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Nature Structural & Molecular Biology Feb 2023Poly(A)-tail-mediated post-transcriptional regulation of maternal mRNAs is vital in the oocyte-to-embryo transition (OET). Nothing is known about poly(A) tail dynamics...
Poly(A)-tail-mediated post-transcriptional regulation of maternal mRNAs is vital in the oocyte-to-embryo transition (OET). Nothing is known about poly(A) tail dynamics during the human OET. Here, we show that poly(A) tail length and internal non-A residues are highly dynamic during the human OET, using poly(A)-inclusive RNA isoform sequencing (PAIso-seq). Unexpectedly, maternal mRNAs undergo global remodeling: after deadenylation or partial degradation into 3'-UTRs, they are re-polyadenylated to produce polyadenylated degradation intermediates, coinciding with massive incorporation of non-A residues, particularly internal long consecutive U residues, into the newly synthesized poly(A) tails. Moreover, TUT4 and TUT7 contribute to the incorporation of these U residues, BTG4-mediated deadenylation produces substrates for maternal mRNA re-polyadenylation, and TENT4A and TENT4B incorporate internal G residues. The maternal mRNA remodeling is further confirmed using PAIso-seq2. Importantly, maternal mRNA remodeling is essential for the first cleavage of human embryos. Together, these findings broaden our understanding of the post-transcriptional regulation of maternal mRNAs during the human OET.
Topics: Humans; RNA, Messenger, Stored; Oocytes; RNA, Messenger; Gene Expression Regulation; Polyadenylation; Poly A
PubMed: 36646905
DOI: 10.1038/s41594-022-00908-2 -
Frontiers in Immunology 2022Eukaryotic mRNA 3´-end processing is a multi-step process beginning with pre-mRNA transcript cleavage followed by poly(A) tail addition. Closely coupled to... (Review)
Review
Eukaryotic mRNA 3´-end processing is a multi-step process beginning with pre-mRNA transcript cleavage followed by poly(A) tail addition. Closely coupled to transcription termination, 3´-end processing is a critical step in the regulation of gene expression, and disruption of 3´-end processing is known to affect mature mRNA levels. Various viral proteins interfere with the 3´-end processing machinery, causing read-through transcription and altered levels of mature transcripts through inhibition of cleavage and polyadenylation. Thus, disruption of 3´-end processing contributes to widespread host shutoff, including suppression of the antiviral response. Additionally, observed features of read-through transcripts such as decreased polyadenylation, nuclear retention, and decreased translation suggest that viruses may utilize these mechanisms to modulate host protein production and dominate cellular machinery. The degree to which the effects of read-through transcript production are harnessed by viruses and host cells remains unclear, but existing research highlights the importance of host 3´-end processing modulation during viral infection.
Topics: DNA Viruses; Polyadenylation; RNA, Messenger; Transcription, Genetic; Viral Proteins
PubMed: 35222412
DOI: 10.3389/fimmu.2022.828665 -
Molecular Cell Jun 2022Viegas et al. (2022) discover that in Trypanosoma brucei the poly(A) tails of the variant surface glycoprotein (VSG) transcripts are methylated, a mechanism that...
Viegas et al. (2022) discover that in Trypanosoma brucei the poly(A) tails of the variant surface glycoprotein (VSG) transcripts are methylated, a mechanism that stabilizes these transcripts and ensures protection against the immune response in mammals.
Topics: Animals; Mammals; Membrane Glycoproteins; Poly A; RNA, Messenger; Trypanosoma brucei brucei; Variant Surface Glycoproteins, Trypanosoma
PubMed: 35659324
DOI: 10.1016/j.molcel.2022.05.017 -
Nucleic Acids Research Aug 2023Differentiation of neural progenitor cells into mature neuronal phenotypes relies on extensive temporospatial coordination of mRNA expression to support the development...
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 positively correlated 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.
Topics: RNA, Messenger; 3' Untranslated Regions; Gene Expression Regulation; Polyadenylation; MicroRNAs; Cell Differentiation
PubMed: 37293985
DOI: 10.1093/nar/gkad499 -
Journal of Experimental Botany Apr 2023To be properly expressed, genes need to be accompanied by a terminator, a region downstream of the coding sequence that contains the information necessary for the... (Review)
Review
To be properly expressed, genes need to be accompanied by a terminator, a region downstream of the coding sequence that contains the information necessary for the maturation of the mRNA 3' end. The main event in this process is the addition of a poly(A) tail at the 3' end of the new transcript, a critical step in mRNA biology that has important consequences for the expression of genes. Here, we review the mechanism leading to cleavage and polyadenylation of newly transcribed mRNAs and how this process can affect the final levels of gene expression. We give special attention to an aspect often overlooked, the effect that different terminators can have on the expression of genes. We also discuss some exciting findings connecting the choice of terminator to the biogenesis of small RNAs, which are a central part of one of the most important mechanisms of regulation of gene expression in plants.
Topics: Terminator Regions, Genetic; Base Sequence; Polyadenylation; RNA, Messenger; Gene Expression; Transcription, Genetic
PubMed: 36477559
DOI: 10.1093/jxb/erac467 -
Nature Communications Jan 2023The molecular causes of deteriorating oocyte quality during aging are poorly defined. Since oocyte developmental competence relies on post-transcriptional regulations,...
The molecular causes of deteriorating oocyte quality during aging are poorly defined. Since oocyte developmental competence relies on post-transcriptional regulations, we tested whether defective mRNA translation contributes to this decline in quality. Disruption in ribosome loading on maternal transcripts is present in old oocytes. Using a candidate approach, we detect altered translation of 3'-UTR-reporters and altered poly(A) length of the endogenous mRNAs. mRNA polyadenylation depends on the cytoplasmic polyadenylation binding protein 1 (CPEB1). Cpeb1 mRNA translation and protein levels are decreased in old oocytes. This decrease causes de-repression of Ccnb1 translation in quiescent oocytes, premature CDK1 activation, and accelerated reentry into meiosis. De-repression of Ccnb1 is corrected by Cpeb1 mRNA injection in old oocytes. Oocyte-specific Cpeb1 haploinsufficiency in young oocytes recapitulates all the translation phenotypes of old oocytes. These findings demonstrate that a dysfunction in the oocyte translation program is associated with the decline in oocyte quality during aging.
Topics: Meiosis; mRNA Cleavage and Polyadenylation Factors; Oocytes; Polyadenylation; Protein Biosynthesis; RNA, Messenger; Aging; Animals; Maternal Age; Female
PubMed: 36697412
DOI: 10.1038/s41467-023-35994-3 -
Development (Cambridge, England) Jul 2022As the microtubule-organizing centers of most cells, centrosomes engineer the bipolar mitotic spindle required for error-free mitosis. Drosophila Pericentrin-like...
As the microtubule-organizing centers of most cells, centrosomes engineer the bipolar mitotic spindle required for error-free mitosis. Drosophila Pericentrin-like protein (PLP) directs formation of a pericentriolar material (PCM) scaffold required for PCM organization and microtubule-organizing center function. Here, we investigate the post-transcriptional regulation of Plp mRNA. We identify conserved binding sites for cytoplasmic polyadenylation element binding (CPEB) proteins within the Plp 3'-untranslated region and examine the role of the CPEB ortholog Oo18 RNA-binding protein (Orb) in Plp mRNA regulation. Our data show that Orb interacts biochemically with Plp mRNA to promote polyadenylation and PLP protein expression. Loss of orb, but not orb2, diminishes PLP levels in embryonic extracts. Consequently, PLP localization to centrosomes and its function in PCM scaffolding are compromised in orb mutant embryos, resulting in genomic instability and embryonic lethality. Moreover, we find that PLP overexpression restores centrosome scaffolding and rescues the cell division defects caused by orb depletion. Our data suggest that Orb modulates PLP expression at the level of Plp mRNA polyadenylation and demonstrates that the post-transcriptional regulation of core, conserved centrosomal mRNAs is crucial for centrosome function.
Topics: Animals; Antigens; Centrosome; Drosophila Proteins; Drosophila melanogaster; Mitosis; Polyadenylation; RNA, Messenger
PubMed: 35661190
DOI: 10.1242/dev.200426 -
FEBS Open Bio Jul 2023During their synthesis in the cell nucleus, most eukaryotic mRNAs undergo a two-step 3'-end processing reaction in which the pre-mRNA is cleaved and released from the... (Review)
Review
During their synthesis in the cell nucleus, most eukaryotic mRNAs undergo a two-step 3'-end processing reaction in which the pre-mRNA is cleaved and released from the transcribing RNA polymerase II and a polyadenosine (poly(A)) tail is added to the newly formed 3'-end. These biochemical reactions might appear simple at first sight (endonucleolytic RNA cleavage and synthesis of a homopolymeric tail), but their catalysis requires a multi-faceted enzymatic machinery, the cleavage and polyadenylation complex (CPAC), which is composed of more than 20 individual protein subunits. The activity of CPAC is further orchestrated by Poly(A) Binding Proteins (PABPs), which decorate the poly(A) tail during its synthesis and guide the mRNA through subsequent gene expression steps. Here, we review the structure, molecular mechanism, and regulation of eukaryotic mRNA 3'-end processing machineries with a focus on the polyadenylation step. We concentrate on the CPAC and PABPs from mammals and the budding yeast, Saccharomyces cerevisiae, because these systems are the best-characterized at present. Comparison of their functions provides valuable insights into the principles of mRNA 3'-end processing.
Topics: Animals; Polyadenylation; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Cell Nucleus; RNA, Messenger; Mammals
PubMed: 36416579
DOI: 10.1002/2211-5463.13528 -
Biomolecules Jun 2020A crucial feature of gene expression involves RNA processing to produce 3' ends through a process termed 3' end cleavage and polyadenylation (CPA). This ensures the... (Review)
Review
A crucial feature of gene expression involves RNA processing to produce 3' ends through a process termed 3' end cleavage and polyadenylation (CPA). This ensures the nascent RNA molecule can exit the nucleus and be translated to ultimately give rise to a protein which can execute a function. Further, alternative polyadenylation (APA) can produce distinct transcript isoforms, profoundly expanding the complexity of the transcriptome. CPA is carried out by multi-component protein complexes interacting with multiple RNA motifs and is tightly coupled to transcription, other steps of RNA processing, and even epigenetic modifications. CPA and APA contribute to the maintenance of a multitude of diverse physiological processes. It is therefore not surprising that disruptions of CPA and APA can lead to devastating disorders. Here, we review potential CPA and APA mechanisms involving both loss and gain of function that can have tremendous impacts on health and disease. Ultimately we highlight the emerging diagnostic and therapeutic potential CPA and APA offer.
Topics: Cardiovascular Diseases; Humans; Neoplasms; Neurodegenerative Diseases; Polyadenylation; RNA; RNA Cleavage
PubMed: 32560344
DOI: 10.3390/biom10060915 -
Nucleic Acids Research May 2023RNA-binding proteins (RPBs) are deeply involved in fundamental cellular processes in bacteria and are vital for their survival. Despite this, few studies have so far...
RNA-binding proteins (RPBs) are deeply involved in fundamental cellular processes in bacteria and are vital for their survival. Despite this, few studies have so far been dedicated to direct and global identification of bacterial RBPs. We have adapted the RNA interactome capture (RIC) technique, originally developed for eukaryotic systems, to globally identify RBPs in bacteria. RIC takes advantage of the base pairing potential of poly(A) tails to pull-down RNA-protein complexes. Overexpressing poly(A) polymerase I in Escherichia coli drastically increased transcriptome-wide RNA polyadenylation, enabling pull-down of crosslinked RNA-protein complexes using immobilized oligo(dT) as bait. With this approach, we identified 169 putative RBPs, roughly half of which are already annotated as RNA-binding. We experimentally verified the RNA-binding ability of a number of uncharacterized RBPs, including YhgF, which is exceptionally well conserved not only in bacteria, but also in archaea and eukaryotes. We identified YhgF RNA targets in vivo using CLIP-seq, verified specific binding in vitro, and reveal a putative role for YhgF in regulation of gene expression. Our findings present a simple and robust strategy for RBP identification in bacteria, provide a resource of new bacterial RBPs, and lay the foundation for further studies of the highly conserved RBP YhgF.
Topics: Chromatin Immunoprecipitation Sequencing; Escherichia coli; Eukaryota; RNA-Binding Proteins; Transcriptome; RNA, Bacterial; Escherichia coli Proteins; Polynucleotide Adenylyltransferase; Polyadenylation; Protein Binding
PubMed: 36987847
DOI: 10.1093/nar/gkad216