MTAIL-seq reveals dynamic poly(A) tail regulation in oocyte-to-embryo development
Ontology highlight
ABSTRACT:
ORGANISM(S): Drosophila melanogaster
SUBMITTER: Jaechul Lim
PROVIDER: E-GEOD-83732 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
mTAIL-seq reveals dynamic poly(A) tail regulation in oocyte-to-embryo development.
Lim Jaechul J Lee Mihye M Son Ahyeon A Chang Hyeshik H Kim V Narry VN
Genes & development 20160721 14
Eukaryotic mRNAs are subject to multiple types of tailing that critically influence mRNA stability and translatability. To investigate RNA tails at the genomic scale, we previously developed TAIL-seq, but its low sensitivity precluded its application to biological materials of minute quantity. In this study, we report a new version of TAIL-seq (mRNA TAIL-seq [mTAIL-seq]) with enhanced sequencing depth for mRNAs (by ∼1000-fold compared with the previous version). The improved method allows us to ...[more]
Similar Datasets
Project description:Eukaryotic mRNAs are subject to multiple types of tailing which critically influence mRNA stability and translatability. To investigate RNA tails at the genomic scale, we previously developed TAIL-seq, but its low sensitivity precluded its application to biological materials of minute quantity. In this study, we report a new version of TAILseq (mRNA TAIL-seq or mTAIL-seq) with enhanced sequencing depth for mRNAs (by ~1000 fold compared to the previous version). The improved method allows us to investigate the regulation of poly(A) tail in Drosophila oocytes and embryos. We find that maternal mRNAs are polyadenylated mainly during late oogenesis, prior to fertilization, and that further modulation occurs upon egg activation. Wispy, a noncanonical poly(A) polymerase, adenylates the vast majority of maternal mRNAs with a few intriguing exceptions such as ribosomal protein transcripts. By comparing mTAILseq data with ribosome profiling data, we find a strong coupling between poly(A) tail length and translational efficiency during egg activation. Our data suggest that regulation of poly(A) tail in oocytes shapes the translatomic landscape of embryos, thereby directing the onset of animal development. By virtue of the high sensitivity, low cost, technical robustness, and broad accessibility, mTAIL-seq will be a potent tool to improve our understanding of mRNA tailing in diverse biological systems. Ten separate sets of TAIL-seq experiments were performed. Two sets of HeLa cells are untransfected normal cells. Eight sets of fly sample include a pair of wild type and mutant.
2016-08-01 | E-GEOD-83731 | biostudies-arrayexpress
Project description:Eukaryotic mRNAs are subject to multiple types of tailing which critically influence mRNA stability and translatability. To investigate RNA tails at the genomic scale, we previously developed TAIL-seq, but its low sensitivity precluded its application to biological materials of minute quantity. In this study, we report a new version of TAILseq (mRNA TAIL-seq or mTAIL-seq) with enhanced sequencing depth for mRNAs (by ~1000 fold compared to the previous version). The improved method allows us to investigate the regulation of poly(A) tail in Drosophila oocytes and embryos. We find that maternal mRNAs are polyadenylated mainly during late oogenesis, prior to fertilization, and that further modulation occurs upon egg activation. Wispy, a noncanonical poly(A) polymerase, adenylates the vast majority of maternal mRNAs with a few intriguing exceptions such as ribosomal protein transcripts. By comparing mTAILseq data with ribosome profiling data, we find a strong coupling between poly(A) tail length and translational efficiency during egg activation. Our data suggest that regulation of poly(A) tail in oocytes shapes the translatomic landscape of embryos, thereby directing the onset of animal development. By virtue of the high sensitivity, low cost, technical robustness, and broad accessibility, mTAIL-seq will be a potent tool to improve our understanding of mRNA tailing in diverse biological systems. Two sets of RNA-seq on 3 developmental stages (immature oocyte, mature oocyte, and activated egg) of wild type and wispy mutant of Drosophila melanogaster.
2016-08-01 | E-GEOD-83730 | biostudies-arrayexpress
Project description:Because maturing oocytes and early embryos lack transcription, posttranscriptional regulatory processes must control their development. To better understand this control, we profiled translational efficiencies and poly(A)-tail lengths throughout Drosophila oocyte maturation and early embryonic development. The correspondence between translational-efficiency changes and tail-length changes indicated that tail-length changes broadly reshape translational activity until gastrulation, when this coupling disappears. Relative changes were largely retained in the absence of poly(A)-tail lengthening, which indicated that selective poly(A)-tail shortening primarily specifies the changes. Many translational changes depended on PAN GU and Smaug, and both acted primarily through tail-length changes. Our results also revealed tail-lengthâindependent mechanisms of translational control that repressed translation regardless of tail-length changes during oocyte maturation, maintained translation despite tail-length shortening during oocyte maturation, and prevented detectable translation of bicoid and several other mRNAs before egg activation. In addition to these fundamental insights, our results provide valuable resources for future studies. 42 samples analyzed using RNA-seq, ribosome footprint profiling, and PAL-seq.
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Project description:Determine the adenylation state of maternal mRNAs during oocyte maturation and early cleavage stages of the xenopus tropicalis embryos
2007-09-30 | E-MEXP-1236 | biostudies-arrayexpress
Project description:The GLD-2 class of poly(A) polymerases regulate the timing of translation of stored transcripts by elongating the poly(A) tails of target mRNAs in the cytoplasm. WISPY is a GLD-2 enzyme that acts in the Drosophila female germline and is required for the completion of the egg-to-embryo transition. Though a handful of WISPY target mRNAs have been identified during both oogenesis and early embryogenesis, we aimed to discover the full range of WISPY targets at each stage. To globally identify these targets, we carried out microarray analysis to look for maternal mRNAs whose poly(A) tails fail to elongate in the absence of WISP function. We examined the polyadenylated portion of the maternal transcriptome in both stage 14 (mature) oocytes and in early embryos that had completed egg activation. Our analysis shows that the poly(A) tails of thousands of maternal mRNAs fail to elongate in wisp-deficient oocytes and embryos. Furthermore, we have identified specific classes of genes that are highly regulated in this manner at each stage. Our study shows that cytoplasmic polyadenylation is a major regulatory mechanism during oocyte maturation and egg activation. Four groups of comparisons: WT vs. wisp total RNA from stage 14 oocytes, WT vs. wisp total RNA from fertilized eggs, WT vs. wisp poly(A)+ RNA from stage 14 oocytes, WT vs. wisp poly(A)+ RNA from fertilized eggs. Each comparison consisted of three independent RNA extractions and each experiment was done with dye-swap pairs as two technical replicates.
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Project description:Mature stage 14 oocytes prior to egg activation were isolated from wild type Oregon Red fly cultures by a combined blender/ sieving method which allows specific and efficient enrichment of stage 14 oocytes. Total RNA was extracted from 4 replicate collections and submitted to Affymetrix microarray hybridisation.
2011-06-02 | E-MEXP-2746 | biostudies-arrayexpress
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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