Project description:We investigated the association of cryptic unstable transcripts (CUTs) to ribosomes in Saccharomyces cerevisiae. We used 5’cap-sequence followed by sucrose gradient fractionation of polyribosome fractions after ultracentrifugation to assess the relative association of transcripts to the ribosomes. We used 5PSeq approach, which measures ribosome dynamics by sequencing the presence of co-translation mRNA degradation intermediates, to assess if cryptic transcripts are engaged in active translation. We performed 5PSeq after glucose depletion to increase the number of ribosomes stop at the 5’UTR and start codon.
Project description:We sequenced both the stable (WT) and unstable (rrp6delta) transcriptomes of three S.cerevisiae strains: S288c, Σ1278b, JAY291 and the S.paradoxus strain N17 for de novo annotation of cryptic unstable transcripts (CUTs). Doing so we have greatly expanded on previous CUTs which were limited to the S.cerevisiae strain S288c and have provided the first assessment of CUT expression conservation in yeast
Project description:Poly(A) and CUT RNA fractions are compared using 3 'Long-SAGE deep-sequencing. ArrayExpress Release Date: 2008-12-19 Publication Title: Widespread bidirectional promoters are the major source of cryptic transcripts in yeast Publication Author List: Helen Neil, Christophe Malabat, Yves d'Aubenton-Carafa, Zhenyu Xu, Lars M. Steinmetz and Alain Jacquier Person Roles: submitter Person Last Name: Malabat Person First Name: Christophe Person Mid Initials: Person Email: christophe.malabat@pasteur.fr Person Phone: Person Address: Unité de Génétique des Interactions Macromoléculaires; CNRS, URA2171,F-75015, Paris, France Person Affiliation: Institut Pasteur
Project description:We investigated cryptic transcription start site usage, chromatin organization and post-transcriptional consequences in Saccharomyces cerevisiae. We used 5PSeq approach, which measures ribosome dynamics by sequencing the presence of co-translation mRNA degradation intermediates, to assess if cryptic transcripts are engaged in active translation. We show that chromatin-dependent cryptic transcripts can be recognized by ribosomes and have the potential to produce truncated polypeptides by using downs-stream, in-frame start codons. Our work suggests that a significant fraction of chromatin-dependent internal cryptic promoters are in fact alternative truncated mRNA isoforms.
Project description:Cryptic transcription is widespread and generates a heterogeneous group of RNA molecules of unknown function. To improve our understanding of cryptic transcription, we investigated their transcription start site usage, chromatin organization and post-transcriptional consequences in Saccharomyces cerevisiae. We show that transcription start sites of chromatin-dependent internal cryptic transcripts resemble those of protein coding genes in terms of DNA sequence, directionality and chromatin accessibility. We define the 5’ and 3’ boundaries of cryptic transcripts and show that, contrary to RNA degradation-dependent ones , they often overlap with the end of the gene thereby using the canonical polyadenylation site and associate to polyribosomes. In fact, we show that chromatin-dependent cryptic transcripts can be recognized by ribosomes and may produce truncated polypeptides from downstream, in-frame start codons. Our work suggests that a significant fraction of chromatin-dependent internal cryptic promoters are in fact alternative truncated mRNA isoforms. The expression of these chromatin-dependent isoforms is conserved from yeast to human expanding the functional consequences of cryptic transcription and proteome complexity.
Project description:mRNA degradation provides a powerful means for controlling gene expression during growth, development, and many physiological transitions in plants and other systems. Rates of decay help define the steady state levels to which transcripts accumulate in the cytoplasm and determine the speed with which these levels change in response to the appropriate signals. When fast responses are to be achieved, rapid decay of mRNAs is necessary. Accordingly, genes with unstable transcripts often encode proteins that play important regulatory roles. Although detailed studies have been carried out on individual genes with unstable transcripts, there is limited knowledge regarding their nature and associations from a genomic perspective, or the physiological significance of rapid mRNA turnover in intact organisms. To address these problems, we have applied cDNA microarray analysis to identify and characterize genes with unstable transcripts in Arabidopsis thaliana (AtGUTs). Our studies showed that at least 1% of the 11,521 clones represented on Arabidopsis Functional Genomics Consortium microarrays correspond to transcripts that are rapidly degraded, with estimated half-lives of less than 60 min. AtGUTs encode proteins that are predicted to participate in a broad range of cellular processes, with transcriptional functions being over-represented relative to the whole Arabidopsis genome annotation. Analysis of public microarray expression data for these genes argues that mRNA instability is of high significance during plant responses to mechanical stimulation and is associated with specific genes controlled by the circadian clock. Set of arrays that are part of repeated experiments Biological Replicate Computed
Project description:mRNA degradation provides a powerful means for controlling gene expression during growth, development, and many physiological transitions in plants and other systems. Rates of decay help define the steady state levels to which transcripts accumulate in the cytoplasm and determine the speed with which these levels change in response to the appropriate signals. When fast responses are to be achieved, rapid decay of mRNAs is necessary. Accordingly, genes with unstable transcripts often encode proteins that play important regulatory roles. Although detailed studies have been carried out on individual genes with unstable transcripts, there is limited knowledge regarding their nature and associations from a genomic perspective, or the physiological significance of rapid mRNA turnover in intact organisms. To address these problems, we have applied cDNA microarray analysis to identify and characterize genes with unstable transcripts in Arabidopsis thaliana (AtGUTs). Our studies showed that at least 1% of the 11,521 clones represented on Arabidopsis Functional Genomics Consortium microarrays correspond to transcripts that are rapidly degraded, with estimated half-lives of less than 60 min. AtGUTs encode proteins that are predicted to participate in a broad range of cellular processes, with transcriptional functions being over-represented relative to the whole Arabidopsis genome annotation. Analysis of public microarray expression data for these genes argues that mRNA instability is of high significance during plant responses to mechanical stimulation and is associated with specific genes controlled by the circadian clock. Set of arrays that are part of repeated experiments Keywords: Biological Replicate
Project description:Genome-wide translational profiling of rng3-65 compared to wild type cells. We used sucrose gradients to separate RNAs according to the number of associated ribosomes (a surrogate for translational efficiency). Preparation of the extracts and fractionation was carried out as described in Lackner et al, 2007 (Mol Cell 26(1):145-55). The fractions were pooled into four groups (1 closest to the top, i.e. not associated with ribosomes and 4 closest to the bottom, i.e., associated with polysomes). RNA was extracted from the pools and the corresponding pools from wild type and mutant cells were directly compared using DNA microarrays. Changes in translation are expected to alter the number of ribosomes associated with specific transcripts, and therefore result in a redistribution of the RNAs across the different fractions.