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Cold Spring Harbor Protocols Oct 2019This is a general protocol for the isolation of mRNA from total RNA using oligo(dT) coupled to magnetic beads. First, total RNA is dissolved in a high-salt buffer and...
This is a general protocol for the isolation of mRNA from total RNA using oligo(dT) coupled to magnetic beads. First, total RNA is dissolved in a high-salt buffer and heated briefly to 65°C-70°C, followed by immediate cooling on ice to disrupt secondary structures. The RNA is subsequently annealed to the oligo(dT)-magnetic beads at room temperature; the high-salt binding buffer stabilizes the poly(A)-oligo(dT) complexes. A high-salt washing buffer is then used to wash away unbound RNAs while retaining oligo(dT)-bound poly(A) mRNAs. To elute the poly(A) mRNAs from the beads, a low-salt buffer (or water) is used to destabilize the poly(A)-oligo(dT) complexes. Alternatively, poly(A) mRNAs can be retained on the beads for downstream applications (e.g., solid-phase cDNA synthesis).
Topics: Cellulose; Chromatography, Affinity; DNA, Complementary; Magnetic Phenomena; Magnetics; Microspheres; Nucleic Acid Hybridization; Oligodeoxyribonucleotides; Poly A; RNA; RNA, Messenger
PubMed: 31575797
DOI: 10.1101/pdb.prot101733 -
Analytical and Bioanalytical Chemistry Feb 2018The 3'-polyadenosine (poly A) tail of in vitro transcribed (IVT) mRNA was studied using liquid chromatography coupled to mass spectrometry (LC-MS). Poly A tails were...
The 3'-polyadenosine (poly A) tail of in vitro transcribed (IVT) mRNA was studied using liquid chromatography coupled to mass spectrometry (LC-MS). Poly A tails were cleaved from the mRNA using ribonuclease T1 followed by isolation with dT magnetic beads. Extracted tails were then analyzed by LC-MS which provided tail length information at single-nucleotide resolution. A 2100-nt mRNA with plasmid-encoded poly A tail lengths of either 27, 64, 100, or 117 nucleotides was used for these studies as enzymatically added poly A tails showed significant length heterogeneity. The number of As observed in the tails closely matched Sanger sequencing results of the DNA template, and even minor plasmid populations with sequence variations were detected. When the plasmid sequence contained a discreet number of poly As in the tail, analysis revealed a distribution that included tails longer than the encoded tail lengths. These observations were consistent with transcriptional slippage of T7 RNAP taking place within a poly A sequence. The type of RNAP did not alter the observed tail distribution, and comparison of T3, T7, and SP6 showed all three RNAPs produced equivalent tail length distributions. The addition of a sequence at the 3' end of the poly A tail did, however, produce narrower tail length distributions which supports a previously described model of slippage where the 3' end can be locked in place by having a G or C after the poly nucleotide region. Graphical abstract Determination of mRNA poly A tail length using magnetic beads and LC-MS.
Topics: Animals; Cell Line; Chromatography, High Pressure Liquid; Mass Spectrometry; Mice; Plasmids; Poly A; Polyadenylation; RNA, Messenger; Transcription, Genetic
PubMed: 29313076
DOI: 10.1007/s00216-017-0840-6 -
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 -
Wiley Interdisciplinary Reviews. RNA Jan 2023The 3'-end poly(A) tail is an important and potent feature of most mRNA molecules that affects mRNA fate and translation efficiency. Polyadenylation is a... (Review)
Review
The 3'-end poly(A) tail is an important and potent feature of most mRNA molecules that affects mRNA fate and translation efficiency. Polyadenylation is a posttranscriptional process that occurs in the nucleus by canonical poly(A) polymerases (PAPs). In some specific instances, the poly(A) tail can also be extended in the cytoplasm by noncanonical poly(A) polymerases (ncPAPs). This epitranscriptomic regulation of mRNA recently became one of the most interesting aspects in the field. Advances in RNA sequencing technologies and software development have allowed the precise measurement of poly(A) tails, identification of new ncPAPs, expansion of the function of known enzymes, discovery and a better understanding of the physiological role of tail heterogeneity, and recognition of a correlation between tail length and RNA translatability. Here, we summarize the development of polyadenylation research methods, including classic low-throughput approaches, Illumina-based genome-wide analysis, and advanced state-of-art techniques that utilize long-read third-generation sequencing with Pacific Biosciences and Oxford Nanopore Technologies platforms. A boost in technical opportunities over recent decades has allowed a better understanding of the regulation of gene expression at the mRNA level. This article is categorized under: RNA Methods > RNA Analyses In Vitro and In Silico.
Topics: Polyadenylation; RNA, Messenger; Cytoplasm; Sequence Analysis, RNA; Cell Nucleus; Poly A
PubMed: 35617484
DOI: 10.1002/wrna.1737 -
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 -
Science Advances Oct 2022Growing oocytes store a large amount of maternal mRNA to support the subsequent "maternal-zygotic transition" process. At present, it is not clear how the growing...
Growing oocytes store a large amount of maternal mRNA to support the subsequent "maternal-zygotic transition" process. At present, it is not clear how the growing oocytes store and process the newly transcribed mRNA under physiological conditions. In this study, we report non-membrane-bound compartments, nuclear poly(A) domains (NPADs), as the hub for newly transcribed mRNA, in developing mouse oocytes. The RNA binding protein PABPN1 promotes the formation of NPAD through its N-terminal disordered domain and RNA-recognized motif by means of liquid phase separation. -null growing oocytes cannot form NPAD normally in vivo and have defects in stability of oocyte growing-related transcripts and formation of long 3' untranslated region isoform transcripts. Ultimately, mice are completely sterile with primary ovarian insufficiency. These results demonstrate that NPAD formed by the phase separation properties of PABPN1-mRNA are the hub of the newly transcribed mRNA and essential for the development of oocytes and female reproduction.
Topics: Animals; Female; Mice; Cell Nucleus; Oocytes; Poly A; RNA, Messenger; RNA-Binding Proteins
PubMed: 36306357
DOI: 10.1126/sciadv.abn9016 -
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 -
Gene Jul 1990Until recently, evidence to support a translational role for the 3'-poly(A) tract of eukaryotic mRNAs has been mostly indirect, including: a correlation between the... (Review)
Review
Until recently, evidence to support a translational role for the 3'-poly(A) tract of eukaryotic mRNAs has been mostly indirect, including: a correlation between the adenylation status of individual mRNAs and their translatability in vivo or in vitro, the demonstration that exogenously added poly(A) is a potent competitive inhibitor of the translation of poly(A)+mRNA, but not poly(A)-mRNAs in vitro, and a correlation between the abundance and stability of poly(A)-binding proteins (PABPs) and the rate of translational initiation in vivo. However, more recent studies demonstrate directly that poly(A)+mRNAs can initiate translation more efficiently than poly(A)-mRNAs, and indicate that this effect is: (i) targeted to the formation of 80S initiation complexes, and (ii) likely to be mediated by the cytoplasmic PABP. We suggest that the 3'-poly(A) tail should be considered a translational enhancer which may stimulate translational initiation in much the same way that transcriptional enhancers are thought to stimulate transcriptional initiation.
Topics: Animals; Carrier Proteins; Cytoplasm; Gene Expression Regulation; Models, Genetic; Poly A; Protein Biosynthesis; RNA, Messenger
PubMed: 1976572
DOI: 10.1016/0378-1119(90)90082-3 -
Trends in Biochemical Sciences Sep 1989This review has focused on the possibility that interactions between mRNA sequences and the poly(A)-nucleoprotein complex play important roles in mRNA turnover. It is... (Review)
Review
This review has focused on the possibility that interactions between mRNA sequences and the poly(A)-nucleoprotein complex play important roles in mRNA turnover. It is important to stress that additional genetic and biochemical tests are necessary to characterize how PABP interacts with mRNA in cells and to determine whether the poly(A) protection hypothesis is accurate. Moreover, there may be a significant number of mRNAs whose half-lives are independent of polyadenylation. For example, the stabilities of poly(A)-containing and deadenylated alpha 2u-globulin and interferon mRNAs are similar in microinjected oocytes. Thus, an important challenge in this field will be to analyse the complex and interactive factors that determine the half-lives of specific mRNAs.
Topics: Carrier Proteins; Poly A; RNA, Messenger
PubMed: 2688202
DOI: 10.1016/0968-0004(89)90011-x -
Briefings in Bioinformatics Jul 2022The poly(A) tail is a dynamic addition to the eukaryotic mRNA and the change in its length plays an essential role in regulating gene expression through affecting...
The poly(A) tail is a dynamic addition to the eukaryotic mRNA and the change in its length plays an essential role in regulating gene expression through affecting nuclear export, mRNA stability and translation. Only recently high-throughput sequencing strategies began to emerge for transcriptome-wide profiling of poly(A) tail length in diverse developmental stages and organisms. However, there is currently no easy-to-use and universal tool for measuring poly(A) tails in sequencing data from different sequencing protocols. Here we established PolyAtailor, a unified and efficient framework, for identifying and analyzing poly(A) tails from PacBio-based long reads or next generation short reads. PolyAtailor provides two core functions for measuring poly(A) tails, namely Tail_map and Tail_scan, which can be used for profiling tails with or without using a reference genome. Particularly, PolyAtailor can identify all potential tails in a read, providing users with detailed information such as tail position, tail length, tail sequence and tail type. Moreover, PolyAtailor integrates rich functions for poly(A) tail and poly(A) site analyses, such as differential poly(A) length analysis, poly(A) site identification and annotation, and statistics and visualization of base composition in tails. We compared PolyAtailor with three latest methods, FLAMAnalysis, FLEPSeq and PAIsoSeqAnalysis, using data from three sequencing protocols in HeLa samples and Arabidopsis. Results show that PolyAtailor is effective in measuring poly(A) tail length and detecting significance of differential poly(A) length, which achieves much higher sensitivity and accuracy than competing methods. PolyAtailor is available at https://github.com/BMILAB/PolyAtailor.
Topics: Gene Expression Profiling; High-Throughput Nucleotide Sequencing; Poly A; Polyadenylation; RNA, Messenger; Sequence Analysis, RNA
PubMed: 35769001
DOI: 10.1093/bib/bbac271