The RNA World Hypothesis suggests that prebiotic life revolved around RNA instead of DNA and proteins. Although modern cells have changed significantly in 4 billion years, RNA has maintained its central role in cell biology. Since the discovery of DNA at the end of the nineteenth century, RNA has been extensively studied. Many discoveries such as housekeeping RNAs (rRNA, tRNA, etc.) supported the messenger RNA model that is the pillar of the central dogma of molecular biology, which was first devised in the late 1950s. Thirty years later, the first regulatory non-coding RNAs (ncRNAs) were initially identified in bacteria and then in most eukaryotic organisms. A few long ncRNAs (lncRNAs) such as H19 and Xist were characterized in the pre-genomic era but remained exceptions until the early 2000s. Indeed, when the sequence of the human genome was published in 2001, studies showed that only about 1.2% encodes proteins, the rest being deemed "non-coding." It was later shown that the genome is pervasively transcribed into many ncRNAs, but their functionality remained controversial. Since then, regulatory lncRNAs have been characterized in many species and were shown to be involved in processes such as development and pathologies, revealing a new layer of regulation in eukaryotic cells. This newly found focus on lncRNAs, together with the advent of high-throughput sequencing, was accompanied by the rapid discovery of many novel transcripts which were further characterized and classified according to specific transcript traits.In this review, we will discuss the many discoveries that led to the study of lncRNAs, from Friedrich Miescher's "nuclein" in 1869 to the elucidation of the human genome and transcriptome in the early 2000s. We will then focus on the biological relevance during lncRNA evolution and describe their basic features as genes and transcripts. Finally, we will present a non-exhaustive catalogue of lncRNA classes, thus illustrating the vast complexity of eukaryotic transcriptomes.

The positioning of nucleosomes within the coding regions of eukaryotic genes is aligned with respect to transcriptional start sites. This organization is likely to influence many genetic processes, requiring access to the underlying DNA. Here we show that the combined action of Isw1 and Chd1 nucleosome spacing enzymes is required to maintain this organization. In the absence of these enzymes regular positioning of the majority of nucleosomes is lost. Exceptions include the region upstream of the promoter, the +1 nucleosome and a subset of locations distributed throughout coding regions where other factors are likely to be involved. These observations indicated that ATP-dependent remodeling enzymes are responsible for directing the positioning of the majority of nucleosomes within the Saccharomyces cerevisiae genome.

Cryptic unstable transcripts (CUTs) are synthesized from intra-and intergenic regions in Saccharomyces cerevisiae and are rapidly degraded by RNA surveillance pathways, but their function(s) remain(s) elusive. Here, we show that an antisense TY1 CUT, starting within the Ty1 retrotransposon and encompassing the promoter 5 long terminal repeat (LTR), mediates RNA-dependent gene silencing and represses Ty1 mobility. We show that the Ty1 regulatory RNA is synthesized by RNA polymerase II, polyadenylated, and destabilized by the cytoplasmic 5 RNA degradation pathway. Moreover, the Ty1 regulatory RNA represses Ty1 transcription and transposition in trans by acting on the de novo transcribed TY1 RNA. Consistent with a transcriptional regulation mechanism, we show that RNA polymerase II occupancy is reduced on the Ty1 chromatin upon silencing, although TBP binding remains unchanged. Furthermore, the Ty1 silencing is partially mediated by histone deacetylation and requires Set1-dependent histone methylation, pointing out an analogy with heterochromatin gene silencing. Our results show the first example of an RNA-dependent gene trans-silencing mediated by epigenetic marks in S. cerevisiae. Recent data have shown that cryptic unstable transcripts (CUTs) are RNA polymerase II (RNAPII)-dependent noncoding RNAs (ncRNAs) corresponding to inter-and intragenic regions of the genome and may represent 10% of intergenic transcripts in Saccharomyces cerevisiae (Wyers et al. 2005). Under normal conditions, CUTs are almost undetectable, as they are rapidly degraded by the activity of Rrp6 and Trf4, members of the nuclear exosome and the TRAMP complex, respectively (LaCava et al. 2005;Vanacova et al. 2005;Wyers et al. 2005). In addition to Trf4 and Rrp6, the cytoplasmic 5Ј-3Ј exoribonuclease Xrn1 also plays an important role in the turnover of CUTs, supporting the idea that some of these transcripts escape the nuclear quality control and might have a cytoplasmic residency (Thompson and Parker 2007).Despite these observations, the function(s) of CUTs remain(s) poorly characterized. Cryptic transcription has been widely described from yeast to human and qualified as "transcriptional noise." Interestingly, it has been proposed that cryptic transcription allows RNA polymerase-dependent chromatin changes but not the production of functional RNA molecules, as those are immediately degraded (Struhl 2007). In agreement with this model, CUT transcription has been shown to interfere with promoters of coding regions and hence regulates gene expression in S. cerevisiae (Martens et al. 2005;Hongay et al. 2006;Uhler et al. 2007). However, alternative models emerged, providing a direct function for cryptic transcripts. Indeed, recent reports have shown that the processing of siRNAs in Schizosaccharomyces pombe is mediated by homologs of the TRAMP and exosome subunits (Buhler et al. 2007;Nicolas et al. 2007), strongly indicating that the fission yeast's siRNAs might originate from CUTs. It is tempting to hypothesize that CUTs in S. cerevisiae are link...

Set1-dependent H3K4 di-and tri-methylation (H3K4me2/3) have been associated with active transcription. Recent data indicate that the H3K4me2/3 also plays a poorly characterized RNA-dependent repressive role. Here, we show that GAL1 promoter is attenuated by the H3K4me2/3 deposited by cryptic transcription. The H3K4me2/3 delay the recruitment of RNA polymerase II (RNAPII) and TBP on GAL1 promoter. Inactivation of RNA decay components revealed the existence of the RNAPII-dependent unstable RNAs, initiating upstream of GAL1 (GAL1ucut). GAL1ucut RNAs are synthesized in glucose and require the Reb1 transcription factor. Consistent with a regulatory function of the cryptic transcription, Reb1 depletion leads to a decrease of H3K4me3 on GAL10-GAL1 locus in glucose and to an acceleration of GAL1 induction. A candidate approach shows that the RPD3 histone deacetylase attenuates GAL1 induction and is tethered at the GAL10-GAL1 locus by H3K4me2/3 upon repression. Strikingly, Set1-dependent Rpd3 recruitment represses also the usage of a hidden promoter within SUC2, suggesting a general function for H3K4me2/3 in promoter fidelity. Our data support a model wherein certain promoters are embedded in a repressive chromatin controlled by cryptic transcription.

SummaryThe circular Escherichia coli chromosome is organized by bidirectional replication into two equal left and right arms (replichores). Each arm occupies a separate cell half, with the origin of replication (oriC) at mid-cell. E. coli MukBEF belongs to the ubiquitous family of SMC protein complexes that play key roles in chromosome organization and processing. In mukBEF mutants, viability is restricted to low temperature with production of anucleate cells, reflecting chromosome segregation defects. We show that in mukB mutant cells, the two chromosome arms do not separate into distinct cell halves, but extend from pole to pole with the oriC region located at the old pole. Mutations in topA, encoding topoisomerase I, do not suppress the aberrant positioning of chromosomal loci in mukB cells, despite suppressing the temperature-sensitivity and production of anucleate cells. Furthermore, we show that MukB and the oriC region generally colocalize throughout the cell cycle, even when oriC localization is aberrant. We propose that MukBEF initiates the normal bidirectional organization of the chromosome from the oriC region.

Although histone H3 Lysine 4 methylation (H3K4me) is strongly associated with active transcription, an increasing number of arguments indicate its repressive role in gene expression. Recent data in the mammalian and budding yeast systems have provided evidence for H3K4me2 and H3K4me3 tethering histone deacetylase complexes (HDACs) to modulate gene expression. In S. cerevisiae, this regulation is mediated by specific subunits within HDACs that recognize the methylation status of H3K4 allowing chromatin reorganization to attenuate or repress transcription. Albeit we are still a long way from understanding the mechanism and biological consequences, it is becoming clear that H3K4me at certain chromatin loci may prevent aberrant gene expression or modulate transcriptional response. This review will provide a brief overview of a novel interpretation of H3K4me and its outcome on transcription regulation and will suggest future challenges for the field of epigenetics.

The SANT domain is a nucleosome recognition module found in transcriptional regulatory proteins, including chromatin-modifying enzymes. It shows high functional degeneracy between species, varying in sequence and copy number. Here, we investigate functions in vivo associated with two SANT motifs, SANT and SLIDE, in the Saccharomyces cerevisiae Isw1 chromatin-remodeling ATPase. We show that differences in the primary structures of the SANT and SLIDE domains in yeast and Drosophila melanogaster reflect their different functions. In yeast, the SLIDE domain is required for histone interactions, while this is a function of the SANT domain in flies. In yeast, both motifs are required for optimal association with chromatin and for formation of the Isw1b complex (Isw1, Ioc2, and Ioc4). Moreover, nucleosome remodeling at the MET16 locus is defective in strains lacking the SANT or SLIDE domain. In contrast, the SANT domain is dispensable for the interaction between Isw1 and Ioc3 in the Isw1a complex. We show that, although defective in nucleosome remodeling, Isw1 lacking the SANT domain is able to repress transcription initiation at the MET16 promoter. Thus, chromatin remodeling and transcriptional repression are distinct activities of Isw1.In eukaryotic organisms, DNA is folded into a highly compacted chromatin structure. This packing restricts the accessibility of DNA to various nuclear factors involved in DNAtemplated processes, including transcription. There are several mechanisms that regulate chromatin structure, among them covalent modifications of histones provided by histone-modifying enzymes and disruption of histone-DNA interactions by ATP-dependent chromatin-remodeling enzymes. Although these two classes of chromatin-modifying factors are functionally distinct, their subunits share conserved motifs, such as the chromodomain, bromodomain, plant homeodomain finger, and SANT domain (56). These domains are involved in histone tail recognition and binding, thus providing recruitment of these enzymes to targeted regions within the chromatin. The bromodomain shows highly selective binding to acetylated lysines (24,25,47). The chromodomain and plant homeodomain finger bind to methylated lysines (17, 27, 28, 34-36, 39, 49). In each case, the specific binding is defined by a hydrophobic pocket, formed by aromatic residues and surrounded by residues determining the specificity of binding.In contrast, our understanding of the function of the SANT domain is still restricted to examples that often prove to be protein specific. Historically, the SANT domain was identified based on its homology to the DNA-binding domain of c-Myb (1). It consists of three alpha-helices arranged in a helix-turnhelix motif; each helix contains a conserved, bulky aromatic residue that plays a key role in helix packing. Although structurally similar, the SANT domain and the canonical Myb DNA-binding domain are functionally divergent (21,26,48).The DNA-contacting residues in Myb are not conserved in SANT and often prove to be incompatible with DNA ...

The human immunodeficiency virus type 1 (HIV-1) integrase is an essential enzyme in the life cycle of the virus and is therefore an attractive target for the development of new antiviral drugs. Among them, inhibitors which are capable of targeting the preassembled integrase/DNA complex are of particular interest, because they could suppress integrase activity in the context of the HIV-1 preintegration complex. Here, we study the mechanism of action of 11-mer oligonucleotides, which are efficient inhibitors of the catalytic activity of integrase, provided that they are conjugated to a hydrophobic compound, acridine. To understand the mechanism of the conjugate inhibitory action, we used a steady-state fluorescence anisotropy assay, which allowed us to study the stability of the integrase/DNA complex in various conditions. We found that oligonucleotide-acridine conjugates induced the efficient dissociation of preassembled integrase/DNA complexes. The simultaneous presence of both acridine and an oligonucleotidic moiety is required for the inhibitory activity of conjugates. However, the dissociation effect is not dependent on the oligonucleotide sequence. Finally, our results suggest that the conjugates bind directly to integrase within its complex with DNA at a site different from the viral DNA binding site.