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A compensatory link between cleavage/polyadenylation and mRNA turnover regulates steady-state mRNA levels in yeast

ABSTRACT: Cells have compensatory mechanisms to coordinate the rates of major biological processes, thereby permitting growth in a wide variety of conditions. Here, we uncover a compensatory link between cleavage/polyadenylation in the nucleus and mRNA turnover in the cytoplasm. On a global basis, same-gene 3’ mRNA isoforms with > two-fold differences in half-lives have steady-state mRNA levels that differ by significantly less than a factor of two. In addition, increased efficiency of cleavage/polyadenylation at a specific site is associated with reduced stability of the corresponding 3’ mRNA isoform. This inverse relationship between cleavage/polyadenylation and mRNA isoform half-life reduces the variability in the steady-state levels of mRNA isoforms, and it occurs in all four growth conditions tested. These observations suggest that during cleavage/polyadenylation in the nucleus, mRNA isoforms are marked in a manner that persists upon translocation to the cytoplasm and affects the activity of mRNA degradation machinery, thus influencing mRNA stability.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE191091 | GEO | 2022/02/02

REPOSITORIES: GEO

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Condition-specific 3’ mRNA isoform half-lives and stability elements in yeast

Project description:Alternative polyadenylation generates numerous 3’ mRNA isoforms that can differ in their stability, structure, and function. These isoforms can be used to map mRNA stabilizing and destabilizing elements within 3’ untranslated regions (3’UTRs). Here, we examine how environmental conditions affect 3’ mRNA isoform turnover and structure in yeast cells on a transcriptome scale. Isoform stability broadly increases when cells grow more slowly, with relative half-lives of most isoforms being well correlated across multiple conditions. Surprisingly, dimethyl sulfate probing reveals that individual 3’ isoforms have similar structures across different conditions, in contrast to the extensive structural differences that can exist between closely related isoforms in an individual condition. Unexpectedly, most mRNA stabilizing and destabilizing elements function only in a single growth condition. The genes associated with some classes of condition-specific stability elements are enriched for different functional categories, suggesting that regulated mRNA stability might contribute to adaptation to different growth environments. Condition-specific stability elements do not result in corresponding condition-specific changes in steady-state mRNA isoform levels. This observation is consistent with a compensatory mechanism between polyadenylation and stability, and it suggests that condition-specific mRNA stability elements might largely reflect condition-specific regulation of mRNA 3’ end formation.

2023-06-01 | GSE228123 | GEO

A compensatory link between cleavage/polyadenylation and mRNA turnover regulates steady-state mRNA levels in yeast

Project description:A compensatory link between cleavage/polyadenylation and mRNA turnover regulates steady-state mRNA levels in yeast

| PRJNA789657 | ENA

Extensive structural differences of closely related 3’ mRNA isoforms: links to Pab1 binding and mRNA stability

Project description:Alternative polyadenylation generates numerous 3' mRNA isoforms per gene; regulation of this process is critical in development and impaired in cancer. Individual isoforms from the same gene can vary greatly in biological properties such as translational efficiency, localization, and half-life. Even closely related isoforms - including those differing by a single nt - can have different biological properties, but the underlying mechanisms are unknown. Here we show that many highly similar yeast isoforms unexpectedly exhibit extensive structural variation and differential Pab1 binding, and that these physical properties serve as a good predictor of relative isoform stability. We developed DREADS, a dimethyl sulphate (DMS)-based technique, to obtain the first transcriptome-scale structural description of individual 3' mRNA isoforms in vivo. Strikingly, near-identical yeast mRNA isoforms arising from the same gene can possess dramatically different DMS modification profiles over hundreds of nt upstream of the poly(A) tail. DREADS analyses of the same mRNAs refolded in vitro or in different species indicate that structural differences in vivo are often due to trans- acting factors. CLIP-READS, a technique we developed to measure isoform-specific binding, reveals that differences in Poly(A) binding protein (Pab1) binding to 3' ends correlate with the extent of structural variation for closely-spaced isoforms. A pattern encompassing single-strandedness near the 3' terminus, double-stranded character of the poly(A) tail, and low Pab1 binding is significantly associated with mRNA stability. Sequences responsible for isoform-specific structures, differential Pab1 binding, and mRNA stability are evolutionarily conserved, and are indicative of biological function.

2018-10-11 | GSE95788 | GEO

Comparative analysis of multiple human cell lines reveals distinct stability regulation of alternative polyadenylation isoforms and mitochondrial RNAs in neuronal cells

Project description:mRNA stability plays an important role in gene expression. Here, using 3’ end sequencing of newly made and steady-state poly(A)+ RNAs, we compare transcript stability in multiple human cell lines, including HEK293T, HepG2, SH-SY5Y. We show that while mRNA stability is generally conserved across the cell lines, specific transcripts having high GC content and likely more stable secondary RNA structures are relatively more stable in SH-SY5Y cells compared to other two. These features also differentiate alternative polyadenylation (APA) 3’UTR isoforms in their stability difference in a cell type-specific manner. By analyzing cell differentiation of a neural stem cell line, we show that mRNA stability difference could contribute to gene expression changes in neurogenesis and confirms the neuronal identity of SH-SY5Y cells at gene expression and APA levels. Through comparison of APA isoforms, we show that mRNA stability control buffer the cleavage and polyadenylation site (PAS) choice in the cells studied. In addition, transcripts using intronic PAS are generally less stable, especially when the intron harboring the PAS is large and has a strong 5’ splice site, suggesting that intronic polyadenylation mostly plays a negative role in gene expression. Moreover, we found that poly(A)+ RNAs encoded by the mitochondrial genome are more stable in SH-SY5Y cells than the other two cell types, suggesting that mitochondrial RNA metabolism is distinct in neurons. Together, our results reveal multiple cell-specific mechanisms by which mRNA stability could contribute to the gene expression and APA programs that are important for cell identity.

2021-12-27 | GSE189899 | GEO

hnRNPC regulates cancer-specific alternative cleavage and polyadenylation profiles

Project description:Alternative cleavage and polyadenylation (APA) can occur at more than half of all human genes, greatly enhancing the cellular repertoire of mRNA isoforms. As these isoforms can have altered stability, localisation and coding potential, deregulation of APA can disrupt gene expression and this has been linked to many diseases including cancer progression. How APA generates cancer-specific isoform profiles and what their physiological consequences are, however, is largely unclear. Here we use a subcellular fractionation approach to determine the nuclear and cytoplasmic APA profiles of successive stages of colon cancer using a cell line-based model. Using this approach, we show that during cancer progression specific APA profiles are established. We identify that overexpression of hnRNPC has a critical role in the establishment of APA profiles characteristic for metastatic colon cancer cells, by regulating poly(A) site selection in a subset of genes that have been implicated in cancer progression including MTHFD1L.

2019-06-01 | GSE102357 | GEO

Alternative Isoform Regulation in Human Tissue Transcriptomes

Project description:Through alternative processing of pre-mRNAs, individual mammalian genes often produce multiple mRNA and protein isoforms that may have related, distinct or even opposing functions. Here we report an in-depth analysis of 15 diverse human tissue and cell line transcriptomes based on deep sequencing of cDNA fragments, yielding a digital inventory of gene and mRNA isoform expression. Analysis of mappings of sequence reads to exon-exon junctions indicated that ~94% of human genes undergo alternative splicing (AS), ~86% with a minor isoform frequency of 15% or more. Differences in isoform-specific read densities indicated that a majority of AS and alternative cleavage and polyadenylation (APA) events exhibit variation between tissues. Variations in alternative mRNA isoform expression between 6 individuals were also detected in cerebellar cortex, with ~2- to 3-fold less isoform variation observed between individuals than between tissues. Extreme or 'switch-like' regulation of splicing between tissues was associated with increased sequence conservation and with generation of full-length open reading frames. Patterns of AS and APA were strongly correlated across tissues, suggesting coordinated regulation, and sequence conservation of known regulatory motifs in both regulated introns and 3' UTRs suggested common involvement of the same factors in regulation of tissue-specific splicing and polyadenylation. Exam mRNA expression in 15 human tissues and cell lines

2008-09-30 | E-GEOD-12946 | biostudies-arrayexpress

Knockdowns of total NFYA and a NFYA long 3’ UTR isoform followed by RNA-seq to determine isoform-specific effects on gene expression

Project description:Alternative cleavage and polyadenylation (APA) produces multiple isoforms of the Nuclear Transcription Factor Y Subunit Alpha (NFYA) gene with variable length 3? untranslated regions (UTRs). These isoforms have been detected in HCT116 colon cancer cells via polyA-seq and northern blots, as well as colon cancer tissue RNA-seq data. NFYA is a transcription factor that may regulate cell growth and proliferation in cancer and development. To determine if there may be transcriptional regulation unique to the long 3’ UTR isoform, we conducted an siRNA knockdown in HCT116 cells specific to either the long isoform (by targeting sequence uniquely present in the longer transcript) or total NFYA mRNA (by targeting sequence shared by all long and short NFYA mRNA isoforms) followed by next generation sequencing of RNA (RNA-seq) to analyze the transcriptome.

2022-12-28 | GSE86179 | GEO

A compendium of conserved cleavage and polyadenylation events in mammalian genes

Project description:Cleavage and polyadenylation is essential for 3’ end processing of almost all eukaryotic mRNAs. Recent studies have shown widespread alternative cleavage and polyadenylation (APA) events leading to mRNA isoforms with different 3’UTRs and/or coding sequences. Here we present a compendium of conserved cleavage and polyadenylation sites (PASs) in mammalian genes, based on ~1.2 billion 3’ end sequencing reads from over 360 human, mouse and rat samples. We show that ~80% of mammalian mRNA genes contain at least one conserved PAS, and ~50% have conserved APA events. PAS conservation generally reduces promiscuous 3’ end processing, stabling gene expression levels across species. Conservation of APA correlates with gene age, gene expression features, and gene functions. Genes with certain functions, such as cell morphology, cell proliferation, and mRNA metabolism, are particularly enriched with APA events. While tissue-specific genes typically have a low APA rate, brain-specific genes tend to evolve APA. We show enrichment of mRNA destabilizing motifs in alternative 3’UTR sequences, leading to substantial differences in mRNA stability between 3’UTR APA isoforms. Using conserved PASs, we reveal sequence motifs surrounding APA sites and a preference of adenosine at the cleavage site. Mutations of the U-rich motif around the PAS often accompany APA profile changes between species. Analysis of lncRNA PASs indicates a mechanism of PAS fixation involving evolution of A-rich motifs. Taken together, our results present a comprehensive view of PAS evolution in mammals, and a phylogenic understanding of APA functions.

2018-08-08 | GSE111134 | GEO

Alternative cleavage and polyadenylation analysis in an isoform-resolved way by Second- and Third-Generation Sequencing technologies

Project description:Alternative cleavage and polyadenylation (APA) is emerging as an important mechanism of gene regulation in eukaryotes and plays important regulatory roles in human development and diseases. Despite the widespread application of Second Generation Sequencing (SGS) technology for polyadenylation site identification, matching each identified polyadenylation site within a gene to its derived isoform remains a major challenge. To achieve the isoform-resolved APA analysis, we developed a tool termed “IDP-APA” that constructs truly expressed isoforms and identifies polyadenylation sites by integrating the respective strengths of Third Generation Sequencing (TGS) long reads and SGS short reads. Compared to existing tools, IDP-APA demonstrated superior performance in both isoform reconstruction and polyadenylation site identification. Applications to human embryonic stem cells, breast cancer cells and brain tissue from a patient with Alzheimer’s disease revealed prevalent APA events and cell-/tissue-specific APA patterns, especially in an isoform-resolved way.

2017-06-01 | GSE97688 | GEO

Transcript buffering and 3ʹUTR lengthening are shaped during human neurodevelopment by shifts in mRNA stability and microRNA load

Project description:The change in abundance of individual mRNAs as cells differentiate through neurodevelopmental stages is generally attributed to transcription level alterations while the potential contribution of mRNA half-life is commonly overlooked. While some genes are regulated solely by transcription, others may be regulated by mRNA half-life only, or genes with changed transcription rates could be buffered by compensating half-life decreases or even boosted by amplifying half-life increases. In addition, during neurodevelopment there is widespread 3ʹUTR lengthening that could be shaped by differential shifts in the stability of transcript isoforms. Here we measure transcription rate and mRNA half-life changes during induced Pluripotent Stem Cell-derived neuronal development using RATE-seq at the resolution of gene isoforms. We found that during transitions to progenitor and neuron stages, transcript buffering occurs in up to half of all genes. Half-life only changes are observed in as many genes as transcription rate only changes, and almost 10% of genes exhibit transcript boosting. Global mRNA half-life decreases two-fold in neurons relative to iPSCs. The observed mRNA destabilization differentially affects gene isoforms and contributes to 3ʹUTR lengthening. Small RNA sequencing further captured an increase in microRNA copy number per cell. We propose that mRNA destabilization and 3ʹUTR lengthening are driven in part by an increase in microRNA load. Our findings identify mRNA stability mechanisms in human neurodevelopment that regulate gene and isoform level abundance and provide a precedent for similar post-transcriptional regulatory events as other tissues develop.

2023-09-01 | GSE212990 | GEO

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