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BMC Genomics Sep 2023While numerous studies have described the transcriptomes of extracellular vesicles (EVs) in different cellular contexts, these efforts have typically relied on...
BACKGROUND
While numerous studies have described the transcriptomes of extracellular vesicles (EVs) in different cellular contexts, these efforts have typically relied on sequencing methods requiring RNA fragmentation, which limits interpretations on the integrity and isoform diversity of EV-targeted RNA populations. It has been assumed that mRNA signatures in EVs are likely to be fragmentation products of the cellular mRNA material, and the extent to which full-length mRNAs are present within EVs remains to be clarified.
RESULTS
Using long-read nanopore RNA sequencing, we sought to characterize the full-length polyadenylated (poly-A) transcriptome of EVs released by human chronic myelogenous leukemia K562 cells. We detected 443 and 280 RNAs that were respectively enriched or depleted in EVs. EV-enriched poly-A transcripts consist of a variety of biotypes, including mRNAs, long non-coding RNAs, and pseudogenes. Our analysis revealed that 10.58% of all EV reads, and 18.67% of all cellular (WC) reads, corresponded to known full-length transcripts, with mRNAs representing the largest biotype for each group (EV = 58.13%, WC = 43.93%). We also observed that for many well-represented coding and non-coding genes, diverse full-length transcript isoforms were present in EV specimens, and these isoforms were reflective-of but often in different ratio compared to cellular samples.
CONCLUSION
This work provides novel insights into the compositional diversity of poly-A transcript isoforms enriched within EVs, while also underscoring the potential usefulness of nanopore sequencing to interrogate secreted RNA transcriptomes.
Topics: Humans; Nanopore Sequencing; Transcriptome; Extracellular Vesicles; RNA; RNA, Messenger; Poly A
PubMed: 37736705
DOI: 10.1186/s12864-023-09552-6 -
Methods in Enzymology 2008This chapter describes several methods for measuring the length of the mRNA poly(A) tail and a novel method for measuring mRNA decay. Three methods for measuring the...
This chapter describes several methods for measuring the length of the mRNA poly(A) tail and a novel method for measuring mRNA decay. Three methods for measuring the length of a poly(A) tail are presented: the poly(A) length assay, the ligation-mediated poly(A) test (LM-PAT), and the RNase H assay. The first two methods are PCR-based assays involving cDNA synthesis from an oligo(dT) primer. The third method involves removing the poly(A) tail from the mRNA of interest. A major obstacle to studying the enzymatic step of mammalian mRNA decay has been the inability to capture mRNA decay intermediates with structural impediments such as the poly(G) tract used in yeast. To overcome this, we combined a standard kinetic analysis of mRNA decay with a tetracycline repressor-controlled reporter with an Invader RNA assay. The Invader RNA assay is a simple, elegant assay for the quantification of mRNA. It is based on signal amplification, not target amplification, so it is less prone to artifacts than other methods for nucleic acid quantification. It is also very sensitive, able to detect attomolar levels of target mRNA. Finally, it requires only a short sequence for target recognition and quantitation. Therefore, it can be applied to determining the decay polarity of a mRNA by measuring the decay rates of different portions of that mRNA.
Topics: Animals; Humans; Mammals; Mutation; Plasmids; Poly A; Proteins; RNA Stability
PubMed: 19111191
DOI: 10.1016/S0076-6879(08)02624-4 -
Nucleic Acids Research Dec 2003The Hfq protein, which shares sequence and structural homology with the Sm and Lsm proteins, binds to various RNAs, primarily recognizing AU-rich single-stranded...
The Hfq protein, which shares sequence and structural homology with the Sm and Lsm proteins, binds to various RNAs, primarily recognizing AU-rich single-stranded regions. In this paper, we study the ability of the Escherichia coli Hfq protein to bind to a polyadenylated fragment of rpsO mRNA. Hfq exhibits a high specificity for a 100-nucleotide RNA harboring 18 3'-terminal A-residues. Structural analysis of the adenylated RNA-Hfq complex and gel shift assays revealed the presence of two Hfq binding sites. Hfq binds primarily to the poly(A) tail, and to a lesser extent a U-rich sequence in a single-stranded region located between two hairpin structures. The oligo(A) tail and the interhelical region are sensitive to 3'-5' exoribonucleases and RNase E hydrolysis, respectively, in vivo. In vitro assays demonstrate that Hfq protects poly(A) tails from exonucleolytic degradation by both PNPase and RNase II. In addition, RNase E processing, which occurred close to the U-rich sequence, is impaired by the presence of Hfq. These data suggest that Hfq modulates the sensitivity of RNA to ribonucleases in the cell.
Topics: Base Sequence; Binding Sites; Electrophoretic Mobility Shift Assay; Endoribonucleases; Escherichia coli Proteins; Exoribonucleases; Host Factor 1 Protein; Molecular Sequence Data; Nucleic Acid Conformation; Poly A; Polyribonucleotide Nucleotidyltransferase; RNA Processing, Post-Transcriptional; RNA, Messenger; Ribosomal Proteins; Substrate Specificity; Thermodynamics
PubMed: 14654705
DOI: 10.1093/nar/gkg915 -
Molecular and Cellular Biology Jul 2015Most human protein-encoding transcripts contain multiple introns that are removed by splicing. Although splicing catalysis is frequently cotranscriptional, some introns...
Most human protein-encoding transcripts contain multiple introns that are removed by splicing. Although splicing catalysis is frequently cotranscriptional, some introns are excised after polyadenylation. Accumulating evidence suggests that delayed splicing has regulatory potential, but the mechanisms are still not well understood. Here we identify a terminal poly(A) tail as being important for a subset of intron excision events that follow cleavage and polyadenylation. In these cases, splicing is promoted by the nuclear poly(A) binding protein, PABPN1, and poly(A) polymerase (PAP). PABPN1 promotes intron excision in the context of 3'-end polyadenylation but not when bound to internal A-tracts. Importantly, the ability of PABPN1 to promote splicing requires its RNA binding and, to a lesser extent, PAP-stimulatory functions. Interestingly, an N-terminal alanine expansion in PABPN1 that is thought to cause oculopharyngeal muscular dystrophy cannot completely rescue the effects of PABPN1 depletion, suggesting that this pathway may have relevance to disease. Finally, inefficient polyadenylation is associated with impaired recruitment of splicing factors to affected introns, which are consequently degraded by the exosome. Our studies uncover a new function for polyadenylation in controlling the expression of a subset of human genes via pre-mRNA splicing.
Topics: Cell Line; Humans; Introns; Poly A; Poly(A)-Binding Protein I; Polynucleotide Adenylyltransferase; RNA Precursors; RNA Splicing
PubMed: 25896913
DOI: 10.1128/MCB.00123-15 -
Methods in Enzymology 2021An increasing number of investigations have established alternative polyadenylation (APA) as a key mechanism of gene regulation through altering the length of 3'...
An increasing number of investigations have established alternative polyadenylation (APA) as a key mechanism of gene regulation through altering the length of 3' untranslated region (UTR) and generating distinct mRNA termini. Further, appreciation for the significance of APA in disease contexts propelled the development of several 3' sequencing techniques. While these RNA sequencing technologies have advanced APA analysis, the intrinsic limitation of 3' read coverage and lack of appropriate computational tools constrain precise mapping and quantification of polyadenylation sites. Notably, Poly(A)-ClickSeq (PAC-seq) overcomes limiting factors such as poly(A) enrichment and 3' linker ligation steps using click-chemistry. Here we provide an updated PolyA-miner protocol, a computational approach to analyze PAC-seq or other 3'-Seq datasets. As a key practical constraint, we also provide a detailed account on the impact of sequencing depth on the number of detected polyadenylation sites and APA changes. This protocol is also updated to handle unique molecular identifiers used to address PCR duplication potentially observed in PAC-seq.
Topics: 3' Untranslated Regions; Poly A; Polyadenylation; RNA, Messenger; Sequence Analysis, RNA
PubMed: 34183121
DOI: 10.1016/bs.mie.2021.04.001 -
PLoS Computational Biology Nov 2020In eukaryotes, polyadenylation (poly(A)) is an essential process during mRNA maturation. Identifying the cis-determinants of poly(A) signal (PAS) on the DNA sequence is...
In eukaryotes, polyadenylation (poly(A)) is an essential process during mRNA maturation. Identifying the cis-determinants of poly(A) signal (PAS) on the DNA sequence is the key to understand the mechanism of translation regulation and mRNA metabolism. Although machine learning methods were widely used in computationally identifying PAS, the need for tremendous amounts of annotation data hinder applications of existing methods in species without experimental data on PAS. Therefore, cross-species PAS identification, which enables the possibility to predict PAS from untrained species, naturally becomes a promising direction. In our works, we propose a novel deep learning method named Poly(A)-DG for cross-species PAS identification. Poly(A)-DG consists of a Convolution Neural Network-Multilayer Perceptron (CNN-MLP) network and a domain generalization technique. It learns PAS patterns from the training species and identifies PAS in target species without re-training. To test our method, we use four species and build cross-species training sets with two of them and evaluate the performance of the remaining ones. Moreover, we test our method against insufficient data and imbalanced data issues and demonstrate that Poly(A)-DG not only outperforms state-of-the-art methods but also maintains relatively high accuracy when it comes to a smaller or imbalanced training set.
Topics: Animals; Deep Learning; Deoxyguanosine; Humans; Neural Networks, Computer; Poly A; Signal Transduction; Species Specificity
PubMed: 33151940
DOI: 10.1371/journal.pcbi.1008297 -
Trends in Biochemical Sciences Dec 1996The signals required for forming 3'-ends of mRNAs from the yeast Saccharomyces cerevisiae differ from the corresponding signals of higher eukaryotes. Yeast signals... (Review)
Review
The signals required for forming 3'-ends of mRNAs from the yeast Saccharomyces cerevisiae differ from the corresponding signals of higher eukaryotes. Yeast signals consist of three elements: (1) the efficiency element, which enhances the efficiency of downstream positioning elements; (2) the positioning element, which positions the poly(A) site; and (3) the actual poly(A) site. These three elements are not only necessary, but also sufficient for mRNA 3'-end formation in yeast.
Topics: Animals; Mammals; Poly A; RNA, Fungal; RNA, Messenger; Saccharomyces cerevisiae
PubMed: 9009831
DOI: 10.1016/s0968-0004(96)10057-8 -
The Journal of Biological Chemistry Feb 1988The murine dihydrofolate reductase (DHFR) gene gives rise to multiple polyadenylated mRNAs displaying heterogeneity in the length of the 3' untranslated region. These...
The murine dihydrofolate reductase (DHFR) gene gives rise to multiple polyadenylated mRNAs displaying heterogeneity in the length of the 3' untranslated region. These species are present in the cytoplasm at levels that vary over 2 orders of magnitude, suggesting that certain poly(A) sites are preferred over others. Previous observations have shown that three out of the four major sites of polyadenylation do not display consensus hexanucleotide (AATAAA, ATTAAA) signals. We have further analyzed the sequences involved in directing multiple polyadenylation events on the DHFR gene by focusing our attention on the 4.1- and 5.6-kilobase mRNAs, the lowest abundance DHFR species observed on RNA blot analysis. Identification and sequence analysis of the poly(A) addition sites corresponding to these species revealed appropriately positioned consensus hexanucleotide signals; additional nearby poly(A) sites were also detected which apparently do not use consensus hexanucleotides to direct poly(A) addition to DHFR mRNAs of relatively lower abundance. We have also identified polyadenylation sites downstream of the 4.1- and 5.6-kilobase sites which display consensus hexanucleotide signals and correspond to messenger species too rare for detection by routine RNA blot analysis. Our data bring to 11 the number of known functional poly(A) addition sites associated with the DHFR gene.
Topics: Animals; Base Sequence; Chromosome Mapping; Mice; Molecular Sequence Data; Nucleic Acid Hybridization; Poly A; RNA, Messenger; Tetrahydrofolate Dehydrogenase
PubMed: 3339015
DOI: No ID Found -
The Journal of Clinical Investigation Sep 2010Posttranscriptional regulation is of critical importance during mammalian spermiogenesis. A set of mRNAs that encode proteins critical to normal sperm formation are...
Posttranscriptional regulation is of critical importance during mammalian spermiogenesis. A set of mRNAs that encode proteins critical to normal sperm formation are synthesized early in the process of male germ cell differentiation and are stored in a repressed state. These mRNAs are subsequently translationally activated during the process of spermatid elongation and maturation. Of note, the translationally repressed mRNAs contain long poly(A) tails that are dramatically shortened during the translational activation process. Understanding the mechanisms that underlie this process of mRNA storage and subsequent translational activation has been a long-standing goal. The relationship of the poly(A) tail to translational control is intimately related to the functions of the cognate poly(A)-binding proteins (PABPs). In this issue of the JCI, Yanagiya and colleagues use a set of knockout mice to demonstrate a novel functional role for a particular modulator of PABP function, PABP-interacting protein 2a (PAIP2A), in the normal terminal differentiation of male germ cells.
Topics: Animals; Gene Expression Regulation; Male; Mice; Mice, Knockout; Models, Biological; Poly A; RNA, Messenger; Repressor Proteins; Spermatids; Spermatogenesis; Trans-Activators
PubMed: 20739750
DOI: 10.1172/JCI44091 -
RNA (New York, N.Y.) Dec 2020Chemical modifications enable preparation of mRNAs with augmented stability and translational activity. In this study, we explored how chemical modifications of...
Chemical modifications enable preparation of mRNAs with augmented stability and translational activity. In this study, we explored how chemical modifications of 5',3'-phosphodiester bonds in the mRNA body and poly(A) tail influence the biological properties of eukaryotic mRNA. To obtain modified and unmodified in vitro transcribed mRNAs, we used ATP and ATP analogs modified at the α-phosphate (containing either O-to-S or O-to-BH substitutions) and three different RNA polymerases-SP6, T7, and poly(A) polymerase. To verify the efficiency of incorporation of ATP analogs in the presence of ATP, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantitative assessment of modification frequency based on exhaustive degradation of the transcripts to 5'-mononucleotides. The method also estimated the average poly(A) tail lengths, thereby providing a versatile tool for establishing a structure-biological property relationship for mRNA. We found that mRNAs containing phosphorothioate groups within the poly(A) tail were substantially less susceptible to degradation by 3'-deadenylase than unmodified mRNA and were efficiently expressed in cultured cells, which makes them useful research tools and potential candidates for future development of mRNA-based therapeutics.
Topics: Adenosine Triphosphate; Animals; DNA-Directed RNA Polymerases; Dendritic Cells; HeLa Cells; Humans; Mice; Phosphorothioate Oligonucleotides; Poly A; Protein Biosynthesis; Protein Processing, Post-Translational; RNA, Messenger; Transcription, Genetic
PubMed: 32820035
DOI: 10.1261/rna.077099.120