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Oppositional Poly(A) Tail Length Regulation by FMRP and CPEB1

ABSTRACT: Poly(A) tail length is regulated in both the nucleus and cytoplasm. One factor that controls polyadenylation in the cytoplasm is CPEB1, an RNA binding protein that associates with specific mRNA 3’UTR sequences to tether enzymes that add and remove poly(A). Two of these enzymes, the noncanonical poly(A) polymerases Gld2 (TENT2, PAPD4, Wispy) and Gld4 (TENT4B, PAPD5, TRF4, TUT3), interact with CPEB1 to extend poly(A). To identify additional RNA binding proteins that might anchor Gld4 to RNA, we expressed double tagged Gld4 in U87MG cells, which was used for sequential immunoprecipitation and elution followed by mass spectrometry. We identified several RNA binding proteins that co-precipitated with Gld4, among which was FMRP. To assess whether FMRP regulates polyadenylation, we performed TAIL-seq from WT and FMRP-deficient HEK293 cells. Surprisingly, loss of FMRP resulted in an overall increase in poly(A), which was also observed for several specific mRNAs. Conversely, loss of CPEB1 elicited an expected decrease in poly(A), which was examined in cultured neurons. We also examined polyadenylation in wild type (WT) and FMRP-deficient mouse brain cortex by direct RNA Nanopore™ sequencing, which identified RNAs with both increased and decreased poly(A). Our data show that FMRP has a role in mediating poly(A) tail length, which adds to its repertoire of RNA regulation.

ORGANISM(S): Mus musculus

PROVIDER: GSE188840 | GEO | 2022/03/01

REPOSITORIES: GEO

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Oppositional Poly(A) Tail Length Regulation by FMRP and CPEB1

Project description:Oppositional Poly(A) Tail Length Regulation by FMRP and CPEB1

| PRJNA780496 | ENA

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Project description:We compared the poly(A) tail length status of mRNAs of HeLa cells expressing a CPEB1 shRNA (CPEB1 knockdown) versus a control shRNA, and expressing a CPEB4 shRNA (CPEB4 knockdown) versus a control shRNA. Results provide insight into the extent of gene regulation mediated by CPEB1 and CPEB4 activity during mitotic cell cycle progression. The different shRNA expressing 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 8 hours release (G2/M phase). For each shRNA expressing HeLa cell line total RNA was purified by two different procedures: poly(U) chromatography and oligo(dT)-chromatography. Poly(U)-chromatography (Jacobson, 1987): 100 μg of total RNA were bound to poly(U)-sepharose (Sigma) and eluted at 35ºC 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 μg total RNA (ALL fraction). Jacobson, A. Purification and fractionation of poly(A)+ RNA. Methods in Enzymology (1987) 152: 254-261. Keywords: knock-down experiment

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Drosophila early embryos LC-MSMS

Project description:Cytoplasmic polyadenylation is a mechanism to promote mRNA translation in a wide variety of biological contexts.The conserved RNA-binding protein family CPEB has been shown to mediate canonical cytoplasmic polyadenylation of target transcripts. We have previously reported evidence for RNA-interference factor Dicer-2 as a component of a non-canonical complex, that operates independent of CPEB in Drosophila. In this study, we investigate Dicer-2 mRNA targets and protein co-factors in cytoplasmic polyadenylation. Using RIP‐Seq analysis we identify hundreds of potential Dicer-2 target transcripts, ~60% of which were previously found as targets of the cytoplasmic poly(A) polymerase Wispy, suggesting widespread roles of Dicer-2 in cytoplasmic polyadenylation. Large-scale immunoprecipitation and mass spectrometry revealed Ataxin-2 and Twenty-four among the high-confidence interactors of Dicer-2. Complex analyses indicated that both factors form an RNA‐independent complex with Dicer‐2, and mediate interactions of Dicer‐2 with Wispy. Functional poly(A)‐test analyses showed that Twenty‐four and Ataxin-2 are required for cytoplasmic polyadenylation of a subset of Dicer‐2 targets. Our results reveal components of a novel cytoplasmic polyadenylation complex that operates during Drosophila early embryogenesis.

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Project description:To obtain global data on polyadenylation of mRNAs, we fractionated the mRNAs according to their poly(A) tail length using a poly-U sepharose column followed by differential elution at five temperatures. Five mRNA fractions with distinct ranges of poly(A) tail length were then hybridized to microarrays using total eluate as a reference.

2007-05-16 | E-TABM-217 | biostudies-arrayexpress

Cytoplasmic polyadenylation is a major regulator of mRNA fate during oogenesis and egg activation.

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.

2016-05-18 | E-GEOD-81495 | biostudies-arrayexpress

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

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

Genome-wide analysis of mRNA polyadenylation and translation during vertebrate oocyte maturation

Project description:Changes in gene expression are required to orchestrate changes in cell state during development. Most cells change patterns of gene expression through transcriptional regulation. In contrast, oocytes are transcriptionally silent and use changes in mRNA poly-A tail length to control protein production. Recent technical advances have enabled genome-wide measurement of poly-A tail length. Poly-A tail lengthening is correlated with translational activation and poly-A tail shortening is correlated with translational repression at a global level during early development. However, it is not clear how poly-A tail changes affect mRNA translation at a global level during vertebrate oocyte maturation. We used TAIL-seq and polyribosome analysis to measure poly-A tail and translational changes during oocyte maturation in Xenopus laevis. We found that the transcriptome undergoes large-scale poly-A and translational changes during oocyte maturation and that poly-A tail length and translation are well-correlated. Interestingly, we found that poly-A tail changes precede translation changes. Additionally, we identified a family of U-rich sequence elements that are enriched near the polyadenylation signal of polyadenylated and translationally activated mRNAs. Interestingly, we found that cytoplasmic polyadenylation is not sufficient to activate translation, which requires a specific density and spacing of U-rich elements. Collectively, our data shows that changes in mRNA polyadenylation are the dominant mechanism controlling protein expression during vertebrate oocyte maturation and that these changes are controlled by a group of cis-acting sequence elements. Our results provide insight into mechanisms of translational control in oocytes and identify novel proteins important for the completion of meiosis.

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Cytoplasmic polyadenylation is a major regulator of mRNA fate during oogenesis and egg activation.

2016-05-18 | GSE81495 | GEO

Extension of mRNA poly(A) tails and 3′UTRs during neuronal differentiation exhibits variable association with post-transcriptional dynamics

Project description:Differentiation of neural progenitor cells into mature neuronal phenotypes relies on extensive temporospatial coordination of mRNA expression to support the development of functional brain circuitry. Cleavage and polyadenylation of mRNA has tremendous regulatory capacity through the alteration of mRNA stability and modulation of microRNA (miRNA) function, however the extent of utilization in neuronal development is currently unclear. Here, we employed poly(A) tail sequencing, mRNA sequencing, ribosome profiling and small RNA sequencing to explore the functional relationship between mRNA abundance, translation, poly(A) tail length, alternative polyadenylation (APA) and miRNA expression in an in vitro model of neuronal differentiation. Differential analysis revealed a strong bias towards poly(A) tail and 3´UTR lengthening during differentiation, both of which were associated with changes in mRNA abundance but not translation. Globally, changes in miRNA expression were predominantly associated with mRNA abundance and translation, however several miRNA-mRNA pairings with potential to regulate poly(A) tail length were identified. Furthermore, 3´UTR lengthening was observed to significantly increase the inclusion of non-conserved miRNA binding sites, potentially enhancing the regulatory capacity of these molecules in mature neuronal cells. Together, our findings suggest poly(A) tail length and APA function as part of a rich post-transcriptional regulatory matrix during neuronal differentiation.

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