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The Plant Journal : For Cell and... Sep 2017CPSF100 is a core component of the cleavage and polyadenylation specificity factor (CPSF) complex for 3'-end formation of mRNA, but it still has no clear functional...
CPSF100 is a core component of the cleavage and polyadenylation specificity factor (CPSF) complex for 3'-end formation of mRNA, but it still has no clear functional assignment. CPSF100 was reported to play a role in RNA silencing and promote flowering in Arabidopsis. However, the molecular mechanisms underlying these phenomena are not fully understood. Our genetics analyses indicate that plants with a hypomorphic mutant of CPSF100 (esp5) show defects in embryogenesis, reduced seed production or altered root morphology. To unravel this puzzle, we employed a poly(A) tag sequencing protocol and uncovered a different poly(A) profile in esp5. This transcriptome-wide analysis revealed alternative polyadenylation of thousands of genes, most of which result in transcriptional read-through in protein-coding genes. AtCPSF100 also affects poly(A) signal recognition on the far-upstream elements; in particular it prefers less U-rich sequences. Importantly, AtCPSF100 was found to exert its functions through the change of poly(A) sites on genes encoding binding proteins, such as nucleotide-binding, RNA-binding and poly(U)-binding proteins. In addition, through its interaction with RNA Polymerase II C-terminal domain (CTD) and affecting the expression level of CTD phosphatase-like 3 (CPL3), AtCPSF100 is shown to potentially ensure transcriptional termination by dephosphorylation of Ser2 on the CTD. These data suggest a key role for CPSF100 in locating poly(A) sites and affecting transcription termination.
Topics: Arabidopsis; Cleavage And Polyadenylation Specificity Factor; Gene Expression Profiling; Poly A; Polyadenylation; RNA Polymerase II; RNA, Messenger; Transcription, Genetic
PubMed: 28621907
DOI: 10.1111/tpj.13611 -
International Journal of Molecular... Oct 2021Mitochondria have their own double-stranded DNA genomes and systems to regulate transcription, mRNA processing, and translation. These systems differ from those... (Review)
Review
Mitochondria have their own double-stranded DNA genomes and systems to regulate transcription, mRNA processing, and translation. These systems differ from those operating in the host cell, and among eukaryotes. In recent decades, studies have revealed several plant-specific features of mitochondrial gene regulation. The polyadenylation status of mRNA is critical for its stability and translation in mitochondria. In this short review, I focus on recent advances in understanding the mechanisms regulating mRNA polyadenylation in plant mitochondria, including the role of poly(A)-specific ribonuclease-like proteins (PARNs). Accumulating evidence suggests that plant mitochondria have unique regulatory systems for mRNA poly(A) status and that PARNs play pivotal roles in these systems.
Topics: Embryophyta; Exoribonucleases; Gene Expression Regulation, Plant; Mitochondria; Poly A; Polyadenylation; RNA Stability; RNA, Messenger; RNA, Mitochondrial
PubMed: 34639116
DOI: 10.3390/ijms221910776 -
Genome Biology Mar 2021Alternative polyadenylation (APA) refers to the regulated selection of polyadenylation sites (PASs) in transcripts, which determines the length of their 3' untranslated...
BACKGROUND
Alternative polyadenylation (APA) refers to the regulated selection of polyadenylation sites (PASs) in transcripts, which determines the length of their 3' untranslated regions (3'UTRs). We have recently shown that SRSF3 and SRSF7, two closely related SR proteins, connect APA with mRNA export. The mechanism underlying APA regulation by SRSF3 and SRSF7 remained unknown.
RESULTS
Here we combine iCLIP and 3'-end sequencing and find that SRSF3 and SRSF7 bind upstream of proximal PASs (pPASs), but they exert opposite effects on 3'UTR length. SRSF7 enhances pPAS usage in a concentration-dependent but splicing-independent manner by recruiting the cleavage factor FIP1, generating short 3'UTRs. Protein domains unique to SRSF7, which are absent from SRSF3, contribute to FIP1 recruitment. In contrast, SRSF3 promotes distal PAS (dPAS) usage and hence long 3'UTRs directly by counteracting SRSF7, but also indirectly by maintaining high levels of cleavage factor Im (CFIm) via alternative splicing. Upon SRSF3 depletion, CFIm levels decrease and 3'UTRs are shortened. The indirect SRSF3 targets are particularly sensitive to low CFIm levels, because here CFIm serves a dual function; it enhances dPAS and inhibits pPAS usage by binding immediately downstream and assembling unproductive cleavage complexes, which together promotes long 3'UTRs.
CONCLUSIONS
We demonstrate that SRSF3 and SRSF7 are direct modulators of pPAS usage and show how small differences in the domain architecture of SR proteins can confer opposite effects on pPAS regulation.
Topics: 3' Untranslated Regions; Alternative Splicing; Animals; Base Sequence; Cleavage And Polyadenylation Specificity Factor; Gene Expression Regulation; Mice; Models, Biological; Monomeric GTP-Binding Proteins; Neurons; Phosphorylation; Poly A; Poly(A)-Binding Proteins; Polyadenylation; Protein Binding; Protein Interaction Domains and Motifs; RNA, Messenger; Serine-Arginine Splicing Factors
PubMed: 33706811
DOI: 10.1186/s13059-021-02298-y -
Genome Biology Nov 2017Drosophila melanogaster has one of best-described transcriptomes of any multicellular organism. Nevertheless, the paucity of 3'-sequencing data in this species precludes...
BACKGROUND
Drosophila melanogaster has one of best-described transcriptomes of any multicellular organism. Nevertheless, the paucity of 3'-sequencing data in this species precludes comprehensive assessment of alternative polyadenylation (APA), which is subject to broad tissue-specific control.
RESULTS
Here, we generate deep 3'-sequencing data from 23 developmental stages, tissues, and cell lines of D. melanogaster, yielding a comprehensive atlas of ~ 62,000 polyadenylated ends. These data broadly extend the annotated transcriptome, identify ~ 40,000 novel 3' termini, and reveal that two-thirds of Drosophila genes are subject to APA. Furthermore, we dramatically expand the numbers of genes known to be subject to tissue-specific APA, such as 3' untranslated region (UTR) lengthening in head and 3' UTR shortening in testis, and characterize new tissue and developmental 3' UTR patterns. Our thorough 3' UTR annotations permit reassessment of post-transcriptional regulatory networks, via conserved miRNA and RNA binding protein sites. To evaluate the evolutionary conservation and divergence of APA patterns, we generate developmental and tissue-specific 3'-seq libraries from Drosophila yakuba and Drosophila virilis. We document broadly analogous tissue-specific APA trends in these species, but also observe significant alterations in 3' end usage across orthologs. We exploit the population of functionally evolving poly(A) sites to gain clear evidence that evolutionary divergence in core polyadenylation signal (PAS) and downstream sequence element (DSE) motifs drive broad alterations in 3' UTR isoform expression across the Drosophila phylogeny.
CONCLUSIONS
These data provide a critical resource for the Drosophila community and offer many insights into the complex control of alternative tissue-specific 3' UTR formation and its consequences for post-transcriptional regulatory networks.
Topics: 3' Untranslated Regions; Animals; Cell Line; Computational Biology; Drosophila; Drosophila melanogaster; Evolution, Molecular; Molecular Sequence Annotation; Organ Specificity; Poly A; Polyadenylation; RNA Isoforms; RNA-Binding Proteins; Species Specificity; Transcription, Genetic
PubMed: 29191225
DOI: 10.1186/s13059-017-1358-0 -
RNA (New York, N.Y.) Jul 2022The poly(A) tail enhances translation and transcript stability, and tail length is under dynamic control during cell state transitions. Tail regulation plays essential...
The poly(A) tail enhances translation and transcript stability, and tail length is under dynamic control during cell state transitions. Tail regulation plays essential roles in translational timing and fertilization in early development, but poly(A) tail dynamics have not been fully explored in post-embryonic systems. Here, we examined the landscape and impact of tail length control during macrophage activation. Upon activation, more than 1500 mRNAs, including proinflammatory genes, underwent distinctive changes in tail lengths. Increases in tail length correlated with mRNA levels regardless of transcriptional activity, and many mRNAs that underwent tail extension encode proteins necessary for immune function and post-transcriptional regulation. Strikingly, we found that , whose protein product destabilizes target transcripts, undergoes tail extension. Our analyses indicate that many mRNAs undergoing tail lengthening are, in turn, degraded by elevated levels of ZFP36, constituting a post-transcriptional feedback loop that ensures transient regulation of transcripts integral to macrophage activation. Taken together, this study establishes the complexity, relevance, and widespread nature of poly(A) tail dynamics, and the resulting post-transcriptional regulation during macrophage activation.
Topics: Gene Expression Regulation; Macrophage Activation; Poly A; Polyadenylation; RNA, Messenger
PubMed: 35512831
DOI: 10.1261/rna.078918.121 -
Cells Oct 2021Extracellular vesicles (EVs) are important for intercellular communication and act as vehicles for biological material, such as various classes of coding and non-coding...
Extracellular vesicles (EVs) are important for intercellular communication and act as vehicles for biological material, such as various classes of coding and non-coding RNAs, a few of which were shown to selectively target into vesicles. However, protein factors, mechanisms, and sequence elements contributing to this specificity remain largely elusive. Here, we use a reporter system that results in different types of modified transcripts to decipher the specificity determinants of RNAs released into EVs. First, we found that small RNAs are more efficiently packaged into EVs than large ones, and second, we determined absolute quantities for several endogenous RNA transcripts in EVs (U6 snRNA, U1 snRNA, Y1 RNA, and GAPDH mRNA). We show that RNA polymerase III (pol III) transcripts are more efficiently secreted into EVs compared to pol II-derived transcripts. Surprisingly, our quantitative analysis revealed no RNA accumulation in the vesicles relative to the total cellular levels, based on both overexpressed reporter transcripts and endogenous RNAs. RNA appears to be EV-associated only at low copy numbers, ranging between 0.02 and 1 molecule per EV. This RNA association may reflect internal EV encapsulation or a less tightly bound state at the vesicle surface.
Topics: Cell Line; Extracellular Vesicles; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); Humans; Poly A; Polyadenylation; RNA Caps; RNA Polymerase III; RNA, Messenger; RNA, Small Nuclear
PubMed: 34685656
DOI: 10.3390/cells10102674 -
Nature Biotechnology Apr 2023Spatial transcriptomics reveals the spatial context of gene expression, but current methods are limited to assaying polyadenylated (A-tailed) RNA transcripts. Here we...
Spatial transcriptomics reveals the spatial context of gene expression, but current methods are limited to assaying polyadenylated (A-tailed) RNA transcripts. Here we demonstrate that enzymatic in situ polyadenylation of RNA enables detection of the full spectrum of RNAs, expanding the scope of sequencing-based spatial transcriptomics to the total transcriptome. We demonstrate that our spatial total RNA-sequencing (STRS) approach captures coding RNAs, noncoding RNAs and viral RNAs. We apply STRS to study skeletal muscle regeneration and viral-induced myocarditis. Our analyses reveal the spatial patterns of noncoding RNA expression with near-cellular resolution, identify spatially defined expression of noncoding transcripts in skeletal muscle regeneration and highlight host transcriptional responses associated with local viral RNA abundance. STRS requires adding only one step to the widely used Visium spatial total RNA-sequencing protocol from 10x Genomics, and thus could be easily adopted to enable new insights into spatial gene regulation and biology.
Topics: Transcriptome; Polyadenylation; RNA, Messenger; Gene Expression Profiling; RNA, Viral
PubMed: 36329320
DOI: 10.1038/s41587-022-01517-6 -
Gene Sep 2019Two core polyadenylation elements (CPE) located in the 3' untranslated region of eukaryotic pre-mRNAs play an essential role in their processing: the polyadenylation...
Two core polyadenylation elements (CPE) located in the 3' untranslated region of eukaryotic pre-mRNAs play an essential role in their processing: the polyadenylation signal (PAS) AAUAAA and the cleavage site (CS), preferentially a CA dinucleotide. Herein, we characterized PAS and CS sequences in a set of cancer predisposition genes (CPGs) and performed an in silico investigation of microRNAs (miRNAs) regulation to identify potential tumor-suppressive and oncogenic miRNAs. NCBI and alternative polyadenylation databases were queried to characterize CPE sequences in 117 CPGs, including 81 and 17 known tumor suppressor genes and oncogenes, respectively. miRNA-mediated regulation analysis was performed using predicted and validated data sources. Based on NCBI analyses, we did not find an established PAS in 21 CPGs, and verified that the majority of PAS already described (74.4%) had the canonical sequence AAUAAA. Interestingly, "AA" dinucleotide was the most common CS (37.5%) associated with this set of genes. Approximately 90% of CPGs exhibited evidence of alternative polyadenylation (more than one functional PAS). Finally, the mir-192 family was significantly overrepresented as regulator of tumor suppressor genes (P < 0.01), which suggests a potential oncogenic function. Overall, this study provides a landscape of CPE in CPGs, which might be useful in development of future molecular analyses covering these frequently neglected regulatory sequences.
Topics: 3' Untranslated Regions; Binding Sites; Cell Line, Tumor; Computer Simulation; Genetic Predisposition to Disease; Humans; MicroRNAs; Neoplasms; Nucleotides; Oncogenes; Poly A; Polyadenylation; RNA Precursors; Signal Transduction
PubMed: 31229581
DOI: 10.1016/j.gene.2019.143943 -
RNA (New York, N.Y.) Dec 2019Most eukaryotic messenger RNA precursors must undergo 3'-end cleavage and polyadenylation for maturation. We and others recently reported the structure of the AAUAAA...
Most eukaryotic messenger RNA precursors must undergo 3'-end cleavage and polyadenylation for maturation. We and others recently reported the structure of the AAUAAA polyadenylation signal (PAS) in complex with the protein factors CPSF-30, WDR33, and CPSF-160, revealing the molecular mechanism for this recognition. Here we have characterized in detail the interactions between the PAS RNA and the protein factors using fluorescence polarization experiments. Our studies show that AAUAAA is recognized with ∼3 nM affinity by the CPSF-160-WDR33-CPSF-30 ternary complex. Variations in the RNA sequence can greatly reduce the affinity. Similarly, mutations of CPSF-30 residues that have van der Waals interactions with the bases of AAUAAA also lead to substantial reductions in affinity. Finally, our studies confirm that both CPSF-30 and WDR33 are required for high-affinity binding of the PAS RNA, while these two proteins alone and their binary complexes with CPSF-160 have much lower affinity for the RNA.
Topics: Cleavage And Polyadenylation Specificity Factor; Fluorescence; Humans; Nuclear Proteins; Poly A; Polyadenylation; Protein Binding; RNA Precursors; RNA, Messenger; mRNA Cleavage and Polyadenylation Factors
PubMed: 31462423
DOI: 10.1261/rna.070870.119 -
Nature Aug 2014Animal transcriptomes are dynamic, with each cell type, tissue and organ system expressing an ensemble of transcript isoforms that give rise to substantial diversity....
Animal transcriptomes are dynamic, with each cell type, tissue and organ system expressing an ensemble of transcript isoforms that give rise to substantial diversity. Here we have identified new genes, transcripts and proteins using poly(A)+ RNA sequencing from Drosophila melanogaster in cultured cell lines, dissected organ systems and under environmental perturbations. We found that a small set of mostly neural-specific genes has the potential to encode thousands of transcripts each through extensive alternative promoter usage and RNA splicing. The magnitudes of splicing changes are larger between tissues than between developmental stages, and most sex-specific splicing is gonad-specific. Gonads express hundreds of previously unknown coding and long non-coding RNAs (lncRNAs), some of which are antisense to protein-coding genes and produce short regulatory RNAs. Furthermore, previously identified pervasive intergenic transcription occurs primarily within newly identified introns. The fly transcriptome is substantially more complex than previously recognized, with this complexity arising from combinatorial usage of promoters, splice sites and polyadenylation sites.
Topics: Alternative Splicing; Animals; Drosophila melanogaster; Female; Gene Expression Profiling; Male; Molecular Sequence Annotation; Nerve Tissue; Organ Specificity; Poly A; Polyadenylation; Promoter Regions, Genetic; RNA, Long Noncoding; RNA, Messenger; Sex Characteristics; Stress, Physiological; Transcriptome
PubMed: 24670639
DOI: 10.1038/nature12962