Graphical Abstract Highlights d GRFs control transcription initiation fidelity and suppress pervasive transcription d Ectopic initiation in the absence of Rap1 has variegated effects on gene expression d Ectopic transcription initiation correlates with altered nucleosome positioning d Rap1 suppresses transcription initiation by a steric hindrance mechanism
The Set1 family of histone H3 lysine 4 (H3K4) methyltransferases is highly conserved from yeast to human. Here we show that the Set1 complex (Set1C) directly binds RNA in vitro through the regions that comprise the double RNA recognition motifs (dRRM) and N-SET domain within Set1 and its subunit Spp1. To investigate the functional relevance of RNA binding, we performed UV RNA crosslinking (CRAC) for Set1 and RNA polymerase II in parallel with ChIP-seq experiments. Set1 binds nascent transcripts through its dRRM. RNA binding is important to define the appropriate topology of Set1C distribution along transcription units and correlates with the efficient deposition of the H3K4me3 mark. In addition, we uncovered that Set1 binds to different classes of RNAs to levels that largely exceed the levels of binding to the general population of transcripts, suggesting the Set1 persists on these RNAs after transcription. This class includes RNAs derived from SET1, Ty1 retrotransposons, specific transcription factors genes and snRNAs (small nuclear RNAs). We propose that Set1 modulates adaptive responses, as exemplified by the post-transcriptional inhibition of Ty1 retrotransposition.
The competition between the connectivity and the local or global order in model fully flexible chain molecules is investigated by molecular-dynamics simulations. States with both missing (melts) and high (crystal) global order are considered. Local order is characterized within the first coordination shell (FCS) of a tagged monomer and found to be lower than in atomic systems in both melt and crystal. The role played by the bonds linking the tagged monomer to FCS monomers (radial bonds), and the bonds linking two FCS monomers (shell bonds) is investigated. The detailed analysis in terms of Steinhardt's orientation order parameters Ql (l = 2 - 10) reveals that increasing the number of shell bonds decreases the FCS order in both melt and crystal. Differently, the FCS arrangements organize the radial bonds. Even if the molecular chains are fully flexible, the distribution of the angle formed by adjacent radial bonds exhibits sharp contributions at the characteristic angles θ ≈ 70°, 122°, 180°. The fractions of adjacent radial bonds with θ ≈ 122°, 180° are enhanced by the global order of the crystal, whereas the fraction with 70° ~/< θ ~/< 110° is nearly unaffected by the crystallization. Kink defects, i.e., large lateral displacements of the chains, are evidenced in the crystalline state.
Highlights d Degradation of ncRNAs by the exosome frees bound termination factors Nab3 and Nrd1 d Exosome inhibition triggers accumulation of ncRNAs complexed with Nab3 and Nrd1 d Out-titration of Nab3 and Nrd1 by ncRNAs decreases NNS co-transcriptional binding d A circular RNA decoy recapitulates the global impairment of NNS-dependent termination
Streptococcus agalactiae (Group B Streptococcus, GBS) causes life-threatening infections in newborns and adults with chronic medical conditions. Serotype IV strains are emerging both among carriers and as cause of invasive disease and recent studies revealed two main Sequence Types (STs), ST-452 and ST-459 assigned to Clonal Complexes CC23 and CC1, respectively. Whole genome sequencing of 70 type IV GBS and subsequent phylogenetic analysis elucidated the localization of type IV isolates in a SNP-based phylogenetic tree and suggested that ST-452 could have originated through genetic recombination. SNPs density analysis of the core genome confirmed that the founder strain of this lineage originated from a single large horizontal gene transfer event between CC23 and the hypervirulent CC17. Indeed, ST-452 genomes are composed by two parts that are nearly identical to corresponding regions in ST-24 (CC23) and ST-291 (CC17). Chromosome mapping of the major GBS virulence factors showed that ST-452 strains have an intermediate yet unique profile among CC23 and CC17 strains. We described unreported large recombination events, involving the cps IV operon and resulting in the expansion of serotype IV to CC23. This work sheds further light on the evolution of GBS providing new insights on the recent emergence of serotype IV.
A large share of the non-coding transcriptome in yeast is controlled by the Nrd1-Nab3-Sen1 (NNS) complex, which promotes transcription termination of noncoding RNA (ncRNA) genes, and by the nuclear exosome, which limits the steady state levels of the transcripts produced. How global ncRNA levels impinge on cellular functions is a longstanding and important question. Here we show that degradation of ncRNAs by the exosome is required for freeing Nrd1 and Nab3 from the released transcript after termination. In exosome mutants, these factors are sequestered by ncRNAs and cannot be efficiently recycled to sites of transcription, which induces termination defects genome-wide. ncRNA-dependent, genome-wide termination defects can be recapitulated by the expression of a degradationresistant, circular RNA containing a natural NNS target in exosome proficient cells.Our results have important implications for the mechanism of termination, the general impact of ncRNAs on cellular physiology and the importance of nuclear ncRNA degradation.Government through its "Investments for the Future" program.This work has benefited from the facilities and expertise of the high throughput sequencing core facility of I2BC (Centre de Recherche de Gif -http://www.i2bc-saclay.fr/). AUTHORS CONTRIBUTION Conceptualization, D.L., T.V., and A.J.; Methodology, T.V., M.B., and M-J.M.N.; Software, M.B.; Formal Analysis, M.B., T.V., and D.L.; Investigation, T.V., M-J.M.N., and D.L.; Writing -Original Draft, T.V. and D.L.; Writing -Review and Editing, T.V. and D.L.; Funding Acquisition, D.L.; Supervision, D.L.
mRNA homeostasis is favored by crosstalk between transcription and degradation machineries. Both the Ccr4-Not and the Xrn1-decaysome complexes have been described to influence transcription. While Ccr4-Not has been shown to directly stimulate transcription elongation, the information available on how Xrn1 influences transcription is scarce and contradictory. In this study we have addressed this issue by mapping RNA polymerase II (RNA pol II) at high resolution, using CRAC and BioGRO-seq techniques in Saccharomyces cerevisiae. We found significant effects of Xrn1 perturbation on RNA pol II profiles across the genome. RNA pol II profiles at 5' exhibited significant alterations that were compatible with decreased elongation rates in the absence of Xrn1. Nucleosome mapping detected altered chromatin configuration in the gene bodies. We also detected accumulation of RNA pol II shortly upstream of polyadenylation sites by CRAC, although not by BioGRO-seq, suggesting higher frequency of backtracking before pre-mRNA cleavage. This phenomenon was particularly linked to genes with poorly positioned nucleosomes at this position.Accumulation of RNA pol II at 3' was also detected in other mRNA decay mutants.According to these and other pieces of evidence, Xrn1 seems to influence transcription elongation at least in two ways: by directly favoring elongation rates and by a more general mechanism that connects mRNA decay to late elongation.
The fidelity of transcription initiation is essential for accurate gene expression, but the determinants of start site selection are not fully understood. Rap1 and other General Regulatory Factors (GRFs) control the expression of many genes in yeast. We show that depletion of these factors induces widespread ectopic transcription initiation within promoters. This generates many novel non-coding RNAs and transcript isoforms with diverse stability, profoundly altering the coding potential of the transcriptome. Ectopic transcription initiation strongly correlates with altered nucleosome positioning. We show that Rap1 sterically constrains nucleosomes as its mere binding to the DNA can be sufficient for restoration normal nucleosome positioning, transcription initiation and gene expression. These results demonstrate an essential role for GRFs in the fidelity of transcription initiation and in the suppression of pervasive transcription, redefining current models of their function. They have general implications for the mechanism of transcription initiation and the control of gene expression.
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