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Cytoplasmic and nuclear fractionation from HEK293T cells

ABSTRACT: The poly(A) tail appended to the 3’ end of many eukaryotic transcripts plays a key role in the stability, nuclear transport, and translation of the RNA. These roles are largely mediated by the Poly(A) Binding Proteins (PABPs) that coat the poly(A) tails and interact with various proteins involved in the biogenesis and function of the RNA. While it is well established that the nuclear PABP (PABPN) binds newly synthesized poly(A) tails and is apparently replaced by the cytoplasmic PABP (PABPC) on transcripts exported to the cytoplasm, the distribution of transcripts for different genes or isoforms of the same gene on these distinct PABPs has not been investigated on a genome-wide scale. Here, we analyzed the identity, splicing status, poly(A) tail size, and translation status of RNAs co-immunoprecipitated with endogenous PABPN or PABPC in human cells. At steady state, many protein coding and non-coding RNAs exhibit strong bias for association with PABPN or PABPC. While PABPN-enriched transcripts more often were incompletely spliced and harbored longer poly(A) tails and PABPC-enriched RNAs tended to have longer half-lives and higher translation efficiency, there are curious outliers. Overall, our study reveals the landscape of RNAs bound by PABPN and PABPC, providing new details that support and advance the current understanding of the roles these proteins play in poly(A) tail synthesis, maintenance and function.

ORGANISM(S): Homo sapiens

PROVIDER: GSE195493 | GEO | 2022/03/30

REPOSITORIES: GEO

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Ribo-STAMP reveals translation status of RNAs bound by PABPN and PABPC

Project description:The poly(A) tail appended to the 3’ end of many eukaryotic transcripts plays a key role in the stability, nuclear transport, and translation of the RNA. These roles are largely mediated by the Poly(A) Binding Proteins (PABPs) that coat the poly(A) tails and interact with various proteins involved in the biogenesis and function of the RNA. While it is well established that the nuclear PABP (PABPN) binds newly synthesized poly(A) tails and is apparently replaced by the cytoplasmic PABP (PABPC) on transcripts exported to the cytoplasm, the distribution of transcripts for different genes or isoforms of the same gene on these distinct PABPs has not been investigated on a genome-wide scale. Here, we analyzed the identity, splicing status, poly(A) tail size, and translation status of RNAs co-immunoprecipitated with endogenous PABPN or PABPC in human cells. At steady state, many protein coding and non-coding RNAs exhibit strong bias for association with PABPN or PABPC. While PABPN-enriched transcripts more often were incompletely spliced and harbored longer poly(A) tails and PABPC-enriched RNAs tended to have longer half-lives and higher translation efficiency, there are curious outliers. Overall, our study reveals the landscape of RNAs bound by PABPN and PABPC, providing new details that support and advance the current understanding of the roles these proteins play in poly(A) tail synthesis, maintenance and function.

2022-03-30 | GSE195494 | GEO

Identification of different transcripts that favor the nuclear or cytoplasmic Poly(A) Binding Proteins (PABPs) through RNA immunoprecipitation

2022-03-30 | GSE195495 | GEO

Poly(A) Tail Length of L4 C. elegans

Project description:Poly(A) tails are important elements in mRNA translation and stability. However, recent genome-wide studies concluded that poly(A) tail length was generally not associated with translational efficiency in non-embryonic cells. To investigate if poly(A) tail size might be coupled to gene expression in an intact organism, we used an adapted TAIL-seq protocol to measure poly(A) tails in Caenorhabditis elegans. Surprisingly, we found that well-expressed transcripts contain relatively short, well-defined tails. This attribute appears dependent on translational efficiency, as transcripts enriched for optimal codons and ribosome association had the shortest tail sizes, while non-coding RNAs retained long tails.

2017-10-19 | GSE104501 | GEO

The molecular basis of coupling between poly(A)-tail length and translational efficiency

Project description:In animal oocytes and early embryos, mRNA poly(A)-tail length strongly influences translational efficiency (TE), but later in development this coupling between tail length and TE disappears. Here, we elucidate how this coupling is first established and why it disappears. Overexpressing cytoplasmic poly(A)-binding protein (PABPC) in frog oocytes specifically improved translation of short-tailed mRNAs, thereby diminishing coupling between tail length and TE. Thus, coupling requires limiting PABPC, implying that in coupled systems longer-tail mRNAs better compete for limiting PABPC. In addition to expressing excess PABPC, post-embryonic cells had two other properties that prevented strong coupling: terminal-uridylation-dependent destabilization of mRNAs lacking bound PABPC, and a regulatory regime wherein PABPC contributes minimally to TE. Thus, these results revealed three fundamental mechanistic requirements for coupling and defined the context-dependent functions for PABPC, in which this protein promotes TE but not mRNA stability in coupled systems and mRNA stability but not TE in uncoupled systems.

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Rapid nuclear deadenylation of mammalian messenger RNA

Project description:Poly(A) tails protect RNAs from degradation and deadenylation rates determine RNA stability. Deadenylation has mostly been investigated within the cytoplasm and the dynamics of poly(A) tail length after transcription are not well understood. Combining long-read sequencing with metabolic labeling and cell fractionations, we investigate deadenylation dynamics of newly synthesized poly(A) tails in vitro and in vivo. We report evidence for genome-wide synthesis of poly(A) tails longer than 200 nt which are enriched upon splicing inhibition. Metabolic labeling reveals rapid deadenylation of poly(A) tails within minutes after transcription. Fractionation experiments show that initial deadenylation is a nuclear process, and that different classes of transcripts, including long noncoding RNAs, have distinctive nuclear poly(A) tail lengths. Modelling deadenylation dynamics predicts that deadenylation in the nucleus is by an order of magnitude faster than in the cytoplasm. In summary, we suggest nuclear deadenylation as a novel regulatory layer which may determine stability and abundance before mRNAs reach the cytoplasm.

2023-01-07 | GSE188539 | GEO

Regulation of poly(A) tail and translation during the somatic cell cycle

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

Regulation of poly(A) tail and translation during the somatic cell cycle

2016-04-04 | GSE79664 | GEO

The 3′ extremity of mRNA unveiled by TAIL-seq

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

The 3′ extremity of mRNA unveiled by TAIL-seq

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Interactome of the two T. brucei Poly(A) binding protein paralogs PABP1and PABP2

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2018-07-16 | PXD008839 | Pride

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