A novel method for genome-wide profiling of poly(A)-tail lengths
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE84287 | GEO | 2020/06/04
REPOSITORIES: GEO
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Project description:Poly(A) tails enhance the stability and translation of most eukaryotic messenger RNAs, but difficulties in globally measuring poly(A)-tail lengths have impeded greater understanding of poly(A)-tail function. Here we describe poly(A)-tail length profiling by sequencing (PAL-seq) and apply it to measure tail lengths of millions of individual RNAs isolated from yeasts, cell lines, Arabidopsis thaliana leaves, mouse liver, and zebrafish and frog embryos. Poly(A)-tail lengths were conserved between orthologous mRNAs, with mRNAs encoding ribosomal proteins and other 'housekeeping' proteins tending to have shorter tails. As expected, tail lengths were coupled to translational efficiencies in early zebrafish and frog embryos. However, this strong coupling diminished at gastrulation and was absent in non-embryonic samples, indicating a rapid developmental switch in the nature of translational control. This switch complements an earlier switch to zygotic transcriptional control and explains why the predominant effect of microRNA mediated deadenylation concurrently shifts from translational repression to mRNA destabilization.
2014-01-29 | GSE52809 | GEO
Project description:Poly(A) tails enhance the stability and translation of most eukaryotic messenger RNAs, but difficulties in globally measuring poly(A)-tail lengths have impeded greater understanding of poly(A)-tail function. Here we describe poly(A)-tail length profiling by sequencing (PAL-seq) and apply it to measure tail lengths of millions of individual RNAs isolated from yeasts, cell lines, Arabidopsis thaliana leaves, mouse liver, and zebrafish and frog embryos. Poly(A)-tail lengths were conserved between orthologous mRNAs, with mRNAs encoding ribosomal proteins and other 'housekeeping' proteins tending to have shorter tails. As expected, tail lengths were coupled to translational efficiencies in early zebrafish and frog embryos. However, this strong coupling diminished at gastrulation and was absent in non-embryonic samples, indicating a rapid developmental switch in the nature of translational control. This switch complements an earlier switch to zygotic transcriptional control and explains why the predominant effect of microRNA mediated deadenylation concurrently shifts from translational repression to mRNA destabilization. 64 samples from a variety of species
2014-01-29 | E-GEOD-52809 | biostudies-arrayexpress
Project description:We report systematical profiling of translation efficiency and mRNA stability dependent on the dynamics of poly(A)-tail length in stress conditions of human cells. In this study, we developed a new feasible method measuring poly(A)-tail length called TED-seq and applied it to investigate the change of mRNA's poly(A)-tail lengths in ER stress pharmacologically induced by thapsigargin (THAP). Combined with other global RNA analyses such as RNA-seq, Ribo-seq and PRO-seq, we observed that ER stress induced lenthening poly(A)-tail length, in particular of ER-stress-regulators, upon ER stress. More specifically, these mRNAs are translationally de-repressed and more stabilized based on increase in poly(A)-tail length. We also identified that insoluble fractions which include stress-induced RNA-granules have overall shorter length of poly(A) tail. Taken together, our data suggest that poly(A)-tail lengths are dynamically regulated and influence both translation efficiency and mRNA stability in ER stress.
2018-01-01 | GSE103719 | GEO
Project description:Poly(A) tails are critical for mRNA stability and translation. However, recent studies have challenged this view, showing that poly(A) tail length and translation efficiency are decoupled in non-embryonic cells. Using TAIL-seq and ribosome profiling, we investigate poly(A) tail dynamics and translational control in the somatic cell cycle. We find dramatic changes in poly(A) tail lengths of cell cycle regulatory genes like CDK1, TOP2A, and FBXO5, explaining their translational repression in M phase. We also find that poly(A) tail length is coupled to translation when the poly(A) tail is <20 nucleotides. However, as most genes have >20 nucleotide poly(A) tails, their translation is regulated mainly via poly(A) tail length-independent mechanisms during the cell cycle. Specifically, we find that terminal oligopyrimidine (TOP) tract-containing transcripts escape global translational suppression in M phase and are actively translated. Our quantitative and comprehensive data provide a revised view of translational control in the somatic cell cycle. HeLa cells were synchronized at S or M phase, and subject to RNA-seq, ribosome profiling and TAIL-seq analysis.
2016-04-04 | E-GEOD-79664 | biostudies-arrayexpress
Project description:Global investigation of the 3′ extremity of mRNA, despite its importance in gene regulation, has not been feasible due to technical challenges associated with homopolymeric sequences and relative paucity of mRNA. By developing a new methodology, TAIL-seq, we find a number of unforeseen features at the 3′ termini of mRNA. Our analyses reveal the transcriptome-wide distribution of poly(A) tail and estimate the median poly(A) length as 55-60 nt in mammalian cells, which is substantially shorter than generally conceived. Poly(A) length correlates with mRNA half-life but not with translation efficiency. Surprisingly, we find widespread uridylation and guanylation at the downstream of poly(A) tail. The U-tails are generally attached to short poly(A) tails (<25 nt) while the G-tails are found mainly on longer poly(A) tails (>40 nt), implicating their generic roles in mRNA stability control. In addition, TAIL-seq identifies, with a single nucleotide resolution, numerous nucleolytic events involved in microRNA processing and mRNA cleavage. Single replicate without any special experimental treatment for each of mouse fibroblast cell line NIH3T3 and human cervical cell line HeLa.
2014-02-28 | E-GEOD-51299 | biostudies-arrayexpress
Project description:Global investigation of the 3′ extremity of mRNA, despite its importance in gene regulation, has not been feasible due to technical challenges associated with homopolymeric sequences and relative paucity of mRNA. By developing a new methodology, TAIL-seq, we find a number of unforeseen features at the 3′ termini of mRNA. Our analyses reveal the transcriptome-wide distribution of poly(A) tail and estimate the median poly(A) length as 55-60 nt in mammalian cells, which is substantially shorter than generally conceived. Poly(A) length correlates with mRNA half-life but not with translation efficiency. Surprisingly, we find widespread uridylation and guanylation at the downstream of poly(A) tail. The U-tails are generally attached to short poly(A) tails (<25 nt) while the G-tails are found mainly on longer poly(A) tails (>40 nt), implicating their generic roles in mRNA stability control. In addition, TAIL-seq identifies, with a single nucleotide resolution, numerous nucleolytic events involved in microRNA processing and mRNA cleavage.
2014-02-28 | GSE51299 | GEO
Project description:Poly(A) tails are critical for mRNA stability and translation. However, recent studies have challenged this view, showing that poly(A) tail length and translation efficiency are decoupled in non-embryonic cells. Using TAIL-seq and ribosome profiling, we investigate poly(A) tail dynamics and translational control in the somatic cell cycle. We find dramatic changes in poly(A) tail lengths of cell cycle regulatory genes like CDK1, TOP2A, and FBXO5, explaining their translational repression in M phase. We also find that poly(A) tail length is coupled to translation when the poly(A) tail is <20 nucleotides. However, as most genes have >20 nucleotide poly(A) tails, their translation is regulated mainly via poly(A) tail length-independent mechanisms during the cell cycle. Specifically, we find that terminal oligopyrimidine (TOP) tract-containing transcripts escape global translational suppression in M phase and are actively translated. Our quantitative and comprehensive data provide a revised view of translational control in the somatic cell cycle.
2016-04-04 | GSE79664 | GEO
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
Project description:Polyadenylation of mRNAs is critical for their export from the nucleus, stability and efficient translation. The Arabidopsis thaliana genome encodes three isoforms of canonical nuclear poly(A) polymerase (PAPS) that redundantly polyadenylate the bulk of pre-mRNAs. However, their distinct mutant phenotypes and transcriptome studies have indicated that subsets of pre-mRNAs are preferentially polyadenylated by either PAPS1 or the two very similar PAPS2 and PAPS4 proteins. Such functional specialization raises the possibility of modulating the balance of activities between the isoforms to alter poly(A) lengths of sets of transcripts, providing an additional level of gene-expression control in plants. Here we test this notion by studying the function of PAPS1 in pollen-tube growth and guidance. Pollen tubes growing through female tissue acquire the competence to find ovules efficiently and upregulate PAPS1 expression more strongly than in vitro grown pollen tubes. Using the temperature-sensitive paps1-1 allele we show that PAPS1 activity during pollen-tube growth is required for full acquisition of competence, resulting in inefficient fertilization by paps1-1 mutant pollen tubes. While these mutant pollen tubes germinate and grow at the same rates as wild-type pollen tubes, they are compromised in locating the micropyles of ovules. Transcriptomic analyses indicate that previously identified competence-associated genes are less expressed in paps1-1 mutant than in wild-type pollen tubes. Estimating the poly(A)-tail lengths of transcripts in mutant and wild-type pollen tubes suggests that polyadenylation by PAPS1 is associated with reduced transcript abundance. Our results therefore suggest that PAPS1 upregulation during pollen-tube growth through the style plays a key role in the acquisition of competence and support the notion that plants can modulate the balance of activity between PAPS isoforms to regulate gene expression.
2023-02-21 | GSE207509 | GEO
Project description:Aim: We question non-templated transcript 3ʹ tails in cde-1 mutants versus WT C. elegans (N2 strain) infected by the Orsay virus Methods: Approximately 200 cde-1 (tm1021) mutants or N2 animals were infected for 48 hours from the L2 stage to the adult stage with 20 µl filtrate of the Orsay virus on 50mm plate seeded with HB101 bacteria, in biological duplicates. Animals were then washed in M9 and resuspended in 1 ml TRIsure (Bioline). Samples were freeze-thaw three times using liquid nitrogen and RNA purification was performed according to Bioline’s guidelines. TAILseq preparation and sequencing was performed as described in Chang, et al. Moll. Cell 2014. Tail-seq libraries were processed using Tailseeker 2 (Chang et al. Moll. Cell 2014). The 5ʹ and 3ʹ libraries were subsequently adapter trimmed using cutadapt 1.10 (Martin. EMBnet.journal 2011) with Illumina small RNA-seq adapters and filtered to a minimum length of 5bp. Trimmed 5’ reads were mapped with STAR 2.5.2a (Dobin, et al. Bioinformatics 2013) against a combined meta-genome consisting of the C. elegans reference genome WBcel235 (Harris, et al. Nucleic Acids Res. 2014) and the Orsay virus genome (Felix, et al. Plos Bio. 2011). Mapping was performed in end-to-end mode allowing no mismatches and a gap opening and extension penalty of 10000. Reads were assigned to genes with bedtools 2.26.0 (Quinlan and Hall. Bioinformatics 2010). Subsequently, 3ʹ reads without poly(A) tail or too many dark cycles were removed from the data. For the subsequent analysis, all C. elegans tags with poly(A) tail length equal to zero were discarded. Average poly(A) tail lengths and uridylation lengths for each sample were calculated as the arithmetic mean weighted by the support for each tag, reported by Tailseeker.
2019-08-19 | GSE85893 | GEO