly (A) tailing in prokaryotic, archeal, and organellar biology. Recently polyadenylated transcripts have also been discovered in rRNA in some eukaryotes including humans and yeast. Here we report the discovery of polyadenylated rRNAs in the ciliate teleost parasite Ichthyophthirius multifiliis, an important fish pathogen. Through large-scale analysis of ESTs, a large contig composed of the 28S rRNA with poly (A) tails was identified. Analysis using multiple sequence alignments revealed four potential polyadenylation sites including three internal regions and the 3′ end of the rRNA. Further analysis using a polyadenylation test, re-sequencing, and gene-specific PCR using primers flanking the presumed poly (A) sites confirmed the presence of polyadenylated rRNA in this parasite. The functions of polyadenylation of rRNA in this organism are largely unknown at present, but the presence of internal polyadenylation sites, along with the presence of truncated segments of the rRNA, may suggest a role of the polyadenylation in the degradation pathway, a function typical of prokaryotes, archaea, and organelles. These results are in congruence with reports of a similar phenomenon in humans and yeast.

Transcription of mRNA-type long non-coding RNAs (mlonRNAs) disrupts chromatin array

Eukaryotic transcriptome analyses have revealed that many transcripts are non-coding RNAs (ncRNAs). In addition, most relatively large (~several kb) polyadenylated mRNA type transcripts are transcribed from regions harboring little coding potential. However the role of such mRNA type long ncRNAs (mlonRNAs) is mostly unknown and has been a matter of debate. Recently, we showed that cascade of RNA polymerase II (RNAPII)-mediated transcriptional initiation of mlonRNA causes stepwise disruption of local chromatin array at the fission yeast Schizosaccharomyces pombe fbp1+ promoter region. Here, we hypothesize that RNAPII transcription of mlonRNA disrupt chromatin array possibly collaborating with histone acetylation mechanism. In addition, conserved action of Atf1, a transcriptional activator, and Tup11-Tup12 corepressors along mlonRNA transcription mediated chromatin regulation is suggested. This idea provides new insight into the biological meaning of mlonRNAs found in various eukaryotes.

Stepwise chromatin remodelling by a cascade of transcription initiation of non-coding RNAs

Recent transcriptome analyses using high-density tiling arrays and data from large-scale analyses of full-length complementary DNA libraries by the FANTOM3 consortium demonstrate that many transcripts are non-coding RNAs (ncRNAs). These transcriptome analyses indicate that many of the non-coding regions, previously thought to be functionally inert, are actually transcriptionally active regions with various features. Furthermore, most relatively large ( approximately several kilobases) polyadenylated messenger RNA transcripts are transcribed from regions harbouring little coding potential. However, the function of such ncRNAs is mostly unknown and has been a matter of debate. Here we show that RNA polymerase II (RNAPII) transcription of ncRNAs is required for chromatin remodelling at the fission yeast Schizosaccharomyces pombe fbp1(+) locus during transcriptional activation. The chromatin at fbp1(+) is progressively converted to an open configuration, as several species of ncRNAs are transcribed through fbp1(+). This is coupled with the translocation of RNAPII through the region upstream of the eventual fbp1(+) transcriptional start site. Insertion of a transcription terminator into this upstream region abolishes both the cascade of transcription of ncRNAs and the progressive chromatin alteration. Our results demonstrate that transcription through the promoter region is required to make DNA sequences accessible to transcriptional activators and to RNAPII.

BARE retrotransposons produce multiple groups of rarely polyadenylated transcripts from two differentially regulated promoters

The BARE retrotransposon family comprises more than 10(4) copies in the barley (Hordeum vulgare) genome. The element is bounded by long terminal repeats (LTRs, 1829 bp) containing promoters and RNA-processing motifs required for retrotransposon replication. Members of the BARE1 subfamily are transcribed, translated, and form virus-like particles. Very similar retrotransposons are expressed as RNA and protein in other cereals and grasses. The BARE2 subfamily is, however, non-autonomous because it cannot produce the GAG capsid protein. The pattern of plant development implies that inheritance of integrated copies should critically depend, in the first instance, on cell-specific and tissue-specific expression patterns. We examined transcription of BARE within different barley tissues and analyzed the promoter function of the BARE LTR. The two promoters of the LTR vary independently in activity by tissue. In embryos TATA1 was almost inactive, whereas transcription in callus appears to be less tightly regulated than in other tissues. Deletion analyses of the LTR uncovered strong positive and negative regulatory elements. The promoters produce multiple groups of transcripts that are distinct by their start and stop points, by their sequences, and by whether they are polyadenylated. Some of these groups do not share the common end structures needed for template switching during replication. Only about 15% of BARE transcripts are polyadenylated. The data suggest that distinct subfamilies of transcripts may play independent roles in providing the proteins and replication templates for the BARE retrotransposon life cycle.