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Trends in Cell Biology Mar 2019Poly(A) tails are non-templated additions of adenosines at the 3' ends of most eukaryotic mRNAs. In the nucleus, these RNAs are co-transcriptionally cleaved at a poly(A)... (Review)
Review
Poly(A) tails are non-templated additions of adenosines at the 3' ends of most eukaryotic mRNAs. In the nucleus, these RNAs are co-transcriptionally cleaved at a poly(A) site and then polyadenylated before being exported to the cytoplasm. In the cytoplasm, poly(A) tails play pivotal roles in the translation and stability of the mRNA. One challenge in studying poly(A) tails is that they are difficult to sequence and accurately measure. However, recent advances in sequencing technology, computational algorithms, and other assays have enabled a more detailed look at poly(A) tail length genome-wide throughout many developmental stages and organisms. With the help of these advances, our understanding of poly(A) tail length has evolved over the past 5 years with the recognition that highly expressed genes can have short poly(A) tails and the elucidation of the seemingly contradictory roles for poly(A)-binding protein (PABP) in facilitating both protection and deadenylation.
Topics: Algorithms; Animals; Cell Nucleus; Computational Biology; Cytoplasm; Humans; Poly A; RNA, Messenger; Sequence Analysis, RNA
PubMed: 30503240
DOI: 10.1016/j.tcb.2018.11.002 -
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 -
Developmental Cell Apr 2024During oocyte maturation and early embryogenesis, changes in mRNA poly(A)-tail lengths strongly influence translation, but how these tail-length changes are orchestrated...
During oocyte maturation and early embryogenesis, changes in mRNA poly(A)-tail lengths strongly influence translation, but how these tail-length changes are orchestrated has been unclear. Here, we performed tail-length and translational profiling of mRNA reporter libraries (each with millions of 3' UTR sequence variants) in frog oocytes and embryos and in fish embryos. Contrasting to previously proposed cytoplasmic polyadenylation elements (CPEs), we found that a shorter element, UUUUA, together with the polyadenylation signal (PAS), specify cytoplasmic polyadenylation, and we identified contextual features that modulate the activity of both elements. In maturing oocytes, this tail lengthening occurs against a backdrop of global deadenylation and the action of C-rich elements that specify tail-length-independent translational repression. In embryos, cytoplasmic polyadenylation becomes more permissive, and additional elements specify waves of stage-specific deadenylation. Together, these findings largely explain the complex tapestry of tail-length changes observed in early frog and fish development, with strong evidence of conservation in both mice and humans.
Topics: Animals; Oocytes; Polyadenylation; Protein Biosynthesis; Poly A; 3' Untranslated Regions; RNA, Messenger; Gene Expression Regulation, Developmental; Mice; Humans; Embryo, Nonmammalian; Embryonic Development; Female; Xenopus laevis; Cytoplasm
PubMed: 38460509
DOI: 10.1016/j.devcel.2024.02.007 -
RNA (New York, N.Y.) May 2022Poly(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...
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 coprecipitated 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.
Topics: Animals; HEK293 Cells; Humans; Mice; Poly A; Polyadenylation; Polynucleotide Adenylyltransferase; RNA, Messenger; RNA-Binding Proteins; Transcription Factors; mRNA Cleavage and Polyadenylation Factors
PubMed: 35217597
DOI: 10.1261/rna.079050.121 -
Biochemical Society Transactions Dec 2015RNA-binding proteins are often multifunctional, interact with a variety of protein partners and display complex localizations within cells. Mammalian cytoplasmic... (Review)
Review
RNA-binding proteins are often multifunctional, interact with a variety of protein partners and display complex localizations within cells. Mammalian cytoplasmic poly(A)-binding proteins (PABPs) are multifunctional RNA-binding proteins that regulate multiple aspects of mRNA translation and stability. Although predominantly diffusely cytoplasmic at steady state, they shuttle through the nucleus and can be localized to a variety of cytoplasmic foci, including those associated with mRNA storage and localized translation. Intriguingly, PABP sub-cellular distribution can alter dramatically in response to cellular stress or viral infection, becoming predominantly nuclear and/or being enriched in induced cytoplasmic foci. However, relatively little is known about the mechanisms that govern this distribution/relocalization and in many cases PABP functions within specific sites remain unclear. Here we discuss the emerging evidence with respect to these questions in mammals.
Topics: Active Transport, Cell Nucleus; Animals; Cell Nucleus; Cytoplasm; Humans; Poly A; Poly(A)-Binding Proteins; Protein Biosynthesis; RNA Stability; RNA, Messenger
PubMed: 26614673
DOI: 10.1042/BST20150171 -
BMC Genomics Jul 2022Genome-wide RNA-sequencing technologies are increasingly critical to a wide variety of diagnostic and research applications. RNA-seq users often first enrich for mRNA,...
BACKGROUND
Genome-wide RNA-sequencing technologies are increasingly critical to a wide variety of diagnostic and research applications. RNA-seq users often first enrich for mRNA, with the most popular enrichment method being poly(A) selection. In many applications it is well-known that poly(A) selection biases the view of the transcriptome by selecting for longer tailed mRNA species.
RESULTS
Here, we show that poly(A) selection biases Oxford Nanopore direct RNA sequencing. As expected, poly(A) selection skews sequenced mRNAs toward longer poly(A) tail lengths. Interestingly, we identify a population of mRNAs (> 10% of genes' mRNAs) that are inconsistently captured by poly(A) selection due to highly variable poly(A) tails, and demonstrate this phenomenon in our hands and in published data. Importantly, we show poly(A) selection is dispensable for Oxford Nanopore's direct RNA-seq technique, and demonstrate successful library construction without poly(A) selection, with decreased input, and without loss of quality.
CONCLUSIONS
Our work expands the utility of direct RNA-seq by validating the use of total RNA as input, and demonstrates important technical artifacts from poly(A) selection that inconsistently skew mRNA expression and poly(A) tail length measurements.
Topics: High-Throughput Nucleotide Sequencing; Poly A; Polyadenylation; RNA; RNA, Messenger; Sequence Analysis, RNA; Transcriptome
PubMed: 35869428
DOI: 10.1186/s12864-022-08762-8 -
Methods in Enzymology 2021Transcription of mRNAs culminates in RNA cleavage and a coordinated polyadenylation event at the 3' end. In its journey to be translated, the resulting transcript is...
Transcription of mRNAs culminates in RNA cleavage and a coordinated polyadenylation event at the 3' end. In its journey to be translated, the resulting transcript is under constant regulation by cap-binding proteins, miRNAs, and RNA binding proteins, including poly(A) binding proteins (PABPs). The interplay between all these factors determines whether nuclear or cytoplasmic exoribonucleases will gain access to and remove the poly(A) tail, which is so critical to the stability and translation capacity of the mRNA. In this chapter, we present an overview of two of the key features of the mRNA life-cycle: cleavage/polyadenylation and deadenylation, and describe biochemical assays that have been generated to study the activity of each of these enzymatic reactions. Finally, we also provide protocols to investigate mRNA's poly(A) length. The importance of these assays is highlighted by the dynamic and essential role the poly(A) tail length plays in controlling gene expression.
Topics: Exoribonucleases; Poly A; Polyadenylation; RNA, Messenger; RNA-Binding Proteins
PubMed: 34183126
DOI: 10.1016/bs.mie.2021.04.005 -
Nature Structural & Molecular Biology Mar 2023Deadenylation generally constitutes the first and pivotal step in eukaryotic messenger RNA decay. Despite its importance in posttranscriptional regulations, the kinetics...
Deadenylation generally constitutes the first and pivotal step in eukaryotic messenger RNA decay. Despite its importance in posttranscriptional regulations, the kinetics of deadenylation and its regulation remain largely unexplored. Here we identify La ribonucleoprotein 1, translational regulator (LARP1) as a general decelerator of deadenylation, which acts mainly in the 30-60-nucleotide (nt) poly(A) length window. We measured the steady-state and pulse-chased distribution of poly(A)-tail length, and found that deadenylation slows down in the 30-60-nt range. LARP1 associates preferentially with short tails and its depletion results in accelerated deadenylation specifically in the 30-60-nt range. Consistently, LARP1 knockdown leads to a global reduction of messenger RNA abundance. LARP1 interferes with the CCR4-NOT-mediated deadenylation in vitro by forming a ternary complex with poly(A)-binding protein (PABP) and poly(A). Together, our work reveals a dynamic nature of deadenylation kinetics and a role of LARP1 as a poly(A) length-specific barricade that creates a threshold for deadenylation.
Topics: Exoribonucleases; RNA-Binding Proteins; Poly(A)-Binding Proteins; Gene Expression Regulation; RNA, Messenger; Poly A
PubMed: 36849640
DOI: 10.1038/s41594-023-00930-y -
Philosophical Transactions of the Royal... Nov 2018Post-transcriptional addition of poly(A) tails to the 3' end of RNA is one of the fundamental events controlling the functionality and fate of RNA in all kingdoms of... (Review)
Review
Post-transcriptional addition of poly(A) tails to the 3' end of RNA is one of the fundamental events controlling the functionality and fate of RNA in all kingdoms of life. Although an enzyme with poly(A)-adding activity was discovered in more than 50 years ago, its existence and role in prokaryotic RNA metabolism were neglected for many years. As a result, it was not until 1992 that poly(A) polymerase I was purified to homogeneity and its gene was finally identified. Further work revealed that, similar to its role in surveillance of aberrant nuclear RNAs of eukaryotes, the addition of poly(A) tails often destabilizes prokaryotic RNAs and their decay intermediates, thus facilitating RNA turnover. Moreover, numerous studies carried out over the last three decades have shown that polyadenylation greatly contributes to the control of prokaryotic gene expression by affecting the steady-state level of diverse protein-coding and non-coding transcripts including antisense RNAs involved in plasmid copy number control, expression of toxin-antitoxin systems and bacteriophage development. Here, we review the main findings related to the discovery of polyadenylation in prokaryotes, isolation, and characterization and regulation of bacterial poly(A)-adding activities, and discuss the impact of polyadenylation on prokaryotic mRNA metabolism and gene expression.This article is part of the theme issue '5' and 3' modifications controlling RNA degradation'.
Topics: Bacteria; Poly A; Polyadenylation; Prokaryotic Cells; RNA
PubMed: 30397102
DOI: 10.1098/rstb.2018.0166 -
Wiley Interdisciplinary Reviews. RNA 2024Most eukaryotic mRNAs and different non-coding RNAs undergo a form of 3' end processing known as polyadenylation. Polyadenylation machinery is present in almost all... (Review)
Review
Most eukaryotic mRNAs and different non-coding RNAs undergo a form of 3' end processing known as polyadenylation. Polyadenylation machinery is present in almost all organisms except few species. In bacteria, the machinery has evolved from PNPase, which adds heteropolymeric tails, to a poly(A)-specific polymerase. Differently, a complex machinery for accurate polyadenylation and several non-canonical poly(A) polymerases are developed in eukaryotes. The role of poly(A) tail has also evolved from serving as a degradative signal to a stabilizing modification that also regulates translation. In this review, we discuss poly(A) tail emergence in prokaryotes and its development into a stable, yet dynamic feature at the 3' end of mRNAs in eukaryotes. We also describe how appearance of novel poly(A) polymerases gives cells flexibility to shape poly(A) tail. We explain how poly(A) tail dynamics help regulate cognate RNA metabolism in a context-dependent manner, such as during oocyte maturation. Finally, we describe specific mRNAs in metazoans that bear stem-loops instead of poly(A) tails. We conclude with how recent discoveries about poly(A) tail can be applied to mRNA technology. This article is categorized under: RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Processing > 3' End Processing RNA Turnover and Surveillance > Regulation of RNA Stability.
Topics: Polyadenylation; Poly A; RNA; RNA, Messenger; Eukaryota
PubMed: 38485452
DOI: 10.1002/wrna.1837