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Proceedings of the National Academy of... Mar 2020Lens transparency is established by abundant accumulation of crystallin proteins and loss of organelles in the fiber cells. It requires an efficient translation of lens...
Lens transparency is established by abundant accumulation of crystallin proteins and loss of organelles in the fiber cells. It requires an efficient translation of lens messenger RNAs (mRNAs) to overcome the progressively reduced transcriptional activity that results from denucleation. Inappropriate regulation of this process impairs lens differentiation and causes cataract formation. However, the regulatory mechanism promoting protein synthesis from lens-expressed mRNAs remains unclear. Here we show that in zebrafish, the RNA-binding protein Rbm24 is critically required for the accumulation of crystallin proteins and terminal differentiation of lens fiber cells. In the developing lens, Rbm24 binds to a wide spectrum of lens-specific mRNAs through the RNA recognition motif and interacts with cytoplasmic polyadenylation element-binding protein (Cpeb1b) and cytoplasmic poly(A)-binding protein (Pabpc1l) through the C-terminal region. Loss of Rbm24 reduces the stability of a subset of lens mRNAs encoding heat shock proteins and shortens the poly(A) tail length of mRNAs encoding lens structural components, thereby preventing their translation into functional proteins. This severely impairs lens transparency and results in blindness. Consistent with its highly conserved expression in differentiating lens fiber cells, the findings suggest that vertebrate Rbm24 represents a key regulator of cytoplasmic polyadenylation and plays an essential role in the posttranscriptional control of lens development.
Topics: Animals; Cataract; Crystallins; Cytoplasm; Lens, Crystalline; Models, Animal; Polyadenylation; RNA Processing, Post-Transcriptional; RNA, Messenger; RNA-Binding Proteins; Transcription Factors; Zebrafish; Zebrafish Proteins
PubMed: 32170011
DOI: 10.1073/pnas.1917922117 -
Genome Research Oct 2019The cleavage and polyadenylation reaction is a crucial step in transcription termination and pre-mRNA maturation in human cells. Despite extensive research, the encoding...
The cleavage and polyadenylation reaction is a crucial step in transcription termination and pre-mRNA maturation in human cells. Despite extensive research, the encoding of polyadenylation-mediated regulation of gene expression within the DNA sequence is not well understood. Here, we utilized a massively parallel reporter assay to inspect the effect of over 12,000 rationally designed polyadenylation sequences (PASs) on reporter gene expression and cleavage efficiency. We find that the PAS sequence can modulate gene expression by over five orders of magnitude. By using a uniquely designed scanning mutagenesis data set, we gain mechanistic insight into various modes of action by which the cleavage efficiency affects the sensitivity or robustness of the PAS to mutation. Furthermore, we employ motif discovery to identify both known and novel sequence motifs associated with PAS-mediated regulation. By leveraging the large scale of our data, we train a deep learning model for the highly accurate prediction of RNA levels from DNA sequence alone ( = 0.83). Moreover, we devise unique approaches for predicting exact cleavage sites for our reporter constructs and for endogenous transcripts. Taken together, our results expand our understanding of PAS-mediated regulation, and provide an unprecedented resource for analyzing and predicting PAS for regulatory genomics applications.
Topics: 3' Untranslated Regions; Deep Learning; Gene Expression Regulation; Genes, Reporter; Genomics; High-Throughput Nucleotide Sequencing; Humans; Mutagenesis; Polyadenylation; RNA Precursors; RNA, Messenger; Transcription Termination, Genetic
PubMed: 31530582
DOI: 10.1101/gr.247312.118 -
Clinical and Translational Medicine Jul 2022Cell-free messenger RNA (cf-mRNA) and long non-coding RNA (cf-lncRNA) are becoming increasingly important in liquid biopsy by providing biomarkers for disease...
BACKGROUND
Cell-free messenger RNA (cf-mRNA) and long non-coding RNA (cf-lncRNA) are becoming increasingly important in liquid biopsy by providing biomarkers for disease prediction, diagnosis and prognosis, but the simultaneous characterization of coding and non-coding RNAs in human biofluids remains challenging.
METHODS
Here, we developed polyadenylation ligation-mediated sequencing (PALM-Seq), an RNA sequencing strategy employing treatment of RNA with T4 polynucleotide kinase to generate cell-free RNA (cfRNA) fragments with 5' phosphate and 3' hydroxyl and RNase H to deplete abundant RNAs, achieving simultaneous quantification and characterization of cfRNAs.
RESULTS
Using PALM-Seq, we successfully identified well-known differentially abundant mRNA, lncRNA and microRNA in the blood plasma of pregnant women. We further characterized cfRNAs in blood plasma, saliva, urine, seminal plasma and amniotic fluid and found that the detected numbers of different RNA biotypes varied with body fluids. The profiles of cf-mRNA reflected the function of originated tissues, and immune cells significantly contributed RNA to blood plasma and saliva. Short fragments (<50 nt) of mRNA and lncRNA were major in biofluids, whereas seminal plasma and amniotic fluid tended to retain long RNA. Body fluids showed distinct preferences of pyrimidine at the 3' end and adenine at the 5' end of cf-mRNA and cf-lncRNA, which were correlated with the proportions of short fragments.
CONCLUSION
Together, PALM-Seq enables a simultaneous characterization of cf-mRNA and cf-lncRNA, contributing to elucidating the biology and promoting the application of cfRNAs.
Topics: Cell-Free Nucleic Acids; Female; Humans; MicroRNAs; Polyadenylation; Pregnancy; RNA, Long Noncoding; RNA, Messenger; Sequence Analysis, RNA
PubMed: 35858042
DOI: 10.1002/ctm2.987 -
International Journal of Molecular... Sep 2020Mature mRNA is generated by the 3' end cleavage and polyadenylation of its precursor pre-mRNA. Eukaryotic genes frequently have multiple polyadenylation sites, resulting... (Review)
Review
Mature mRNA is generated by the 3' end cleavage and polyadenylation of its precursor pre-mRNA. Eukaryotic genes frequently have multiple polyadenylation sites, resulting in mRNA isoforms with different 3'-UTR lengths that often encode different C-terminal amino acid sequences. It is well-known that this form of post-transcriptional modification, termed alternative polyadenylation, can affect mRNA stability, localization, translation, and nuclear export. We focus on the alternative polyadenylation of pre-mRNA for vascular endothelial growth factor receptor-1 (VEGFR-1), the receptor for VEGF. VEGFR-1 is a transmembrane protein with a tyrosine kinase in the intracellular region. Secreted forms of VEGFR-1 (sVEGFR-1) are also produced from the same gene by alternative polyadenylation, and sVEGFR-1 has a function opposite to that of VEGFR-1 because it acts as a decoy receptor for VEGF. However, the mechanism that regulates the production of sVEGFR-1 by alternative polyadenylation remains poorly understood. In this review, we introduce and discuss the mechanism of alternative polyadenylation of VEGFR-1 mediated by protein arginine methylation.
Topics: Arginine; Methylation; Polyadenylation; RNA Precursors; RNA Stability; RNA, Messenger; Vascular Endothelial Growth Factor Receptor-1
PubMed: 32899690
DOI: 10.3390/ijms21186460 -
Molecules and Cells Jan 2023In eukaryotic cells, a key RNA processing step to generate mature mRNA is the coupled reaction for cleavage and polyadenylation (CPA) at the 3' end of individual... (Review)
Review
In eukaryotic cells, a key RNA processing step to generate mature mRNA is the coupled reaction for cleavage and polyadenylation (CPA) at the 3' end of individual transcripts. Many transcripts are alternatively polyadenylated (APA) to produce mRNAs with different 3' ends that may either alter protein coding sequence (CDS-APA) or create different lengths of 3'UTR (tandem-APA). As the CPA reaction is intimately associated with transcriptional termination, it has been widely assumed that APA is regulated cotranscriptionally. Isoforms terminated at different regions may have distinct RNA stability under different conditions, thus altering the ratio of APA isoforms. Such differential impacts on different isoforms have been considered as post-transcriptional APA, but strictly speaking, this can only be considered "apparent" APA, as the choice is not made during the CPA reaction. Interestingly, a recent study reveals sequential APA as a new mechanism for post-transcriptional APA. This minireview will focus on this new mechanism to provide insights into various documented regulatory paradigms.
Topics: Polyadenylation; Transcription, Genetic; Protein Isoforms; RNA, Messenger; 3' Untranslated Regions
PubMed: 36697238
DOI: 10.14348/molcells.2023.2176 -
Science Advances Feb 2023Alternative polyadenylation (APA) creates distinct transcripts from the same gene by cleaving the pre-mRNA at poly(A) sites that can lie within the 3' untranslated...
Alternative polyadenylation (APA) creates distinct transcripts from the same gene by cleaving the pre-mRNA at poly(A) sites that can lie within the 3' untranslated region (3'UTR), introns, or exons. Most studies focus on APA within the 3'UTR; however, here, we show that CPSF6 insufficiency alters protein levels and causes a developmental syndrome by deregulating APA throughout the transcript. In neonatal humans and zebrafish larvae, CPSF6 insufficiency shifts poly(A) site usage between the 3'UTR and internal sites in a pathway-specific manner. Genes associated with neuronal function undergo mostly intronic APA, reducing their expression, while genes associated with heart and skeletal function mostly undergo 3'UTR APA and are up-regulated. This suggests that, under healthy conditions, cells toggle between internal and 3'UTR APA to modulate protein expression.
Topics: Animals; Humans; Infant, Newborn; 3' Untranslated Regions; Exons; Introns; Polyadenylation; Zebrafish; Embryo, Nonmammalian
PubMed: 36800428
DOI: 10.1126/sciadv.ade4814 -
Biomolecules Oct 2021Signal transduction pathways transmit the information received from external and internal cues and generate a response that allows the cell to adapt to changes in the... (Review)
Review
Signal transduction pathways transmit the information received from external and internal cues and generate a response that allows the cell to adapt to changes in the surrounding environment. Signaling pathways trigger rapid responses by changing the activity or localization of existing molecules, as well as long-term responses that require the activation of gene expression programs. All steps involved in the regulation of gene expression, from transcription to processing and utilization of new transcripts, are modulated by multiple signal transduction pathways. This review provides a broad overview of the post-translational regulation of factors involved in RNA processing events by signal transduction pathways, with particular focus on the regulation of pre-mRNA splicing, cleavage and polyadenylation. The effects of several post-translational modifications (i.e., sumoylation, ubiquitination, methylation, acetylation and phosphorylation) on the expression, subcellular localization, stability and affinity for RNA and protein partners of many RNA-binding proteins are highlighted. Moreover, examples of how some of the most common signal transduction pathways can modulate biological processes through changes in RNA processing regulation are illustrated. Lastly, we discuss challenges and opportunities of therapeutic approaches that correct RNA processing defects and target signaling molecules.
Topics: Alternative Splicing; Humans; Methylation; Phosphorylation; Polyadenylation; RNA; RNA Processing, Post-Transcriptional; RNA-Binding Proteins; Signal Transduction; Sumoylation; Ubiquitination
PubMed: 34680108
DOI: 10.3390/biom11101475 -
The New Phytologist Jul 2022Alternative splicing (AS) and alternative polyadenylation (APA) contribute significantly to the regulation of gene expression in higher eukaryotes. Their biological...
Alternative splicing (AS) and alternative polyadenylation (APA) contribute significantly to the regulation of gene expression in higher eukaryotes. Their biological impact in filamentous fungi, however, is largely unknown. Here we combine PacBio Isoform-Sequencing and strand-specific RNA-sequencing of multiple tissues and mutant characterization to reveal the landscape and regulation of AS and APA in Fusarium graminearum. We generated a transcript annotation comprising 51 617 isoforms from 17 189 genes. In total, 4997 and 11 133 genes are alternatively spliced and polyadenylated, respectively. Majority of the AS events alter coding sequences. Unexpectedly, the AS transcripts containing premature-termination codons are not sensitive to nonsense-mediated messenger RNA decay. Unlike in yeasts and animals, distal APA sites have strong signals, but proximal APA isoforms are highly expressed in F. graminearum. The 3'-end processing factors FgRNA15, FgHRP1, and FgFIP1 play roles in promoting proximal APA site usage and intron splicing. A genome-wide increase in intron inclusion and distal APA site usage and downregulation of the spliceosomal and 3'-end processing factors were observed in older and quiescent tissues, indicating intron inclusion and 3'-untranslated region lengthening as novel mechanisms in regulating aging and dormancy in fungi. This study provides new insights into the complexity and regulation of AS and APA in filamentous fungi.
Topics: 3' Untranslated Regions; Alternative Splicing; Animals; Fungi; Polyadenylation; Protein Isoforms
PubMed: 35451076
DOI: 10.1111/nph.18164 -
International Journal of Molecular... May 2020The 3' untranslated regions (3' UTRs) of mRNAs serve as hubs for post-transcriptional control as the targets of microRNAs (miRNAs) and RNA-binding proteins (RBPs).... (Review)
Review
The 3' untranslated regions (3' UTRs) of mRNAs serve as hubs for post-transcriptional control as the targets of microRNAs (miRNAs) and RNA-binding proteins (RBPs). Sequences in 3' UTRs confer alterations in mRNA stability, direct mRNA localization to subcellular regions, and impart translational control. Thousands of mRNAs are localized to subcellular compartments in neurons-including axons, dendrites, and synapses-where they are thought to undergo local translation. Despite an established role for 3' UTR sequences in imparting mRNA localization in neurons, the specific RNA sequences and structural features at play remain poorly understood. The nervous system selectively expresses longer 3' UTR isoforms via alternative polyadenylation (APA). The regulation of APA in neurons and the neuronal functions of longer 3' UTR mRNA isoforms are starting to be uncovered. Surprising roles for 3' UTRs are emerging beyond the regulation of protein synthesis and include roles as RBP delivery scaffolds and regulators of alternative splicing. Evidence is also emerging that 3' UTRs can be cleaved, leading to stable, isolated 3' UTR fragments which are of unknown function. Mutations in 3' UTRs are implicated in several neurological disorders-more studies are needed to uncover how these mutations impact gene regulation and what is their relationship to disease severity.
Topics: 3' Untranslated Regions; Alternative Splicing; Animals; Axons; Dendrites; Humans; Neurons; Polyadenylation; RNA Isoforms; RNA-Binding Proteins
PubMed: 32408514
DOI: 10.3390/ijms21103413 -
Molecules and Cells Jan 2023Genomic information stored in the DNA is transcribed to the mRNA and translated to proteins. The 3' untranslated regions (3'UTRs) of the mRNA serve pivotal roles in... (Review)
Review
Genomic information stored in the DNA is transcribed to the mRNA and translated to proteins. The 3' untranslated regions (3'UTRs) of the mRNA serve pivotal roles in posttranscriptional gene expression, regulating mRNA stability, translation, and localization. Similar to DNA mutations producing aberrant proteins, RNA alterations expand the transcriptome landscape and change the cellular proteome. Recent global analyses reveal that many genes express various forms of altered RNAs, including 3'UTR length variants. Alternative polyadenylation and alternative splicing are involved in diversifying 3'UTRs, which could act as a hidden layer of eukaryotic gene expression control. In this review, we summarize the functions and regulations of 3'UTRs and elaborate on the generation and functional consequences of 3'UTR diversity. Given that dynamic 3'UTR length control contributes to phenotypic complexity, dysregulated 3'UTR diversity might be relevant to disease development, including cancers. Thus, 3'UTR diversity in cancer could open exciting new research areas and provide avenues for novel cancer theragnostics.
Topics: Humans; 3' Untranslated Regions; RNA, Messenger; Polyadenylation; Transcriptome; Alternative Splicing
PubMed: 36697237
DOI: 10.14348/molcells.2023.0003