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MTAIL-seq reveals dynamic poly(A) tail regulation in oocyte-to-embryo development [RNA-seq Drosophila oocyte stages]

ABSTRACT: 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.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Jaechul Lim

PROVIDER: E-GEOD-83730 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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mTAIL-seq reveals dynamic poly(A) tail regulation in oocyte-to-embryo development [RNA-seq and mTAIL-seq Human and Drosphila]

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

mRNA Poly(A)-tail Changes Specified by Deadenylation Broadly Reshape Translation in Drosophila Oocytes and Early Embryos

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.

2016-08-08 | E-GEOD-83616 | biostudies-arrayexpress

mTAIL-seq reveals dynamic poly(A) tail regulation in oocyte-to-embryo development [RNA-seq Drosophila oocyte stages]

2016-08-01 | GSE83730 | GEO

mTAIL-seq reveals dynamic poly(A) tail regulation in oocyte-to-embryo development [RNA-seq and mTAIL-seq Human and Drosphila]

2016-08-01 | GSE83731 | GEO

Pentatricopeptide repeat poly(A) binding protein from mitochondria of trypanosomes

Project description:In Trypanosoma brucei, most mitochondrial mRNAs undergo U-insertion/deletion editing, and 3′ adenylation and uridylation. The internal sequence changes and terminal extensions are coordinated: Pre-editing addition of the short (A) tail protects the edited transcript against 3′-5′ degradation, while post-editing A/U-tailing renders mRNA competent for ribosome recruitment. Participation of a poly(A) binding protein (PABP) in coupling of editing and 3′ modification processes has been inferred, but its identity and mechanism of action remained elusive. We report identification of KPAF4, a pentatricopeptide repeat-containing PABP which sequesters the A-tail and impedes exonucleolytic degradation. Conversely, KPAF4 inhibits uridylation of A-tailed transcripts and, therefore, premature A/U-tailing of partially-edited mRNAs. This quality check point prevents translation of incompletely edited mRNAs. Our findings also implicate the RNA editing substrate binding complex (RESC) in mediating the interaction between the 5′-end bound pyrophosphohydrolase MERS1 and 3′-end associated KPAF4 to enable mRNA circularization. This event is critical for transcript stability during the editing process.

2019-04-02 | PXD012008 | Pride

Calibrated total RNAseq (cRNA-seq) of E14_Scc1_AID mESCs mixed with Drosophila SG4 cells with and without a 6-hour auxin treatment

Project description:We performed calibrated total RNAseq on mESC (Scc1-AID) for both untreated and treated cells with a 6-hour auxin exposure to assess the effects of cohesin removal on gene expression. For calibration purposes 2x10ˆ6 Drosophila SG4 cells were spiked in.

2020-01-23 | E-MTAB-7818 | biostudies-arrayexpress

Genome-wide analysis of PAPS1-dependent polyadenylation identifies novel roles for functionally specialized poly(A) polymerases in Arabidopsis thaliana

Project description:The poly(A) tail at 3' ends of eukaryotic mRNAs promotes their nuclear export, stability and translational efficiency, and changes in its length can strongly impact gene expression. The Arabidopsis thaliana genome encodes three canonical nuclear poly(A) polymerases, PAPS1, PAPS2 and PAPS4. As shown by their different mutant phenotypes, these three isoforms are functionally specialized, with PAPS1 modifying organ growth and suppressing a constitutive immune response. However, the molecular basis of this specialization is largely unknown. Here, we have estimated poly(A)-tail lengths on a transcriptome-wide scale in wild-type and paps1 mutants. This identified categories of genes as particularly strongly affected in paps1 mutants, including genes encoding ribosomal proteins, cell-division factors and major carbohydrate-metabolic proteins. We experimentally verified two novel functions of PAPS1 in ribosome biogenesis and redox homoeostasis that were predicted based on the analysis of poly(A)-tail length changes in paps1 mutants. When overlaying the PAPS1-dependent effects observed here with coexpression analysis based on independent microarray data, the two clusters of transcripts that are most closely coexpressed with PAPS1 show the strongest change in poly(A)-tail length and transcript abundance in paps1 mutants in our analysis. This suggests that their coexpression reflects at least partly the preferential polyadenylation of these transcripts by PAPS1 versus the other two poly(A)-polymerase isoforms. Thus, transcriptomewide analysis of poly(A)-tail lengths identifies novel biological functions and likely target transcripts for polyadenylation by PAPS1. Data integration with large-scale coexpression data suggests that changes in the relative activities of the isoforms are used as an endogenous mechanism to co-ordinately modulate plant gene expression. Analysing long and short poly(A)-tail fractions from 4 paps1 mutant and 4 Ler samples.

2015-07-30 | E-GEOD-57690 | biostudies-arrayexpress

PAT-seq - a high-throughput polyadenylation assay for the Illumina platform

Project description:Polyadenylation - the cleavage of the nascent mRNA and the addition of a tract consisting of adenine bases in a non-template-dependent manner - is a post-transcriptional modification characteristic of the majority of eukaryotic transcripts. In this study we explore PAT-seq, an Illumina-based poly(A) tail assay, based on the low-throughput G-tailing approach. Briefly, our approach is based on G-tailing poly(A) selected mRNA followed by fragmentation and reverse transcription using a mixture of primers targeting the poly(A)-tail/G-tag junction and the polyadenylation site and the body of the transcripts. The resulting cDNA fragments are sequenced using standard Illumina RNA-seq protocol.

2014-04-05 | E-MTAB-2456 | biostudies-arrayexpress

Global analysis of differential polyadenylation of mRNAs during mitotic cell cycle: S phase versus G2/M phase.

Project description:We compared the poly(A) tail length status of mRNAs of HeLa cells between two phases of the mitotic cell cycle: S and G2/M phases. Hundreds of mRNAs were found to be regulated by changes in their poly(A) tail length during mitotic cell cycle in a phase specific manner. Many of these differentially polyadenylated mRNAs encode proteins related to cell death, cell cycle and cellular growth and proliferation. HeLa cells were synchronized with double thymidine blockade (12 hours with 2 mM thymidine, 12 hours release, and 12 hours with 2 mM thymidine), and samples were taken after 2 hours release (S phase) and 8 hours release (G2/M phase). For each condition total RNA was purified by two different procedures: poly(U) chromatography and oligo(dT)-chromatography. Poly(U)-chromatography (Jacobson, 1987): 100 Î¼g of total RNA were bound to poly(U)-sepharose (Sigma) and eluted at 35ÂºC to isolate mRNAs with short poly(A) tail (<30As, SHORT fraction). Oligo(dT) chromatography: mRNAs were purified independently of their poly(A) tail length with Ambion Poly(A)Purist kit from 20 Î¼g total RNA (ALL fraction). Jacobson, A. Purification and fractionation of poly(A)+ RNA. Methods in Enzymology (1987) 152: 254-261. Keywords: time course Comparison of ALL fraction mRNAs and SHORT fraction mRNAs measured after 2 hours (S phase) and 8 hours release (G2/M) from double thymidine blockade; 3 biological replicates at each of the two time points; two technical replicates with dye swapping per comparison.

2010-05-07 | E-GEOD-15399 | biostudies-arrayexpress

mRNA Poly(A)-tail Changes Specified by Deadenylation Broadly Reshape Translation in Drosophila Oocytes and Early Embryos

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-length–independent 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.

2016-08-08 | GSE83616 | GEO

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