Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Roles for Transcript Leaders in Translation and mRNA Decay Revealed by Transcript Leader Sequencing

ABSTRACT: Transcript leaders (TLs) can have profound effects on mRNA translation and stability. To map TL boundaries genome-wide, we developed TL-Sequencing (TL-Seq), a technique combining enzymatic capture of m7G-capped mRNA 5'-ends with high-throughput sequencing. TL-Seq identified mRNA start sites for the majority of yeast genes and revealed many examples of intragenic TL heterogeneity. Transcription initiation sites occur in at least 5% of protein-coding regions and are concentrated near the 5'ends of ORFs. These truncated mRNAs are translated, based on ribosome density analysis. Translation Associated TL-Seq (TATL-Seq), which combines TL-Seq with polysome fractionation, revealed substantial differences in translation of alternative TL isoforms. Globally, while some TL features are associated with poor translation and nonsense-mediated mRNA decay (uAUGs, very short length), others (secondary structure, long length) have much less impact than predicted from analyses of individual genes. TL-Seq and TATL-Seq can be applied to any eukaryote to investigate TL-mediated regulation of gene expression. TL-Seq (+/- pyrophosphatase), TATL-Seq (TL-Seq libraries from polysome gradient)

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Joshua Arribere

PROVIDER: E-GEOD-39074 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:We use mRNA-seq in combination with polysome profiling to determine translational status for all mRNAs in Drosophila mature oocytes and activated eggs. Puromycin-treated lysates are used as a negative control in polysome profiling experiments. Additionally, we use ribosome footprinting to globally measure translational efficiency of mRNAs in wild type mature oocytes as well as wild type and png mutant activated eggs. Lysates of hand-dissected Drosophila mature oocytes (containing ~540 M-NM-<g of total RNA) were subjected to separation by velocity sedimentation through sucrose gradients. In this way, free mRNAs (present in RNPs fraction) or those comigrating with ribosomal subunits (40S or 60S+80S fractions) or with varying numbers of bound ribosomes (low polysomes (2-4 ribosomes), medium polysomes (5-9 ribosomes), and heavy polysomes (more than 10 ribosomes) can be separated based on their size and collected as sucrose gradient fractions. To compare quantitatively the levels of every mRNA across the polysome gradient fractions, we added 5ng of S. cerevisiae mRNA as an exogenous spike-in to each of the six fractions of interest: RNPs, 40S, 60S+80S, low polysomes, medium polysomes and heavy polysomes. RNA was extraced from these fractions, follwing proteinase K treatment, by hot acid phenol method. In case of unfractionated lysates, RNA was extracted using TRIzol (Invitrogen) according to manufacturerM-bM-^@M-^Ys instructions. mRNA-seq samples were prepared from 1 M-NM-<g of total RNA (in case of sucrose gradient fractions and unfractionated lysates) and subject to Illumina based sequencing. Puromycin-treated lysates of mature oocytes or 0-2h Drosophila activated eggs (containing ~540 M-NM-<g of total RNA) were also subjected to separation by velocity sedimentation through sucrose gradients. Puromycin causes premature termination of elongating ribosomes and thus it can be used to determine whether the mRNAs co-sedimenting with the polysomal peaks (defined here as M-bM-^IM-%5 ribosomes) were actively engaged in translation. As an independent approach to assess translation and obtain information on the position of ribosomes on mRNAs, we employed ribosome footprinting. In addition to analyzing the same samples, as by polysome profiling, we also analyzed png mutant activated eggs by ribosome footprinting. Ribosome footprint profiling measures the number of ribosome-protected fragments (RPFs) derived from the mRNAs of each gene, resulting in a singular value of translational efficiency (TE) for each gene (TE=RPF/RNA).

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Roles for Transcript Leaders in Translation and mRNA Decay Revealed by Transcript Leader Sequencing

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