Sexual development in Schizosaccharomyces pombe culminates in meiosis and sporulation. We used ribosome profiling to investigate the translational landscape of this process. We show that the translation efficiency of hundreds of genes is regulated in complex patterns, often correlating with changes in RNA levels. Ribosome-protected fragments show a three-nucleotide periodicity that identifies translated sequences and their reading frame. Using this property, we identified 46 new translated genes ...[more]
Project description:CHX is an inhibitor of translation elongation often used in ribosome profiling experiments. There is evidence that CHX treatment of cells may cause artefacts in the distribution of ribosomes on mRNAs. We investigate this possibility in S. pombe by performing ribosome profiling in the presence and absence of this drug.
Project description:We present a combined experimental/computational technology to reveal a catalogue of functional regulatory elements embedded in 3’UTRs of human transcripts. We used a bidirectional reporter system coupled with flow cytometry and high-throughput sequencing to measure the effect of short, non-coding vertebrate-conserved RNA sequences on transcript stability and translation. Information-theoretic motif analysis of the resulting sequence-to-gene-expression mapping revealed linear and structural RNA cis-regulatory elements that positively and negatively modulate the post-transcriptional fates of human transcripts. We explored the functional role of 16332 short human 3'UTR sequences (C3U Library) evolutionarily conserved with P. troglodytes, M. musculus and G. gallus. For sorting the C3U library into subpopulations, we gated the population using FACS into bins each containing 10% of the total number of cells. We collected cells for the top four high expression bins (H10, H20, H30, H40) and the bottom four low expression bins (L10, L20, L30, L40) Multiple samples (C3U library subpopulations) were pooled for each illumina index (decribed n the 'library construction protocol' field). For example, six samples (A-L10 I & II, B-L10 I & II, A-L20 I & II) were all pooled in illumina index CGATGTAT. Therefore, each individual fastq file corresponds to multiple samples. The individual samples were sorted in the processing step using identifiers inside the sequence reads. One single pooled sample was run on both lanes (the extra lane was run for additional reads). The expression level (H: high, L:low or background) and library replicates (A or B) of the pooled library subpopulations are indicated in the sample title.
Project description:To estimate mRNA steady-state levels we used RNA extracted from logarithmically growing fisson yeast cells on Affymetrix Yeast 2.0 Genechip arrays. The signal intensities from two independent biological repeats were averaged, resulting in measurements for 4818 out of 4962 nuclear protein-coding genes.
Project description:Regulatory elements in the 3 untranslated regions (UTRs) of eukaryotic mRNAs influence mRNA localization, translation, and stability. The length of these regions is determined by the location at which mRNAs are cleaved and polyadenylated. The use of alternative polyadenylation sites is common, and can be regulated in different situations. I present here a new method to identify cleavage and polyadenylation sites (CSs) at the genome-wide level. The approach is strand-specific, avoids RNA enzymatic modification steps that can introduce sequence-specific biases, and uses molecular barcodes to ensure that every identified CS originates from an individual RNA molecule. I applied to create the first comprehensive genome-wide map of polyadenylation sites in the fission yeast Schizosaccharomyces pombe, comprising the analysis of over two million individual mRNAs that defined 10,422 major CSs. CSs were identified for 90% of coding genes and 15% of non-coding RNAs. Alternative polyadenylation was prevalent in both groups, with 61% and 49% of all detected genes, respectively, displaying more than one CS. The specificity of the cleavage reaction was gene-specific, resulting in highly variable levels of heterogeneity in the length of the 3' UTRs. Finally, I show that for both coding and non-coding genes the most common regulatory motif associated with CSs in fission yeast is the canonical human AAUAAA sequence.