We also highlighted the implications of biogenesis and gene expression deregulation of sncRNAs and lncRNAs in human diseases like cancer.In the last years it has become increasingly clear that the mammalian transcriptome is highly complex and includes a large number of small non-coding RNAs (sncRNAs) and long noncoding RNAs (lncRNAs). Here we review the biogenesis pathways of the three classes of sncRNAs, namely short interfering RNAs (siRNAs), microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs). These ncRNAs have been extensively studied and are involved in pathways leading to specific gene silencing and the protection of genomes against virus and transposons, for example. Also, lncRNAs have emerged as pivotal molecules for the transcriptional and post-transcriptional regulation of gene expression which is supported by their tissue-specific expression patterns, subcellular distribution, and developmental regulation. Therefore, we also focus our attention on their role in differentiation and development. SncRNAs and lncRNAs play critical roles in defining DNA methylation patterns, as well as chromatin remodeling thus having a substantial effect in epigenetics. The identification of some overlaps in their biogenesis pathways and functional roles raises the hypothesis that these molecules play concerted functions  Tetrahymena intron of the 26S rRNA that is a self-sufficient catalytic unit capable of autoexcision and autocyclization [in vivo, and led to the re-evaluation of RNAs as crucial molecules in the evolution of life. In view of the ability of RNAs to catalyze biological reactions, it is conceivable that the first organisms could rely only on RNA molecules and that only later an evolution of a more complex system based on proteins was established. This hypothesis gave support to the model of a primordial “RNA World”  that is then translated into proteins with the help of other RNA molecules like transfer RNA (tRNA) and ribosomal RNAs (rRNAs). The idea that RNAs are much more than molecules involved in storage/transfer of information emerged with the discovery of ribozymes, RNA molecules that have, like proteins, active roles as catalysts of chemical reactions in cells. The two ribozymes identified first have RNAs as substrates and were the r review ,4).Progressively, the participation of RNAs in other critical molecular processes in eukaryotic cells was revealed, as in the case of DNA replication , protein translation and RNA transcript maturation. For example, several ribosome functions required for protein synthesis were shown to be, at least in part, RNA-mediated, including peptidyl transferase activity , decodinFurthermore, the information content of tRNA, rRNA and mRNA molecules can be biochemically altered after transcription by different molecular mechanisms that are generally designated by RNA editing . These iIn the mid-1980s Blackburn and Greider, demonstrated the existence of an enzymatic activity within cell extracts that added tandem hexanucleotides to chromosome ends and led to the discovery of telomerase . Today iin vivo. Importantly, the observations that exogenously introduced double stranded RNA (dsRNA) molecules and plasmids expressing short hair-pin RNA (shRNA) specifically base-pairing with target mRNA molecules were able to trigger mRNA degradation (RNA interference -RNAi) [At this point the growing descriptions of the importance of RNA molecules for cell function started to push them to the limelight, but the complexity of their roles and the wide variety of molecular mechanisms where RNA molecules are critical players was still far from clear. In recent years, the use of genome wide approaches and the large output of genome sequencing technologies have revealed that the mammalian transcriptome is much more complex than previously thought since it includes a large number of small non-coding RNAs (sncRNAs) and long noncoding RNAs (lncRNAs) ,15. In me -RNAi) ,20 reveaThe aim of this review is to give a summarized overview of the biogenesis pathways of distinct classes of sncRNAs, including miRNA, piRNA, and siRNA, as well as lncRNAs, focusing on the miRNA and lncRNAs gene regulatory roles in distinct cellular functions and developmental regulatory programs. We will highlight the implications of the deregulation of miRNA and lncRNAs biogenesis pathways further illustrating the role of these molecules in the establishment of human diseases such as cancer. Finally, we will bring to discussion the fact that the pathways where distinct family members of sncRNAs and lncRNA funtion are probably interconnected, establishing a complex network of interactions and actions required for rapid and fine-tuned gene expression regulation at multiple levels.Three classes of sncRNAs, namely short interfering RNAs (siRNAs), microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs), have been extensively studied in the last decade and have been associated with pathways that lead to silencing of specific genes and to the protection of the cell/genome against viruses, mobile repetitive DNA sequences, retro-elements and transposons .The siRNAs and miRNAs (~20–30 nucleotides long) originate from double-stranded RNA (dsRNA) precursors that are introduced into, or produced endogenously by gene transcription of both sense and anti-sense DNA strands and of pseudogenes and inverted repeats. These molecules are critical in pathways involved in mRNA degradation, translational repression, or both, therefore regulating gene expression.In the case of siRNAs, they are small RNA duplex molecules produced by the action of Dicer, a ribonuclease III (RNaseIII) enzyme that creates RNA duplexes with 2-nt overhangs at their 3′ ends and phosphate groups at their 5′ ends .The miRNAs are mostly transcribed by RNA polymerase II as primary-miRNA (pri-miRNA) molecule precursors that possess a characteristic stem loop structure and are subsequently subjected to processing mechanisms . In animAn alternative nuclear pathway for miRNA biogenesis was described in invertebrates  where thThe next step, for both siRNA and miRNA production, is the subsequent association with members of the Argonaute protein family that have diverged into specialized clades (or subfamilies), each recognizing different sncRNA types and conferring the specific features of the various silencing pathways operating in cells . ArgonauO-methyl group at the 3′ ends of small RNAs bound to Ago2, but not those bound to Ago1 [The selection of the different Argonaute proteins seems to be based on the small interfering RNA duplex structure. For example, siRNAs that are perfect duplexes in terms of base pairing are loaded into Argonaute 2 (Ago2), whereas duplexes presenting mismatches, as in the case of miRNAs, are generally driven to Argonaute 1 (Ago1) ,28. When to Ago1 . This me to Ago1 . These mDrosophila Loqs-PB Dicer-partner cleaves pre-miR-307a, generating a longer miRNA isoform with a distinct seed sequence and target specificity [In the small RNA duplex of the siRNA the guide strand seems to be the one whose 5′ end is less tightly paired to its complement . In bothcificity . The mamcificity .In fission yeast a specialized nuclear complex, known as the RNA-induced transcriptional silencing complex (RITS), mediates transcriptional gene silencing by inducing heterochromatin formation . The RITDrosophila melanogaster [The piRNAs are the least characterized class of sncRNAs and, contrary to the siRNAs and miRNAs that are widely expressed in different tissues and cell types, the piRNAs have been essentially detected in the germline cells of mammals, fish and nogaster ,39 wherenogaster .O-methyl (2′-O-Me) modification on the nucleotide at the 3′ end  got their name from the fact that they only associate to the PIWI subfamily of the Argonaute protein family (Piwi proteins). These sncRNAs usually have a uridine at the 5′ end, hold a 5′ monophosphate, and present a 2′-iew, see ).Although not much is known concerning the intervening factors involved in piRNAs biogenesis pathways and transcription regulation, it is now well documented that they diverge from siRNAs and miRNAs by being generated by RNaseIII-independent pathways that do not involve dsRNA precursors. These sncRNAs are generated from long single-stranded precursors ,42 that Drosophila, as in mammals, the majority of piRNAs are transcribed from discrete genomic loci that are clustered in large pericentromeric or subtelomeric domains, generally spanning from 50–100 kb, and that comprise mainly various transposable DNA elements and their remnants [flamenco) impels transposon silencing in the soma. Interestingly, if new transposons are introduced into piRNA clusters, and if they are heritable by the progeny, novel piRNAs will be produced that can lead to the control of the new transposons indicating that the mechanisms that drive adaptation to transposon invasion might be mediated by the piRNA pathway [In remnants . Other premnants ,43. Most pathway .Drosophila piRNAs and genes coding for the proteins involved in their biogenesis. In the germline these mutations cause a retro-transposition up-regulation causing the loss of germ cells and a variety of defects due to alterations in microtubule cytoskeleton polarization, with consequences to the polarized localization of specific proteins and mRNAs required for normal oogenesis [The critical role of piRNAs on transposon silencing was demonstrated by loss-of function mutations in ogenesis . Howeverogenesis  suggestiogenesis ).Other studies also reveal that besides being involved in keeping genome integrity, a subset of piRNA genes have been implicated in the assembly of the telomere protection complex .Drosophila female germline showed that these sncRNAs have in their structure and sequences, signatures that give clues about their biogenesis. The most abundant piRNAs are mainly generated from the antisense strand of retro-transposons and these preferentially associate with Piwi and Aubergine proteins [Detailed analysis of the small RNAs associated with the Piwi sub-family  ,47 in thproteins ,47. ThosThe piRNAs from the germ cells seem to be generated by a self-amplifying loop designated by ping-pong cycle. Specifically, PIWI and/or Aubergine form complexes with antisense piRNAs that direct the slicing of sense strand transposon transcripts ,47. The Drosophila, mouse and rhesus macaque samples demonstrated that piRNAs are widespread and are abundant in other tissues as much as in the germline [Drosophila, where the degradation of a subset of maternal RNAs, i.e., embryonic posterior morphogen Nanos (Nos), at the maternal-to-zygotic transition, was shown to require the zygotic expression of a piRNA cluster [Recent studies have shown that, in addition to their role in germ line transposon regulation and genome stability, piRNAs have a broader function in heterochromatin formation and developmental gene regulation. The analysis of a high-throughput small RNA sequencing data in germline . In fact cluster . When th cluster .et al. [Drosophila ovarian somatic cells piRNAs mediate the silencing of hundreds of transposon copies at the transcriptional level by establishing heterochromatic methylation of H3K9 on transposons and their genomic surroundings. The involvement of the piRNA pathway in de novo methylation of the differentially methylated region of the imprinted mouse Rasgrf1 locus [The importance of the piRNAs pathway in the nervous system and in epigenetic regulation has also been gaining support. In the hippocampus, the inhibition of piRNAs causes a decrease of the dendrite spine area suggesting that these sncRNAs are required for spine morphogenesis . More reet al.  have alsf1 locus  shows thDrosophila that a protein complex composed of Hsp90, Piwi and Hop, is involved in canalization, probably through phosphorylation regulation of the Piwi protein by Hsp90 and Hop [There is also growing evidence that piRNA-pathway dependent mechanisms may have been critical during evolution, in the establishment of developmental robustness. In fact, the piRNA-pathway seems to be required for preventing phenotypic variation despite genotypic variation and environmental influences  . The Hsp and Hop . Therefo and Hop .The piRNAs, contrary to miRNAs, are less conserved through the eukaryotic lineage. This difference has been explained by the possible co-evolvement of miRNA with their RNA targets which have created sequence divergence constraints. There are increasing examples that piRNAs play roles in somatic cells regulating protein encoding genes. It is possible that piRNAs are more likely to be involved in epigenetic regulation rather than post-transcriptional regulation . These pThe role of piRNAs in protecting genomes against parasitic nucleic acids seems to have developed early in evolution since ciliates present a mechanism that resembles that of piRNAs. Ciliates are single celled organisms that present a polyploid macronucleus that guarantees the vegetative growth of cells (the somatic nucleus) and the diploid micronucleus that is only active during sexual conjugation and constitutes the germline . After cIt is now clear that the mammalian genome produces a large transcriptome of long noncoding RNA . The number of gene members integrating this class of ncRNAs is still under debate and ranges from 10,000 to >200,000 .The lncRNAs can be transcribed from intergenic regions, promoter regions or be interleaved, overlapping or antisense to annotated protein-coding genes . There iThe referred transcriptional origins have been used to establish classification classes for lncRNA, as for example promoter-associated long RNAs (lpaRNAs) , naturalMammalian genomes encode a large number of natural antisense transcripts (NATs) ,73. For cis-NATs. On the other hand those transcribed from different genomic loci may have imperfect sequence complementarity and are named trans-NATs [NATs have been defined as endogenous RNA molecules at least partially complementary to transcripts of known function . NATs caans-NATs . Sense aans-NATs . Antisenans-NATs .Studies performed in various organisms have suggested that NATs can participate in a broad range of regulatory events that will be discussed later.LncRNAs have emerged as pivotal molecules for the regulation of gene expression . These tLncRNAs encompass a wide variety of functions which include almost all levels of gene expression regulation, ranging from epigenetic to translational regulation, including transcriptional and post-transcriptional control. The main functions of lncRNAs are summarized below.lncRNAs modulate chromatin through the specific recruitment of histone and chromatin modifying complexes on one hand and by the recruitment of transcription factors on the other hand. X chromosome inactivation (XCI) is the classic example of the former type of regulation and is caused by the lncRNA “Xist” which physically associates with the Polycomb repressive complex 2 (PRC2) recruiting it to the X chromosome ultimately leading to its inactivation . More pri.e., Air, Kcnq1ot1, HOTAIR) can control chromatin states in cis and/or in trans, thereby regulating gene expression through the association with chromatin-modifying complexes [trans-acting lncRNA that serves as a scaffold for two histone modification complexes: it binds both to polycomb repressive complex 2 (PRC2) and to LSD1 (in complex with CoREST/REST). This coordinates targeting of PCR2 and LSD1 to chromatin for coupled histone H3 at lysine 27 methylation and lysine 4 demethylation leading to subsequent gene silencing [Arabidopsis it was demonstrated that environmental conditions, such as cold, are able to induce the transcription of related NATs  that are involved in the silencing of a flower repressor locus designated by flowering locus c (FLC) [FLC mRNA transcription, interacts directly with PRC2 and targets it to FLC, establishing an epigenetic memory [FLC and it was shown that COLDAIR is induced by cold, demonstrating that lncRNas participate in the integration of signals from the environment to cell signaling pathways.Other lncRNAs  . More rec memory . Interestrans-acting lncRNAs have different functions some of which remain incompletely defined. For example, the p21-associated ncRNA DNA damage-activated (PANDA) lncRNA is induced upon DNA damage in a p53-dependent manner and it interacts with the transcription factor NF-YA to limit expression of pro-apoptotic genes [Other ic genes . Mistralic genes .Another group of lncRNAs that play a role in mammalian genomes are the long intergenic non-coding RNAs (lincRNAs) that range in size from ~300 nucleotides to several thousands and that, in humans, have been estimated to be around 3300, although a more correct number may be closer to 4500 . This grSaccharomyces cerevisiae [The discovery and characterization of several ncRNAs that are able to associate with promoters (promoter associated RNAs—paRNA) is also changing the traditional view of how genes encoding proteins are regulated at the transcriptional level. Promoter associated RNAs paRNAs are transcribed approximately from the start of or within the promoter, and include long, short and tiny RNA molecules (for review ). The lorevisiae  and generevisiae .Interestingly, long (antisense) paRNAs have the potential to form double stranded molecules that can be processed into endo-siRNAs, and that, due to their sequence complementarity to that of a promoter, are able to induce transcriptional gene silencing –99 or acOn the other hand, lncRNAs can modulate the function of transcription factors by acting as co-regulators, modulators of transcription factors activity or by regulating the association and activity of co-regulators, among others. The ncRNA Evf-2, for example, functions as a co-activator for the homeobox transcription factor Dlx2, which plays important roles in forebrain development and neurogenesis . Local nFinally, lncRNAs also regulate the basal transcription machinery by targeting transcription factors required for the RNAP II transcription of all genes . These gTRα) is an example where the antisense transcript RevErbAα influences splicing of TRα1 and TRα2 mRNAs [PU.1 mRNA. PU.1 mRNA translation is inhibited by a noncoding antisense transcript, which is a polyadenylated RNA with a lower concentration but a half-life longer than the sense PU.1 transcript [LncRNAs can act on splicing, on mRNA stability and translation. It has been shown that lnc antisense RNA may bind to the sense RNA, masking the splice sites and thereby changing the balances between splice variants. Thyroid hormone receptor alpha gene (α2 mRNAs . Recentlα2 mRNAs . The autα2 mRNAs . By contα2 mRNAs . TINCR-mα2 mRNAs . Anotherα2 mRNAs . Therefoanscript . On the anscript . InteresNRON is a non-coding repressor of nuclear factor of the activated T cells (NFAT), which interacts with multiple proteins including members of the importin-beta superfamily and likely functions as a specific regulator of NFAT nuclear trafficking .foci that are coincident with “paraspeckles” [The lncRNA nuclear-enriched autosomal transcript 1 (NEAT1), and abundant 4 kb ncRNA, is retained in nuclei peckles” . It has peckles” .loci (cis-NATs) or from different loci (trans-NATs). Interestingly, it was observed that certain trans-NATs are produced from pseudogene transcription. For example, in rice, a small number of pseudogenes are transcribed and processed into siRNAs, after pairing with the coding gene or a paralogous pseudogene transcript [It has been described that NATs can originate siRNAs that will be involved in mRNA down-regulation. This mechanism requires the formation of a sense:anti-sense pair of transcripts that are then processed into siRNAs. This pair can be originated directly from the same anscript . A similanscript ,118. Theanscript . Pseudoganscript ; (2) dupanscript ; and (3)anscript ,122. Theanscript .et al. [It is evident that the critical roles of lncRNAs at different levels of gene expression regulation will largely contribute to establish differential profiles of gene expression required for development –127. Thiet al.  using a et al. . Accordiet al. .Tbca pseudogene that is transcribed in both directions [Tbca pseudogene is down-regulated leading to the increase of the Tbca mRNA, during testis maturation suggesting that this Tbca lncRNA is required for the undifferentiated state of spermatids. Similarly, the gene encoding the nitric oxide synthase protein (NOS2A) is transcribed into a noncoding RNA containing a region of significant antisense homology with the NOS2A mRNA. As in the case of Tbca lncRNA, the expression patterns of the anti-NOS2A RNA and the NOS2A mRNA exhibit opposite changes in undifferentiated human embryonic stem cells (hESCs) and in hESCs induced to differentiate into neurogenic precursors [Spermatogenesis is a very complex developmental process that requires precise microtubule cytoskeleton remodeling, creating complex microtubule structures such as the manchette and the flagellum of the sperm . During rections . The Tbcecursors . In concmiRNAs act as sequence-specificity guides for the RNAi machinery to mediate repression of target gene expression. First identified as regulators of larval development in nematodes , miRNAs It is still unclear whether miRNAs act mainly at the mRNA translational or transcriptional levels. The miRNA repression, at the level of transcriptional inhibition, can occur as a consequence of mRNA decay, direct mRNA cleavage or through miRNA-mediated chromatin reorganization. Decay of targeted mRNA occurs without direct cleavage at the binding site. Unlike in translational inhibition where only a slight protein decrease can be obtained, protein level reductions greater than 33% indicate that mRNA decay is the major component of miRNA-driven silencing . miRNA-mFinally, the repressed mRNAs, Ago proteins and miRNAs are frequently accumulated in processing bodies (P-bodies), which are cytoplasmic structures enriched in the mRNA degradation machinery but where the translational machinery is normally absent .The second major mechanism of miRNAs activity includes repression of translation initiation and/or elongation, premature termination and nascent polypeptide degradation. Inhibition of translation initiation can occur at the level of cap-40S association or via 40S-AUG-60S association. Endogenous let-7 micro-ribonucleoproteins (miRNPs) or the tethering of Ago proteins to reporter mRNAs in human cells inhibit m(7)G-cap-dependent translation initiation, suggesting that miRNPs interfere with the recognition of the cap . The capIntriguingly, there is also evidence for transcriptional  and tranin vitro) and embryos (in vivo) [While the global importance of miRNAs is clearly illustrated by the developmental failure of Dicer-deficient embryonic stem cells (in vivo) , unique in vivo) ). Severain vivo)  were up-in vivo)  is exprein vivo) . Detailein vivo) .In the immune system, miRNAs mediate the regulation of T cell development and function, as confirmed by the observation of defective thymic and peripheric T cell subsets in Dicer deficient mice ,159.Individual miRNAs play different roles at distinct developmental stages. For example, miR-125b and miR-132 regulate dendritic spine development. More specifically, miR-125b and miR-132  are associated with fragile X mental retardation protein (FMRP) in mouse brain. The miR-125b overexpression results in longer, thinner processes of hippocampal neurons. FMRP knockdown is shown to ameliorate the effect of overexpressed miR-125b and miR-132 on spine morphology. It has been proposed that miR-125b negatively regulates its target, NR2A, along with FMRP and AGO1 .Focusing on T cells, miRNA expression patterns vary among stages of development and T cell subsets, which indicate that these molecules may contribute to the identity of the cell subsets or their functional state . ConsistLike lncRNAs, miRNAs are required to regulate differentiation programs during development. However, they are associated with both undifferentiated and differentiated states repressing target genes involved in maintaining those programs.The deregulation of gene expression networks, responsible for normal cellular identity, growth and differentiation leads to cancer. The large majority of genome-wide association studies (GWAS) identify cancer risk loci outside of protein-coding regions. Of 301 single-nucleotide polymorphisms (SNPs) currently linked to cancer, only 12 (3.3%) change the protein amino-acid sequence. Most are located in the introns of protein-coding genes (40%) or intergenic regions (44%), raising the question of the function of these noncoding loci and their role in cancer development . These fMany studies have also shown that miRNA and LncRNAs themselves can function as tumor suppressor genes or oncogenes, –176. SevSimilarly to miRNAs, some lincRNAs are transcriptional targets of p53 like the lincRNA-p21 that plays a role as a transcriptional repressor in the p53 pathway by triggering apoptosis. The lincRNA-p21 binds to hnRNP-K that allows for the correct localization of hnRNP-K, probably by influencing their target preference, and therefore the transcriptional repression of p53-regulated genes . The prein vitro [Although most of the mechanisms that implicate lncRNA in cancer biology are uncovered, the growing available data show that they are probably linked for example to chromatin remodeling. For example lncRNAs that are known to be involved in the recruitment of epigenetic modifiers to specific loci such as ANRIL, XIST, HOTAIR and KCNQ1OT1 were observed to have modified expression in a variety of cancers . Also thin vitro  is downrThe broad functional classes of genes and regulatory pathways that involve ncRNA participation clearly justifies that the deregulation of their biogenesis and roles could are not restricted to cancer development . PerturbThe presented examples directly implicate long ncRNA and miRNAs in cancer biology and other human diseases and indicate that a complex interplay between their biogenesis pathways, their regulatory mechanisms and their targets should be seriously taken into consideration not only in cancer research but in other human pathologies and also in the definition of future strategies of diagnostics and therapeutics.In the last years we have witnessed an unprecedented discovery of numerous functions of non-coding RNAs in eukaryotic cells ranging from gene expression regulation to genome imprinting roles that were previously attributed to proteins.This means that proteins are likely to cooperate with ncRNAs to control gene expression at different levels of regulation. They cooperate in the regulation of the transcription of genes encoding proteins, to process and maturate their transcripts and finally to regulate their mRNA stability and translation. Moreover, upstream of these regulatory steps, cooperation will also be required for altering DNA methylation profiles and the remodeling of chromatin contributing to epigenetic regulation. This means that complex networks between proteins and RNAs have been established during the course of evolution. We can envisage and speculate that due to their biochemical nature and biogenesis, ncRNAs will contribute to speed-up, make more flexible, transform and ultimately make more accurate the regulatory pathways conducted by regulatory proteins, pushing gene expression regulation to a new level. It is predictable that this complex regulatory web will have several hubs that will be composed of ncRNAs and proteins or alternatively only proteins or ncRNAs, which will also allow a rapid and better integration of different environmental signals. Although, the field of ncRNAs has been growing fast we are still far from understanding the complexity and the mechanisms underlying the establishment of the regulatory networks between RNAs and proteins.From the evolutionary point of view it seems that the “invention” of proteins like telomerase was a critical step in the establishment of accurate spatial and temporal regulatory processes which probably allowed the evolution of eukaryotic complexity and later on, the appearance of multicellularity. In the view of an “RNA World hypothesis” it is tempting to speculate that the first RNA activities were to maintain the viability and integrity of “cell precursors” defending them from destructive invader molecules; these ancestral defense functions of ncRNAs that are still present and operate in “modern cells” seem to have been extended to mechanisms of gene regulation.It should also be pointed out that the close analysis of different classes of ncRNAs (sncRNA and lncRNA), and the fact that we can detect biogenesis see  and funcFrom what has been compiled, the deregulation of biogenesis and functional roles of ncRNAs were, as expected at the crossroads of different human pathologies ranging from cancer to neurodegenerative and immune diseases. Finally the continued understanding of the molecular mechanisms and signaling pathways where ncRNAs participate should offer new insights to define new diagnostic strategies and open new avenues for therapies.


Arabidopsis with a special focus on their role in vascular development and functioning.In higher plants phloem and xylem are responsible for long-distance transport of water, nutrients, and signals that act systemically at short or long-distance to coordinate developmental processes. The formation of the plant vascular system is a complex process that integrates signaling events and gene regulation at transcriptional and posttranscriptional levels. Thanks to transcriptomic and proteomic analysis we start to better understand the mechanisms underlying the formation and the functioning of the vascular system. The role of the DNA-binding with one finger (Dof TFs), a group of plant-specific transcription factors, recently emerged as part of the transcriptional regulatory networks acting on the formation and functioning of the vascular tissues. More than half of the members of this TF family are expressed in the vascular system. In addition some of them have been proposed to be mobile proteins, suggesting a possible role in the control of short- or long-distance signaling as well. This review summarizes the current knowledge on Dof TFs family in  The gradual colonization of the land through plant evolution has been possible thanks to the differentiation of complex vascular tissues that provide mechanical support and allow long-distance transport of water and nutrients. In land plants, vascular tissues are comprised of two conducting tissues the phloem and the xylem, and intervening cambium that can generate the different conducting cell types  transcription factor has been identified in maize (ZmDof1) and was shown to be involved in light response and transcriptional regulation of genes involved in carbon metabolism AAAG-3′ core . Along plant evolution, Dof transcription factors might have originated from a common ancestor, likely represented by the single Chlamydomonas reinhardtii gene, and then expanded in the different taxonomic groups of vascular plants through recurrent duplication events  so far analyzed eight to nine Dof TF genes are found     present only in the phloem of infected plants allowed the identification of a virus promoter functional in plants  to confer phloem-specific expression   potentially expressed in vascular tissue at different step of its development.The mining of RNA profiling datasets at the resolution of the vascular tissue  and vascular cell type  , or through plasmodesmata in a short distance (between cell types)  are potential mobile transcription factor RNAs.In addition, cell-type specific expression maps from A direct experimental proof of the intercellular movement of a non-cell-autonomous Dof TFs has recently been brought by Chen et al.  who showDof genes have been found expressed in the phloem, there is so far no evidence for long-distance transport since neither RNAs nor Dof proteins have been found in transcriptome and proteome datasets of phloem sap ], a differentiation-promoting transcription factor of the vascular system , which shows a precocious formation of the interfascicular cambium and its subsequent cell division, Guo et al. cambium initiation and activity  are suspected to be involved in the control of the maintenance of the cambium and/or cambial activity by controlling the cell cycle , and while looking for direct targets of AtDof3.4/OBP1 several core cell cycle genes and transcription factors were identified , CONSTANS (CO), and FLOWERING LOCUS T (FT) genes  have been shown to be expressed in the vasculature throughout the plant and were shown to negatively regulate the transcription of CO  specific H+-sucrose symporter  . Altogether these data suggest that interactions between Dof TFs and TFs belonging to other families are theoretically possible. Experimentally, OBP1 was shown to directly interact with the promoter of a bZIP transcription factor but also with the promoter of a member of the same family AtDof2.3 (Zhang et al., Arabidopsis (Zhao et al., REVOLUTA have also been demonstrated (Kim et al., Arabidopsis vein formation (Gardiner et al., Structurally, like for other zinc fingers, the Dof domain is known to be a bi-functional domain that mediates not only DNA-binding but also protein–protein interactions (Yanagisawa, In the context of the vascular system development and functioning, which integrates a large variety of internal or external stimuli, regulators are needed to fine-tune the responses to changes in the environment. In this review, we present evidences that the Dof TFs family is likely to participate in such fine-tuning of the responses to the different stimuli in the vascular system. By modulating a variety of transcription factors, by protein-DNA and/or protein–protein interactions, the Dof TFs could therefore act at the cross-talk of various developmental pathways directly or indirectly linked to the vascular development and/or functioning. One of the next challenges, besides the functional characterization of the still undescribed Dofs TFs, will therefore be identifying the actors involved in an integrated regulatory network. Combination of the high-throughput data collection at the resolution of the vascular cell types and developmental stages as well as the use of the next generation sequencing technology will certainly allow identifying genes network regulating the fundamental features of the vascular development.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Arabidopsis thaliana protein FPA is a rare example of a trans-acting regulator of poly(A) site choice. Analysing fpa mutants therefore provides an opportunity to reveal generic consequences of disrupting this process. We used direct RNA sequencing to quantify shifts in RNA 3′ formation in fpa mutants. Here we show that specific chimeric RNAs formed between the exons of otherwise separate genes are a striking consequence of loss of FPA function. We define intergenic read-through transcripts resulting from defective RNA 3′ end formation in fpa mutants and detail cryptic splicing and antisense transcription associated with these read-through RNAs. We identify alternative polyadenylation within introns that is sensitive to FPA and show FPA-dependent shifts in IBM1 poly(A) site selection that differ from those recently defined in mutants defective in intragenic heterochromatin and DNA methylation. Finally, we show that defective termination at specific loci in fpa mutants is shared with dicer-like 1 (dcl1) or dcl4 mutants, leading us to develop alternative explanations for some silencing roles of these proteins. We relate our findings to the impact that altered patterns of 3′ end formation can have on gene and genome organisation.Alternative cleavage and polyadenylation influence the coding and regulatory potential of mRNAs and where transcription termination occurs. Although widespread, few regulators of this process are known. The  Arabidopsis thaliana. Studying FPA therefore provides an opportunity to understand not only its roles in plant biology but also the generic consequences of disrupting alternative polyadenylation. In this study, we use a technique called direct RNA sequencing to quantify genome-wide shifts in poly(A) site selection in plants that lack FPA function. One of our most striking findings is that in the absence of FPA we detect chimeric RNAs formed between two otherwise separate and well-characterised genes.The ends of almost all eukaryotic protein-coding genes are defined by a poly(A) signal. When genes are transcribed into mRNA by RNA polymerase II, the poly(A) signal guides cleavage of the precursor mRNA at a particular site; this is accompanied by the addition of a poly(A) tail to the mRNA and termination of transcription. Many genes have more than one poly(A) signal and the regulated choice of which to select can effectively determine what the gene will code for, how the gene can be regulated and where transcription termination occurs. We discovered a rare example of a regulator of poly(A) site choice, called FPA, while studying flower development in the model plant  Eukaryotic mRNA 3′ ends are defined by a protein complex that cleaves pre-mRNA in close association with RNA polymerase II (Pol II) and adds a poly(A) tail to the free 3′ end The selection of alternative cleavage and polyadenylation sites defines different 3′ ends within pre-mRNAs transcribed from a single gene and can therefore affect function by determining coding potential and the inclusion of regulatory sequence elements. Termination efficiency can also affect transcript levels, possibly because termination facilitates recycling of transcription complexes A less well-studied phenomenon, which suggests an additional role for regulated 3′ end formation and transcription termination, is the existence of chimeric transcripts formed between exons of neighbouring genes encoded on the same chromosomal strand trans-acting regulator of RNA 3′ end formation, but is not a conserved component of either the splicing or the cleavage and polyadenylation apparatus A. thaliana mutants disrupted in factors that mediate RNA 3′ end formation are late flowering as a result of the specific misregulation of FLCFLC is unknown, but increased FLC transcription in fpa mutants is accompanied by increased levels of alternatively processed non-coding antisense RNAs (asRNAs) at the FLC locus fpa mutants were isolated in a screen designed to identify factors required for RNA silencing AtSN1) in fpa mutants can be explained by read-through resulting from defective 3′ end formation at an upstream gene The spen family protein FPA is a A. thaliana using direct RNA sequencing (DRS), thereby refining our understanding of 3′ end formation in this model organism fpa mutants with DRS: (1) could we clarify the roles of FPA in plant biology (particularly in relation to flowering and RNA silencing)?; and (2) could we define generic consequences of defective RNA 3′ end regulation that would be of broad relevance? Here we identify the abundance and sites of 3′ end formation of RNAs transcribed antisense to the floral regulator FLC, but do not detect evidence of a widespread role for FPA in RNA-mediated chromatin silencing. We identify the generic consequences of disrupting regulated RNA 3′ end formation, prominent among which is the formation of specific chimeric RNAs between exons of otherwise separate and well-characterised genes. In addition, we make the unexpected but related discovery that transcription termination defects in fpa are shared at some of the same loci in both dcl1 and dcl4 mutants. Consequently, we suggest an alternative explanation involving defective upstream termination for the previously reported DCL1-mediated silencing of overlapping gene pairs There is intense interest in determining how the widespread alternative patterns of RNA 3′ end formation can be regulated and what the consequences of disrupting specific regulators may be. We recently defined genome-wide patterns of cleavage and polyadenylation in A. thaliana [Columbia-0 (Col-0) accession] and fpa-7 loss-of-function mutants to DRS. RNA was prepared from 14-day-old whole seedlings. A total of 22,560,508 WT and 24,383,585 fpa-7 reads that map polyadenylated RNA 3′ ends were aligned uniquely to the most recent A. thaliana genome release (currently TAIR10). A summary of the read statistics is given in www.compbio.dundee.ac.uk/polyADB/.We subjected total RNA purified from three biological replicates of WT fpa mutant DRS datasets. The expression of 18,406 protein-coding genes was detected in WT A. thaliana; DESeq analysis suggested that 1,114 genes were differentially expressed in the fpa-7 mutant , down-regulation of the floral pathway integrator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1  and up-regulation of the floral repressor FLC  were readily detected  target sites Although FPA has been reported to play a widespread role in RNA-mediated chromatin silencing n TAIR10 . Since wfpa-7 (P = 2.63e−32). Recent re-annotation of At5g35935 defined a newly arisen pseudogene psORF  within this sequence fpa-7 of psORF, rather than the transposon, that is detected by DRS , but not FCA were dependent on FPA function FPA RNA itself, but only in the transfer (T)-DNA-induced fpa-7 allele , a gene encoding a histone demethylase specific for H3K9 dimethylation and monomethylation IBM1 distal site in fpa mutants (P = 0.03); this was confirmed by RNA gel blot hybridisation  signals located within intergenic regions.DRS extends previous studies by identifying the cleavage and polyadenylation sites of read-through RNAs. We analysed the sequence features of intergenic cleavage sites and, although the relatively small size of the detected . We prevdetected . Becausefpa mutants that result in transcription of novel RNAs antisense to expressed protein-coding genes  in which sense strand gene expression was increased and 13 cases in which it was decreased when increased DRS reads mapping antisense to promoters in fpa-7 were detected. Overall, we conclude that novel asRNAs generated in fpa mutants are not necessarily associated with gene silencing. However, we also identify loci where detailed experimental analysis is required to uncover potentially distinct regulatory consequences of novel asRNAs.We identified 14 read-through RNAs in ng genes , but in ng genes . Since ianalysis  identififpa-7 mutant we found a reduction in the number of reads aligning to the 3′ end of At3g59060  compared to WT, but the number of reads mapping to the 3′ end of the downstream gene At3g59050  was increased  detected the formation of chimeric RNAs specifically in fpa mutants  or the intergenic sequence (γ′)  . The difnce (γ′) . None of mutants . The 5′ F5 3′UTR  but, as F5 3′UTR . This suin fpa-8 . These dfpa-7 and fpa-8 mutant alleles, chimeric PIF5–PA03 RNAs were detected in an early-flowering flc-3 fpa-7 double mutant but were generally not found in other late-flowering mutants  that have conserved peptide open reading frames in their 5′UTRs . Despite these similarities, chimeric RNAs were found at PIF5, but not PIF4  in fpa mutants. Failsafe termination at this site was recently shown to depend upon DCL4 FCA read-through RNAs similar to those previously found in dc14 mutants were detected here in fpa mutants , are also found fpa mutants, using RT-PCR to reveal read-through at the 3′ end of At1g51390, for example  site choice reveal it to be an important level at which gene expression can be regulated. Here we used DRS to assess the consequences of disrupting regulated 3′ end formation dependent on the spen family protein FPA.FLCFLC expression, in which other factors that mediate RNA cleavage and polyadenylation are also disrupted, suggests a critical role for RNA 3′ end formation in FLC regulation FLC mRNA itself appears to be largely unaffected by these mutations. Instead, attention has focused on a potential role for asRNA processing in FLC regulation because polyadenylated asRNA expression at the FLC locus is also changed in these mutants FLC regulation. For example, the positive correlation of sense and asRNA expression detected here may be a consequence of gene-looping events that juxtapose promoters and terminators that are each characterised by nucleosome-free regions FLC locus makes experimental separation of these events particularly challenging. DRS sheds new light on these complex issues because it can unequivocally score the strand of origin and simultaneously define and quantify multiple sites of RNA 3′ end formation fpa mutants, while increased levels of asRNAs that read-through to cleavage sites antisense to the FLC promoter were clearly detected in fpa mutants. These findings are not easily reconciled with a model that suggests proximal processing of asRNA triggers FLC chromatin silencing FLC regulation and, while the DRS data might restrict some models of interpretation, greater sequencing depth and characterisation of further RNA species are also required. For example, it is unclear whether RNAs engaged in R-loops at this locus  The function of FPA in flowering ultimately depends on its control of the floral repressor fpa mutants, was not obligatorily associated with reduced sense strand expression, but we also identified cases in which increased cleavage and polyadenylation antisense to promoter regions in fpa-7 mutants was associated with either increased or reduced sense strand expression. Therefore, one explanation for the identification of FPA as a factor required for RNA silencing may be that defective 3′ end formation, arising either within the transgenes used for the screen or at endogenous genes close to the site of transgene insertion, affects the efficacy of the silencing trigger from the transgene itself psORF, a recently acquired pseudogene fpa mutant allele. The silencing of such recently duplicated sequences in different backgrounds is poorly studied, but recent work suggests that epialleles may exist in either different accessions or different generations. In other words, de novo originated genes might be prone to epigenetic variation in the early stages of their formation psORF in fpa-7 and not in fpa-8, and also did not detect misregulation of asRNAs at the newly acquired At1TE93275 transposable element in this fpa-7 dataset, it is unclear whether FPA is genuinely involved in the control of such newly arisen sequences.A second proposed function for FPA is in RNA-mediated chromatin silencing A. thaliana, we also asked whether this analysis could reveal generic consequences of defective RNA 3′ end formation. We detected changes in intronic cleavage sites, intergenic read-through and chimeric RNA formation, thus defining a range of events that inform future analyses of the impact of such regulators in other species. These findings also have broader implications because they suggest how 3′ end formation might affect the evolution of gene structure and gene order through regulated 3′UTR sequences, new exon combinations or de novo evolution of new genes. We  signals (activated here in fpa mutants) may derive from genuine alternative polyadenylation, unannotated genes or transposons or may instead reflect the evolution of high quality intergenic poly(A) sites to trap runaway read-through RNAs. Such read-through to intergenic poly(A) signals may contribute to the de novo evolution of new or orphan genes from non-coding genomic regions Besides addressing specific biological roles of FPA in IBM1. Alternative polyadenylation of IBM1 pre-mRNA controls the activity of the histone demethylase encoded by this gene IBM1 poly(A) site choice is also dependent on DNA methylation: the IBM1 intronic DNA sequence downstream of the proximal poly(A) site is heavily methylated and alternative polyadenylation is profoundly altered in met1 and cmt3 mutants IBM1 still needs to be established. However, the opposing alternative polyadenylation phenotypes of met1 and cmt3, on the one hand, and fpa mutants, on the other, suggest that A. thaliana IBM1 could be a genetically tractable model system to dissect the interplay between DNA methylation, intragenic heterochromatin and poly(A) site selection. It will be interesting to determine whether other FPA-dependent alternative polyadenylation events are also related to DNA methylation. It may be relevant that methyl-CpG binding domain protein 9 (MDB9) binds to regions of FLC chromatin coincident with the sites of alternative polyadenylation of FLC asRNAs and influences FLC expression and DNA methylation One strikingly clear shift in poly(A) site choice was detected at fpa mutants was the formation of chimeric RNAs. Conceptually related chimeric RNAs have previously been discovered trans-acting factor mediating their formation has been found. Therefore, one of the advances we make here is to reveal that a consequence of losing regulated 3′ end formation can be the transcription of specific chimeric RNAs. We do not yet know whether FPA-dependent chimeric RNAs are regulated in vivo or are simply the consequence of loss-of-function in fpa mutants. Regardless, chimeric RNAs have been overlooked in genome-wide studies of 3′ end formation until now trans-acting factor that affects specific chimeric RNAs, it should now also become possible to design switches that reversibly convert two genes into one in a controllable way and that may have biotechnological applications.A striking consequence of read-through we discovered in fpa mutants, it is likely that our algorithm identifying reciprocal changes in expression at tandem protein-coding genes underestimated the number of potential chimeric RNAs. For example, Pol II read-through could result in complex transcripts comprised of more than two genes (including non-protein-coding RNAs), which terminate at a site other than the 3′ end of an annotated protein-coding gene. Indeed, a transcript previously detected in fpa fca double mutants Although it is clear that chimeric RNAs were formed at only a limited number of sites in fpa mutants is the stable accumulation of RNAs that are cleaved and polyadenylated in intergenic regions, revealing that FPA ultimately promotes transcription termination. Our analysis of these read-through events led us to make the unexpected discovery that FPA and DCL proteins share termination targets. DCL4, an RNase III-like protein previously established to function in processing small RNAs, was recently also shown to function in transcription termination at the FCA locus FCA locus in fpa mutants, revealing that termination at this locus requires not only DCL4 but also FPA. Since FPA does not affect DCL4 expression, this suggests that termination at certain loci requires multiple, specific trans-acting factors in addition to the torpedo exonuclease and the cleavage and polyadenylation machinery FCA locus in dc14 mutants also occurs antisense to an overlapping gene, At4g16270 trans to silence At1g51402 gene expression dc11 mutants can explain these findings. This set of discoveries raises the general question of whether some aspects of dcl mutant phenotypes are a direct consequence of defects in Pol II transcription termination.A clear molecular phenotype associated with loss-of-function By focusing our analysis on polyadenylated RNA 3′ ends, we set out to investigate the generic consequences of disrupting a regulator of alternative polyadenylation. We have documented examples of such alterations here, but it will now be interesting to study the mechanism by which FPA mediates this control. Crucial to this is the identification of the immediate, not simply ultimate, targets of FPA function. Furthermore, sequencing RNA from individual cells of different tissues and across circadian rhythms of expression is likely to be required for the comprehensive identification of transcripts affected by FPA.A. thaliana genes require re-annotation A. thaliana genome annotation will require diverse datasets that include DRS and, for example, RNA-seq and chromatin modification data. The meta-analysis of data from these different technologies nevertheless requires appropriate alignment software specific for each sequencing technology. Single molecule sequencing is more susceptible to mismatches and deletions and so requires indel-aware alignment software. A recent analysis of DRS data is fundamentally flawed because it used a version of the Bowtie alignment software with parameters that are wholly inappropriate for DRS data analysis A. thaliana transcriptome, it is likely that de novo annotation of control transcriptomes prepared in parallel, rather than improved genome annotation alone, will be required to comprehensively analyse changes in the sequences and levels of RNA molecules resulting from altered RNA processing.We recently proposed that the 3′ ends of thousands of fpa-7 (SALK_021959) and the flc-3 mutant were provided by R. Amasino (Madison). The fpa-8 mutant (induced by EMS and containing a point mutation leading to a premature stop codon) and fy-2 were provided by C. Dean (John Innes Centre), and fve-3 was provided by J. Martínez-Zapater (Madrid). The WT strain Col-0 and the T-DNA insertion lines upf1–5 (SALK_112922), sr45–1 (SALK_004132) and flk-1 (SALK_007750), as well as ld-1, were obtained from NASC (UK). fld-3 and ref6–3 were provided by S. Michaels (Indiana University) and Y.S. Noh , respectively. A. thaliana WT Col-0, fpa-7 and fpa-8 mutant seeds were sown in MS10 plates, stratified for 2 days at 4°C and germinated in a controlled environment at a constant temperature of 24°C under 16 h light/8 h dark conditions. Seedlings were harvested 14 days after transfer to 24°C.The T-DNA insertion line fpa-8 backgrounds using the FirstChoice RLM-RACE Kit (Ambion), with or without TAP treatment, according to the manufacturer's instructions. Fragments obtained by 5′RACE were cloned into the pGEM-T Easy (Promega) vector and then sequenced.Samples were prepared for DRS as described previously http://sourceforge.net/projects/openhelisphere/files/helisphere/).Sequencing datasets described in this study have been deposited at the European Nucleotide Archive (ENA): Study, PRJEB3993; accession no, ERP003245. Raw DRS sequences were aligned using open source HeliSphere software  normalisation, i.e. each DRS read was assigned a weight in such a way that the sum of all weights in a sample condition was equal to 1×106. We called the weights ‘normalised reads’. All images of normalised read alignments were made using the Integrated Genome Browser In order to compare WT and An algorithm that accommodates intergenic DRS reads in automated gene re-annotation was carried out as previously described P values for the features to be non-DE based on the hypothesis that replicated expression levels of the features are distributed according to negative binomial distribution. These P values were adjusted using Benjamini-Hochberg multiple testing corrections fpa-7.The DESeq (version 1.8.2) package fpa). A total of 18,406 protein-coding genes were identified. Once the requirement of a minimum raw read count of 15 was applied to all replicates, 15,081 protein-coding genes remained for the DE analysis. P values calculated by DESeq for each coding gene with a P value of <0.01 to be considered as differentially expressed. Counts of DRS reads mapping to re-annotated protein-coding genes were prepared for three replicates of each genotype  peaks for intergenic read-through analysis and our list of DE coding genes . P valuefpa). We selected gene pairs with expression levels higher than 0.5 RPM (this corresponds to roughly 11–12 raw reads within a gene) for both genes in the pair and for both genotypes. Pairs with a DESeq P value of <0.01 for which the upstream gene was down-regulated and the downstream gene was up-regulated in the fpa mutant were considered as candidates for chimeric RNA formation.Uniquely aligned and filtered reads and re-annotated coding genes were used for this analysis. We prepared a list of neighbouring genes with normalised read counts for every gene for both genotypes (WT and We prepared a dataset of poly(A) sites, corresponding to the 3′ ends of aligned and filtered DRS reads, by applying smoothing and peak-finding algorithms as previously described centroid = ΣEiPi/ΣEi, where Ei is the expression level of ith poly(A) peak within each region and Pi is the genomic coordinate of the peak. P values between WT and fpa were calculated for intergenic regions with at least 11 raw reads in a replicate. At the final step, intergenic regions with P values<0.05 and centroid positions not within 30 bp downstream of the coding gene 3′ end in the fpa mutants were classified as DE. The 30-bp offset corresponds to the median length of DRS reads; by applying this additional filter we therefore excluded situations where intergenic reads were mainly grouped near the 3′ end of the upstream gene and hence may belong to the gene.We defined intergenic regions as regions between protein-coding genes and excluded genomic regions of transposable gene elements, pseudogenes and non-coding genes. The analysis was based on uniquely aligned, filtered and smoothed poly(A) sites and our re-annotation of coding genes. We separately prepared poly(A) peaks for each replicate, clustered poly(A) peaks within a 4-bp window and selected poly(A) peaks with at least 0.5 RPM expression. We removed poly(A) peaks within protein-coding genes, transposable gene elements, pseudogenes and non-coding genes. Since we did not re-annotate the transposable gene elements, pseudogenes and non-coding genes, we also excluded poly(A) peaks within 50 bp downstream of the 3′ ends of these genomic features. We assigned two parameters to every intergenic region: the total sum of poly(A) peaks within the region (and for each replicate); and the position of the centroid of the poly(A) peaks in a region as Pfpa-7.In this analysis, we compared expression levels of individual poly(A) peaks in the two genotypes rather than the total reads aligned to a gene. The comparison of peaks is complicated by over-calling peaks (i.e. peaks being identified where there should be no peak) and the fact that the centre of biologically equivalent peaks may be called in slightly different positions in different genotypes as a consequence of the varied read depth and the peak-calling algorithm. P value of <0.01 were considered to be DE in the fpa-7 mutant. These comprised 6% of the selected poly(A) peaks: 2,538 down-regulated and 2,671 up-regulated. Due to the data partition between two neighbouring peaks in one genotype and/or incorrect peak matching, our algorithms can produce ‘false positives’. For example, in fpa-7. The peak-finding algorithm incorrectly combined poly(A) sites belonging to the bump on the right-hand slope of the peak to the most highly expressed poly(A) peak in fpa-7 and created a new peak in WT. Since this WT peak was matched to 0 in fpa-7, it was wrongly classified as DE by DESeq. We therefore designed an algorithm to identify and exclude these ‘false positive’ peaks: if a poly(A) peak was up-regulated in one genotype, we looked for poly(A) peaks within an 8-bp window in the other genotype; if we found poly(A) peaks in the genotype that were not associated with the DE poly(A) peak and the summed expression of the peaks was significantly different (20% higher than the associated poly(A) peak only), then we defined this DE poly(A) peak as a ‘false positive’. This led to 37% of down-regulated and 32% of up-regulated DE peaks being excluded from further analysis. We also applied this method to select intronic cleavage sites that were differentially used in fpa-7 compared to WT. We then only considered peaks that comprised >10% of expression at a particular gene and manually excluded sites that mapped to 3′UTR introns.Having identified peak pairs, we applied DESeq to all pairs with at least 8 raw reads in a replicate. This gave 86,699 poly(A) peak pairs. Peak pairs with a 3′RACE was performed using the FirstChoice RLM-RACE Kit Protocol (Ambion) according to the manufacturer's instructions. The reaction was started with 250 ng poly(A)+ RNA. Multiple PCR products were purified, cloned into the pGEM-T Easy vector (Promega) and sequenced.ChIP was performed as previously described All primers used in this study are listed in Figure S1fpa mutant backgrounds in polyadenylated RNAs transcribed in the vicinity of the FLC locus. (A) DRS Reads mapping to FLC locus. Normalised reads are presented for wild-type (WT) and fpa A. thaliana. Genes are orientated 5′–3′; exons are denoted by rectangles, UTRs by adjoining narrower rectangles and introns by lines. The image of normalised read alignments was made using the Integrated Genome Browser FLC lncasRNAs in fpa-7 and WT was based on a low number of reads at surrounding sites, while the distal ‘D’ cleavage sites were clearly defined. In contrast to our findings, reduced levels of proximally polyadenylated lncasRNAs in fpa mutants relative to WT have been reported et al. expressed proximally polyadenylated transcripts as a fraction of the total lncasRNA transcripts fpa mutants, the proportion of total lncasRNA polyadenylated at the proximal site is indeed reduced, but the DRS data indicate that there is no clear change to proximal cleavage site usage. (B) Enlarged image of normalised DRS reads that mapped to lncasRNAs cleaved and polyadenylated antisense to the FLC promoter. The enlarged region is boxed in et al. (2010) is shown by a green line and sites selected by this primer are coloured green. The single nucleotide polymorphism (SNP) described by Coustham et al. (2012) is also displayed (red triangle). The preferred distal cleavage site has cis-elements typical of preferred cleavage sites in A. thaliana 3′UTRs FLC transcription start site that is associated with changes in vernalisation requirement, flowering time and the abundance of cold-induced distally polyadenylated lncasRNA Changes between WT and (PDF)Click here for additional data file.Figure S2fpa-7. (A) Analysis of DNA methylation pattern in fpa-7 at At5g35935 from data published previously psORF can be observed in fpa-7psORF is lost in fpa-7 (see B) Analysis of DRS reads at the At1TE93275 locus. Misregulation of antisense RNAs at the newly acquired helitron transposable element At1TE93275 has previously been reported in fpa mutants fpa-7 dataset. Normalised reads mapping to the different loci are presented for wild-type (WT) and fpa. The top panel displays the reads corresponding to the (+) strand while the bottom panel displays the reads corresponding to the (−) strand. Exons are denoted by rectangles, UTRs by adjoining narrower rectangles and introns by lines. Transposable elements are in red and the UA228 segment in green.Differentially expressed transposons between wild-type and pa-7 see . CG meth(PDF)Click here for additional data file.Figure S3A) Location of the poly(A) sites ‘T’ due to the insertion in fpa-7. (B) Additional cleavage sites found within intron 4 upstream of the fpa-7 T-DNA insertion. DRS reads that map to fpa-7 T1 sites are displayed. The positions of 3′RACE products are indicated above the DNA sequence by red triangles (numbers indicate how many 3′RACE clones were obtained). Images of normalised read alignments were made using the Integrated Genome Browser C) Additional cleavage sites found within intron 5 upstream of the fpa-7 T-DNA insertion. DRS reads that map to fpa-7 T2 sites are displayed. Positions of 3′RACE products are indicated on the DNA sequence by red triangles (the numbers indicate how many 3′RACE clones were obtained). (D) Additional cleavage sites found within intron 5 upstream of the fpa-7 T-DNA insertion. Positions of 3′RACE products are marked above the DNA sequence by red triangles (the numbers indicate how many 3′RACE clones were obtained), while DRS reads that aligned to the FPA T-DNA junction are marked by blue triangles (with the number of raw reads shown above). A black line indicates the T-DNA sequence. (E) 3′RACE analysis of the mom1–3 mutant. Positions of 3′RACE products are marked on the DNA sequence by red triangles (numbers indicate how many 3′RACE clones were obtained). As the T-DNA mutant mom1–3 also displayed cryptic cleavage sites upstream of its T-DNA insertion, it is possible that premature cleavage and polyadenylation may form part of the mechanism by which T-DNAs disrupt gene function; this raises the general question of how cryptic sites are triggered. (F) Structure of the T-DNA. The left and right borders  are indicated by red boxes. NPTII is the kanamycin resistance gene used for selection.FPA-dependent alternative polyadenylation events. ((PDF)Click here for additional data file.Figure S4A) Predicted IBM1 protein domain organisation encoded by mRNAs cleaved and polyadenylated at promoter proximal and distal poly(A) sites. (B) Reads mapping to the locus encoding IBM1. Promoter proximal ‘P’ and distal ‘D’ alternative poly(A) sites are indicated. The probe used for RNA gel blot analysis of alternatively polyadenylated transcripts is indicated in red C) RNA gel blot analysis of IBM1 alternatively polyadenylated transcripts. β-TUBULIN (β-TUB.) was used as an internal control. (D) Reads mapping to At1g56500. Promoter proximal ‘P’ and distal ‘D’ alternative poly(A) sites are indicated. The probe used for RNA gel blot analysis of alternatively polyadenylated transcripts is indicated by a red line. (E) The predicted protein domain organisation encoded by At1g56500 mRNAs cleaved and polyadenylated at promoter proximal and distal poly(A) sites. At1g56500 encodes a putative hydrolase, but RNA cleavage at the proximal site eliminates a sequence encoding the putative TlpA-like TRX domain and three NHL  repeats, while the sequence encoding the hydrolase domain (HAD-like) is retained. (F) RNA gel blot analysis of At1g56500 alternatively polyadenylated transcripts. β-TUBULIN (β-TUB.) was used as an internal control. Quantification of the bands revealed that At1g56500 polyadenylation at the distal and proximal sites is 18% and 82%, respectively, in fpa-7; 15% and 85% in fpa-7 flc-3; and 30% and 70% in wild-type (WT). (G) Reads mapping to At5g35170. Promoter proximal ‘P’ and distal ‘D’ alternative poly(A) sites are indicated. The probe used for RNA gel blot analysis of alternatively polyadenylated transcripts is indicated by a red line. (H) The predicted protein domain organisation encoded by At5g35170 mRNAs cleaved and polyadenylated at promoter proximal and distal poly(A) sites. At5g35170 encodes a putative adenylate kinase, but RNA cleavage at the proximal site eliminates a domain of unknown function (DUF1995). (I) RNA gel blot analysis of At5g35170 alternatively polyadenylated transcripts. β-TUBULIN (β-TUB.) was used as an internal control. The asterisk indicates a non-specific hybridisation signal. Quantification of the bands revealed that At5g35170 polyadenylation at the distal and proximal sites is 81% and 19%, respectively, in fpa-7; 92% and 8% in WT; 73% and 27% in fpa-7; and 75% and 25% fpa-7 flc-3. (J) Distally polyadenylated transcripts at At5g35170 exhibit various cryptic splicing sites in the region located between exons 7 and 12 (purple box). Details of the region located between exons 7 and 12 are shown for each isoform. Images of normalised read alignments were made using the Integrated Genome Browser FPA affects intronic cleavage site selection. ((PDF)Click here for additional data file.Figure S5A) Histogram of the read-through percentage in fpa-7 for protein-coding genes with differentially expressed downstream intergenic regions. (B-E) Characterisation of MKK5 intergenic read-through RNAs. (B) Normalised reads mapping to the MKK5 locus. The red dashed line indicates the region amplified by RT-PCR. (C) RNA gel blot analysis of MKK5 read-through transcripts. The probe used is indicated by a red solid line in (B). (D) Transcripts are either cleaved and polyadenylated in the annotated 3′UTR or at the intergenic sites, as determined by sequencing the cloned RT-PCR products. Red rectangles represent the 3′UTR specific to the read-through transcript and red lines represent 3′UTR introns. (E) Identification of contiguous RNA from the MKK5 gene upstream to the intergenic cleavage sites. RT-PCR products were separated on agarose gels and stained with ethidium bromide. Amplification controls for genomic DNA contamination (RT-) are included. Three biological replicates  were used for each genotype: wild-type (WT) and fpa-7. (F–Q) Selection of tested read-through RNAs.  Normalised reads mapping to the analysed loci. Red dashed lines indicate the region amplified by RT-PCR. Images of normalised read alignments were made using the Integrated Genome Browser G, J, M, P) Identification of contiguous RNAs from the upstream gene to the intergenic poly(A) sites. RT-PCR products were separated on agarose gels and stained with ethidium bromide. Amplification controls for genomic DNA contamination (RT-) are included. Three biological replicates  were used for each genotype: WT and fpa-7.  Transcripts are either cleaved and polyadenylated in the annotated 3′UTR or at the intergenic sites, as determined by sequencing the cloned RT-PCR products. Narrower red rectangles represent 3′UTRs specific to the read-through transcripts. (Q) Read-through at At5g66450 is accompanied by cryptic splicing that alters the protein-coding sequence.FPA affects intergenic read-through. ((PDF)Click here for additional data file.Figure S6A–F) Left panels represent normalised reads mapping to the analysed loci. Red dashed lines indicate the regions amplified by RT-PCR. Images of normalised read alignments were made using the Integrated Genome Browser fpa-7. Bottom right panels schematically show transcripts either cleaved and polyadenylated in the annotated 3′UTR or at the intergenic sites, as determined by sequencing the cloned RT-PCR products. Narrower red rectangles represent the 3′UTR section specific to the read-through transcript and red lines indicate the 3′UTR introns.Schematic representation of a selection of tested read-through RNAs. ((PDF)Click here for additional data file.Figure S7PIF5–PA03 chimeric RNA formation. (A) RNA gel blot analysis of PIF5–PA03 chimeric RNAs in fpa and flc mutants. P, PIF5; C, PA03 transcripts. β-TUBULIN (β-TUB.) was used as an internal control. Probes used are shown in B,C) RT-qPCR analysis of PIF5–PA03 chimeric RNAs in fpa and flc (B) and late-flowering (C) mutants. Location of the RT-qPCR amplicon is displayed in (D) by a red dotted line and labelled qPCR. PIF5–PA03 chimeric RNAs were quantified by RT-qPCR analysis of the expression of the intergenic fragment between PIF5 and PA03. Data are the means ± SEM obtained for three independent PCR amplifications of three biological replicates. The y-axis shows the fold change relative to wild-type  after normalisation to UBC21 gene expression. *, P<0.05; Student's t-test. (D) Structure of the PIF5–PA03 locus: exons are denoted by yellow rectangles, UTRs by narrower black rectangles and introns by grey lines; regions analysed for splicing efficiency and accuracy are indicated . The red dashed line indicates the location of the RT-qPCR amplicon. (E) Expression levels of PIF5 spliced RNA for primer sets 1, 2, 3 and 4; their location is displayed in (D). Histograms show means ± SEM obtained from three PCR reactions for each of three biological replicates. (F) Electropherograms for primer sets 1, 2, 3 and 4. The forward primer in each set was 6-FAM labelled. Numbers on the x-axis represent size markers in nucleotides; numbers on the y-axis represent relative fluorescence ×103, reflecting transcript abundance. Electropherograms are representative of three biological and three PCR replicates. The main splicing product is identified by its size. No significant changes in the ratios of alternatively spliced transcripts were observed between WT and fpa-8. (G) Normalised reads mapping to loci encoding PIF4 and PA02. Images of normalised read alignments on the left panel were made using the Integrated Genome Browser PIF4 and PA02 is displayed but does not reveal formation of chimeric RNAs at this locus in fpa-7. The region amplified by RT-PCR is indicated by a red dashed line. RT-PCR products were separated on agarose gels and stained with ethidium bromide. Amplification controls for genomic DNA contamination (RT-) are included. Three biological replicates  were used for each genotype: WT and fpa-7. UBIQUITIN LIGASE 21 (UBC21) was used as a control, as well as gDNA for the chimeric RNA analysis. (H) RT-PCR analysis of the contiguous RNA between PIF5 and PA03 detected in fpa-7 but not in dc11–11. UBIQUITIN LIGASE 21 (UBC21) was used as a control. Three biological replicates  were used for each genotype: WT and fpa-7.Analysis of the potential causes of (PDF)Click here for additional data file.Figure S8A) Normalised reads mapping to the At1g02475–At1g02470 loci. Exons are denoted by rectangles, UTRs by adjoining narrower rectangles and introns by lines. Images of normalised read alignments were made using the Integrated Genome Browser (B) Chimeric RNAs identified by RT-PCR. The region amplified by RT-PCR is indicated by a red dashed line. RT-PCR products were separated on agarose gels and stained with ethidium bromide. Amplification controls for genomic DNA contamination (RT-) are included. Three biological replicates  were used for each genotype: wild-type (WT) and fpa-7. (C) Schematic representation of the chimeric RNAs, as determined by sequencing the cloned RT-PCR products. A red line depicts splicing events. (D) RT-qPCR analysis of the different RNAs expressed at the At1g02475–At1g02470 loci in fpa mutants. Data are the means ± SEM obtained for three independent PCR amplifications on three biological replicates. The y-axis shows the fold change relative to wild-type (set to 1) after normalisation to UBC21 gene expression.Analysis of chimeric RNAs formed between At1g02475 and At1g02470. ((PDF)Click here for additional data file.Figure S9A) Normalised reads mapping to the At2g32320–At2g32340 loci. Exons are denoted by rectangles, UTRs by adjoining narrower rectangles and introns by lines. Images of normalised read alignments were made using the Integrated Genome Browser B) Chimeric RNAs identified by RT-PCR. The region amplified by RT-PCR is indicated by a red dashed line. RT-PCR products were separated on agarose gels and stained with ethidium bromide. Amplification controls for genomic DNA contamination (RT-) are included. Three biological replicates  were used for each genotype: wild-type (WT) and fpa-7. (C) Schematic representation of the chimeric RNAs, as determined by sequencing the cloned RT-PCR products. A red line depicts splicing events. (D) RT-qPCR analysis of the different RNAs expressed at the At2g32320–At2g32340 locus in fpa mutants. Data are the means ± SEM obtained for three independent PCR amplifications of three biological replicates. The y-axis shows the fold change relative to WT (set to 1) after normalisation to UBC21 gene expression. Chimeric RNAs formed between At2g32320 and At2g32340 did not alter the coding potential of the upstream gene but changed the effective 3′UTR.Analysis of chimeric RNAs formed between At2g32320 and At2g32340. ((PDF)Click here for additional data file.Figure S10A) Normalised reads mapping to the At3g06700–At3g06690 loci. Exons are denoted by rectangles, UTRs by adjoining narrower rectangles and introns by lines. Images of normalised read alignments were made using the Integrated Genome Browser B) Chimeric RNAs identified by RT-PCR. The region amplified by RT-PCR is indicated by a red dashed line. RT-PCR products were separated on agarose gels and stained with ethidium bromide. Amplification controls for genomic DNA contamination (RT-) are included. Three biological replicates  were used for each genotype: wild-type (WT) and fpa-7. (C) Schematic representation of the chimeric RNAs, as determined by sequencing the cloned RT-PCR products. A red line depicts splicing events. (D) RT-qPCR analysis of the different RNAs expressed at the At3g06700–At3g06690 loci in fpa mutants. Data are the means ± SEM obtained for three independent PCR amplifications of three biological replicates. The y-axis shows the fold change relative to WT (set to 1) after normalisation to UBC21 gene expression. Chimeric RNAs formed between At3g06700 and At3g06690 did not alter the coding potential of the upstream gene but changed the effective 3′UTR.Analysis of chimeric RNAs formed between At3g06700 and At3g06690. ((PDF)Click here for additional data file.Figure S11A) Normalised reads mapping to the At1g74880–At1g74875 locus. Exons are denoted by rectangles, UTRs by adjoining narrower rectangles and introns by lines. Images of normalised read alignments were made using the Integrated Genome Browser B) Chimeric RNAs were identified by RT-PCR. The region amplified by RT-PCR is indicated by a red dashed line. RT-PCR products were separated on agarose gels and stained with ethidium bromide. Amplification controls for genomic DNA contamination (RT-) are included. (C) Schematic representation of the chimeric RNAs, as determined by sequencing of cloned RT-PCR products. A red line depicts splicing events. (D) RT-qPCR analysis of the different RNAs expressed at the At1g74880–At1g74875 locus in fpa mutants. Data are the means ± SEM obtained for three independent PCR amplifications on three biological replicates. The y-axis shows the fold change relative to wild-type  after normalisation to UBC21 gene expression. The significance of this validated example of chimeric RNA formation (At1g74875 and At1g74880) is less clear because the downstream gene is relatively poorly characterised; it may reflect annotation errors rather than chimeric RNA formation between two well-characterised genes.Analysis of putative chimeric RNAs formed between At1g74880 and At1g74875. ((PDF)Click here for additional data file.Figure S12fpa-7 is identified if it is within +/−5 bp. Peaks A and B were matched. If two peaks are found, then the first is taken as the equivalent peak and the second is assigned to zero. Peaks C1 and D were matched; peak C2 is matched with 0 in WT. Images of normalised read alignments were made using the Integrated Genome Browser Illustration of the peak combination and peak-matching algorithms. (A) Grouping algorithm: (i) stylised smoothed read profile; (ii) peaks called by our algorithm as previously described (PDF)Click here for additional data file.Figure S13Example of a ‘false positive’ differentially expressed poly(A) peak and matched poly(A) peaks at At2g28000. Top and bottom panels, smoothed raw poly(A) sites mapping to At2g28000; middle panels, poly(A) peaks called from these smoothed poly(A) profiles. All data were subject to read-per-million (RPM) normalisation. Images of normalised read alignments were made using the Integrated Genome Browser (PDF)Click here for additional data file.Table S1A. thaliana genome release, TAIR10.Classification of reads mapping to (XLSX)Click here for additional data file.Table S2fpa-7. Normalised reads  are displayed for wild-type (WT) and fpa-7. * When WT read number is ‘0’, fold change is set to ‘1,000,000’.List of protein-coding genes differentially expressed in (XLSX)Click here for additional data file.Table S3fpa-7. Normalised reads  are displayed. * When wild-type (WT) read number is ‘0’, fold change is set to ‘1,000,000’.List of transposons differentially expressed in (XLSX)Click here for additional data file.Table S4fpa-7. Normalised reads  are displayed. * When wild-type (WT) read number is ‘0’, fold change is set to ‘1,000,000’.List of Poly(A) peaks differentially expressed in (XLSX)Click here for additional data file.Table S5fpa-7. Normalised reads  are displayed. * When wild-type (WT) intergenic read number is ‘0’, distance for WT is set to ‘−1’.List of the intergenic regions differentially expressed in (XLSX)Click here for additional data file.Table S6fpa-7. Normalised reads  are displayed. * When the antisense gene is not expressed, N/A is indicated.List of the intergenic regions with differentially expressed peaks in (XLSX)Click here for additional data file.Table S7fpa-7. Normalised reads  are displayed. * When wild-type (WT) read number is ‘0’, fold change is set to ‘1,000,000’.List of differentially expressed peaks located in promoters of genes differentially expressed in (XLSX)Click here for additional data file.Table S8List of putative chimeric RNAs. Normalised reads  are displayed.(XLSX)Click here for additional data file.Table S9List of tested putative chimeric RNAs. Normalised reads  are displayed.(XLSX)Click here for additional data file.Table S10List of primers used in this study.(XLSX)Click here for additional data file.


Repressible promoters are a useful tool for down-regulating the expression of genes, especially those that affect cell viability, in order to study cell physiology. They are also popular in biotechnological processes, like heterologous protein production.Pichia pastoris promoters of different strength: PSER1, PMET3, PTHR1, PPIS1 and PTHI11. eGFP was expressed under the control of each of these promoters and its fluorescence could be successfully decreased in liquid culture by adding different supplements. We also expressed the essential genes with different native promoter strength, ERO1 and PDI1, under the control of two of the novel promoters. In our experiments, a clear down-regulation of both repressible promoters on transcriptional level could be achieved. Compared to the transcript levels of these two genes when expressed under the control of their native promoters, only ERO1 was significantly down-regulated.Here we present five novel repressible Our results show that all of the novel promoters can be used for repression of genes in liquid culture. We also came to the conclusion that the choice of the repressible promoter is of particular importance. For a successful repression experiment it is crucial that the native promoter of a gene and the repressible promoter in its non-repressed state are of similar strength. Pichia pastoris is a favored microorganism for the production of heterologous proteins in biotechnology. It has gained its popularity, because this yeast is easy to manipulate, can grow to high cell densities on cheap cultivation media and is well suited for the production of secreted proteins [P. pastoris has also gained increasing interest as a model for cell biological studies of higher eukaryotic cells [The methylotrophic yeast proteins ,2. Recenic cells -5.For biotechnological processes as well as for fundamental research, controlling of gene expression is required. In the industrial production of proteins inducible or repressible promoters are desirable, as they enable the separation of the cell growth phase from the protein production phase. Inducible/repressible promoters are also a useful tool for studying the function of essential genes, where the cells would not survive a complete knock-out.Saccharomyces cerevisiae. There, the most prominent candidates are the promoters of the genes MET3 (repressed by methionine), PHO5 (down-regulated by inorganic phosphate), CUP1 (responsive to Cu2+ ions), and the GAL genes  [P. pastoris are those of the methanol utilization pathway, which enable high expression levels. These promoters are well suited for recombinant protein production, but not for studying gene function in cell biology. The best known is the alcohol oxidase 1 (AOX1) promoter which is activated on methanol and repressed on glucose [P. pastoris promoters by cultivating strains on different carbon sources . Among them, especially PTHI11 has been shown to be independent from regulation by the tested carbon sources, but was repressed in presence of thiamine [S. cerevisiae[P. pastoris we identified other potential repressible promoters , which all could be of interest for studying physiology of the cell.Studies on repressible promoters are quite rare in yeast, and have mostly been performed with glucose) . However glucose . As the thiamine . Based oerevisiae-12 and oTHI11, SER1, MET3, THR1 and PIS1 (Table P. pastoris genomic DNA (wild type X-33) and fused to the gene of the enhanced green fluorescent protein (eGFP) as a reporter. P. pastoris strains expressing eGFP under the control of these promoters were cultivated in the synthetic M2 medium either without or with respective repressing supplements, in order to test the regulation of the chosen promoters by these compounds. Fluorescence of all strains was measured by flow cytometry during the exponential growth phase.In this study we tested five different promoters for their ability to be repressed under certain cultivation conditions. Promoter sequences of the genes P. pastoris promoter PMET3 responded to addition of L-methionine and the initial fluorescence was fully down-regulated in the presence of this amino acid. The ability of the MET3 promoter to be repressed by addition of methionine was already shown for S. cerevisiae and Ashbia gossypii[MET3 promoter was used for short-term studies on cell physiology in S. cerevisiae as shown in Dünkler and Wendland [MET3 for the regulation of the promoter expression in liquid culture cultivations for a period of ~20 h with 10 mM methionine. The higher methionine concentration is possibly the explanation for the longer repression period of this promoter.The  gossypii,12. DünkSER1 promoter can be repressed by addition of L-serine to the medium, but only to 30% under the cultivation conditions of these experiments. In case of the thiamine repressible promoter PTHI11 there was no residual fluorescence in presence of thiamine after background correction with the fluorescence of the wild type X-33  promoter. The fluorescence of the promoter PGAP was set to the value 100%, and that of the others was normalized to this value. The promoters of the genes THR1 and MET3 appear to have similar strength (13% of PGAP) and show ~10 times higher expression of eGFP than the PSER1 promoter under the described cultivation conditions. Under non-repressing conditions, the PPIS1 promoter was determined to be three times stronger than PTHR1 and PMET3. The promoter of THI11 was the strongest among the analyzed repressible promoters according to eGFP fluorescence measurements. The PTHI1 promoter exhibits ~63% of the GAP promoter strength under the described cultivation conditions. The fluorescence achieved with the weakest (PSER1), compared to the strongest (PTHI11) promoter, is almost 50 times lower  than PDI1 (~35% of the GAP promoter) in P. pastoris . In each case addition of the repressing supplements caused a reduction of the transcription of the respective gene. The measured residual expression was ~10% for PDI1 under the control of PTHI11 and ~40% for ERO1 when expressed under the control of PTHR1 , but not of the promoter PTHR1 (data not shown).We could also observe a significant transcript level down-regulation of the promoters PP. pastoris (wild type strain X-33) promoters (1000 bp upstream of the ATG start codon) of the genes THI11 , MET3 , SER1 , THR1  and PIS1  were amplified from X-33 genomic DNA and cloned into the pPuzzle vector [AOX1 terminator locus of the P. pastoris genome after linearization with AscI in the respective sequence. After transformation by electroporation positive transformants were selected on YPD agar plates with 25 mg/L Zeocin.P. pastoris genes ERO1 (PAS_chr1-1_0011) and PDI1 (PP7435_Chr4-0183), and 500 bp of their respective promoter regions (starting −200 bp upstream from the ATG) were amplified by PCR from X-33 genomic DNA 2HPO4, 0.03 g of CaCl2.2H2O, 0.8 g of KCl, 0.5 g of MgSO4.7H2O, 2 mL of biotin (0.2 g L-1), 1.5 mL of trace salts stock solution. The pH was set to 5.0 with 5 M KOH solution. Trace salts stock solution contained per liter: 6.0 g of CuSO4.5H2O, 0.08 g of NaI, 3.0 g of MnSO4.H2O, 0.2 g of Na2MoO4.2H2O, 0.02 g of H3BO3, 0.5 g of CoCl2, 20.0 g of ZnCl2, 5.0 g of FeSO4.7H2O, and 5.0 mL of H2SO4 (95-98% w/w).M2 minimal medium contained per liter: 20 g of glucose, 20 g of citric acid, 3.15 g of (NH600 = 0.1 and incubated in 100 mL shake flasks at 28°C with 170 rpm (rotations per minute). Fluorescence of the cells was measured after 24 h of cultivation. All clones were supplemented two more times (after ~12 h) with the respective components (concentration as described below).All analyzed clones were grown over night in 5 mL M2 medium as pre-culture. Main cultures were inoculated to an ODTHI11 10 mM of thiamine hydrochloride (Merck), for PSER1 10 mM of L-serine (Serva), for PMET3 10 mM of L-methionine (Serva), for PTHR1 10 mM of L-threonine (Serva), L-leucine (Serva), L-isoleucine (Serva) and L-valine (Serva), and for PPIS1 100 μM inositol or 1.5 μM zinc sulfate (Merck).For comparing the non-repressed and repressed state of a promoter, cultivations of same clones in the M2 medium with and without supplements were performed. For repressing the promoters following concentrations of supplements were added to the medium: for PFor selection on plates, 2% agar was added to the M2 medium and the pH was set to ~ 7.600 of 0.4 were harvested by centrifugation and resuspended in 2 mL PBS. Flow cytometry analysis was performed using a FACS Canto (Becton Dickinson) with these settings: excitation wavelength at 488 nm, emission wavelength at 530 nm . Measured fluorescence was referred to the cell size.1 mL cells of an ODACT1) was used as reference gene for normalization. Each gene was correlated to actin as internal control, and the respective non-repressed strain served as the reference strain for each relative transcript level determination with the delta-delta Ct method [RNA isolation, cDNA synthesis and measurement of mRNA transcript levels using real time PCR was performed as described in . Samplest method . RT PCR The authors declare to have no competing interests.MD contributed to selecting the promoter sequences, performed the experimental work and drafted the manuscript. DM coordinated the project and contributed to drafting the manuscript. BG designed the study and contributed to drafting the manuscript. All authors read and approved the final manuscript.