Rational design of an artificial tethered enzyme for non-templated post-transcriptional mRNA polyadenylation by the second generation of the C3P3 system.

We have previously introduced the first generation of C3P3, an artificial system that allows the autonomous in-vivo production of mRNA with m7GpppN-cap. While C3P3-G1 synthesized much larger amounts of capped mRNA in human cells than conventional nuclear expression systems, it produced a proportionately much smaller amount of the corresponding proteins, indicating a clear defect of mRNA translatability. A possible mechanism for this poor translatability could be the rudimentary polyadenylation of the mRNA produced by the C3P3-G1 system. We therefore sought to develop the C3P3-G2 system using an artificial enzyme to post-transcriptionally lengthen the poly(A) tail. This system is based on the mutant mouse poly(A) polymerase alpha fused at its N terminus with an N peptide from the λ virus, which binds to BoxBr sequences placed in the 3'UTR region of the mRNA of interest. The resulting system selectively brings mPAPαm7 to the target mRNA to elongate its poly(A)-tail to a length of few hundred adenosine. Such elongation of the poly(A) tail leads to an increase in protein expression levels of about 2.5-3 times in cultured human cells compared to the C3P3-G1 system. Finally, the coding sequence of the tethered mutant poly(A) polymerase can be efficiently fused to that of the C3P3-G1 enzyme via an F2A sequence, thus constituting the single-ORF C3P3-G2 enzyme. These technical developments constitute an important milestone in improving the performance of the C3P3 system, paving the way for its applications in bioproduction and non-viral human gene therapy.

Poly(A) tale: From A to A; RNA polyadenylation in prokaryotes and eukaryotes.

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.

Measuring Poly-Adenosine Tail Length of RNAs by High-Resolution Northern Blotting Coupled with RNase H Cleavage.

The poly-adenosine, or poly(A) tail, plays key roles in controlling the stability and translation of messenger RNAs in all eukaryotes, and, as such, facile assays that can measure poly(A) length are needed. This chapter describes an approach that couples RNase H-mediated cleavage of an RNA of interest with high-resolution denaturing gel electrophoresis and northern blot-based detection. Major advantages of this method include the ability to directly measure the abundance of any RNA and the length of its poly(A) tail without amplification steps. The assay provides high specificity, sensitivity, and reproducibility for accurate quantitation using standard molecular biology equipment and reagents. Overall, the high-resolution northern blotting approach offers a cost-effective means of poly(A) RNA analysis that is especially useful for small numbers of transcripts and comparisons between experimental conditions or time points.

Profiling the polyadenylated transcriptome of extracellular vesicles with long-read nanopore sequencing.

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.

BESST: a novel LncRNA knockout strategy with less genome perturbance.

Long noncoding RNAs (lncRNAs) are >200 nt RNA transcripts without protein-coding potential. LncRNAs can be categorized into intergenic, intronic, bidirectional, sense, and antisense lncRNAs based on the genomic localization to nearby protein-coding genes. The current CRISPR-based lncRNA knockout strategy works efficiently for lncRNAs distant from the protein-coding gene, whereas it causes genomic perturbance inevitably due to technical limitations. In this study, we introduce a novel lncRNA knockout strategy, BESST, by deleting the genomic DNA fragment from the branch point to the 3' splicing site in the last intron of the target lncRNA. The BESST knockout exhibited comparable or superior repressive efficiency to RNA silencing or conventional promoter-exon1 deletion. Significantly, the BESST knockout strategy minimized the intervention of adjacent/overlap protein-coding genes by removing an average of ∼130 bp from genomic DNA. Our data also found that the BESST knockout strategy causes lncRNA nuclear retention, resulting in decapping and deadenylation of the lncRNA poly(A) tail. Further study revealed that PABPN1 is essential for the BESST-mediated decay and subsequent poly(A) deadenylation and decapping. Together, the BESST knockout strategy provides a versatile tool for investigating gene function by generating knockout cells or animals with high specificity and efficiency.

Only fourteen 3'-end poly(A)s sufficient for rescuing Senecavirus A from its cDNA clone, but inadequate to meet requirement of viral replication.

Senecavirus A (SVA) belongs to the genus Senecavirus in the family Picornaviridae. Its genome is a positive-sense, single-strand RNA that has 5' and 3' untranslated regions. There is a poly(A) tail at the 3' end of viral genome. Although the number of poly(A)s is variable, the length of poly(A) tail generally has the minimum nucleotide limit for picornaviral replication. To identify a range limit of poly(A)s for SVA recovery, five SVA cDNA clones, separately containing 25, 20, 15, 10 and 5 poly(A)s, were constructed for rescuing viruses. Replication-competent SVAs could be rescued from the first three cDNA clones, implying the range limit of poly(A)s was (A)15 to (A)10. To recognize the precise limit, four extra cDNA clones, separately containing 14, 13, 12 and 11 poly(A)s, were constructed to rescue SVAs further. The replication-competent SVA was rescued only from the poly(A)14-containing plasmid, indicating that the precise limit was poly(A)14 at the 3' end of cDNA clone for SVA recovery. The rescued SVA was serially passaged in cells. The passage-5 and -10 progenies were independently subjected to the analysis of 3'-rapid amplification of cDNA ends. Both progenies showed their own poly(A) tails far more than 14 (A)s, implying extra (A)s added to the poly(A)14 sequence during viral passaging. It can be concluded that fourteen (A)s are sufficient for rescuing a replication-competent SVA from its cDNA clone, but inadequate for maintaining viral propagation in cells.

Measuring the tail: Methods for poly(A) tail profiling.

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.

Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore cDNA sequencing.

RNA polyadenylation plays a central role in RNA maturation, fate, and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translation efficiency and stability of mRNAs. Here we develop Nanopore 3' end-capture sequencing (Nano3P-seq), a method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance, tail composition, and tail length dynamics at per-read resolution. By employing a template-switching-based sequencing protocol, Nano3P-seq can sequence RNA molecule from its 3' end, regardless of its polyadenylation status, without the need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P-seq provides quantitative estimates of RNA abundance and tail lengths, and captures a wide diversity of RNA biotypes. We find that, in addition to mRNA and long non-coding RNA, polyA tails can be identified in 16S mitochondrial ribosomal RNA in both mouse and zebrafish models. Moreover, we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform-specific level, correlating with mRNA decay. Finally, we demonstrate the ability of Nano3P-seq in capturing non-A bases within polyA tails of various lengths, and reveal their distribution during vertebrate embryogenesis. Overall, Nano3P-seq is a simple and robust method for accurately estimating transcript levels, tail lengths, and tail composition heterogeneity in individual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome.

Nucleotide-level linkage of transcriptional elongation and polyadenylation.

Alternative polyadenylation yields many mRNA isoforms whose 3' termini occur disproportionately in clusters within 3' untranslated regions. Previously, we showed that profiles of poly(A) site usage are regulated by the rate of transcriptional elongation by RNA polymerase (Pol) II (Geisberg et al., 2020). Pol II derivatives with slow elongation rates confer an upstream-shifted poly(A) profile, whereas fast Pol II strains confer a downstream-shifted poly(A) profile. Within yeast isoform clusters, these shifts occur steadily from one isoform to the next across nucleotide distances. In contrast, the shift between clusters - from the last isoform of one cluster to the first isoform of the next - is much less pronounced, even over large distances. GC content in a region 13-30 nt downstream from isoform clusters correlates with their sensitivity to Pol II elongation rate. In human cells, the upstream shift caused by a slow Pol II mutant also occurs continuously at single nucleotide resolution within clusters but not between them. Pol II occupancy increases just downstream of poly(A) sites, suggesting a linkage between reduced elongation rate and cluster formation. These observations suggest that (1) Pol II elongation speed affects the nucleotide-level dwell time allowing polyadenylation to occur, (2) poly(A) site clusters are linked to the local elongation rate, and hence do not arise simply by intrinsically imprecise cleavage and polyadenylation of the RNA substrate, (3) DNA sequence elements can affect Pol II elongation and poly(A) profiles, and (4) the cleavage/polyadenylation and Pol II elongation complexes are spatially, and perhaps physically, coupled so that polyadenylation occurs rapidly upon emergence of the nascent RNA from the Pol II elongation complex.

PABPN1 functions as a hub in the assembly of nuclear poly(A) domains that are essential for mouse oocyte development.

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. Pabpn1-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, Pabpn1fl/fl;Gdf9-Cre 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.

3' Untranslated Regions Are Modular Entities That Determine Polyadenylation Profiles.

The 3' ends of eukaryotic mRNAs are generated by cleavage of nascent transcripts followed by polyadenylation, which occurs at numerous sites within 3' untranslated regions (3' UTRs) but rarely within coding regions. An individual gene can yield many 3'-mRNA isoforms with distinct half-lives. We dissect the relative contributions of protein-coding sequences (open reading frames [ORFs]) and 3' UTRs to polyadenylation profiles in yeast. ORF-deleted derivatives often display strongly decreased mRNA levels, indicating that ORFs contribute to overall mRNA stability. Poly(A) profiles, and hence relative isoform half-lives, of most (9 of 10) ORF-deleted derivatives are very similar to their wild-type counterparts. Similarly, in-frame insertion of a large protein-coding fragment between the ORF and 3' UTR has minimal effect on the poly(A) profile in all 15 cases tested. Last, reciprocal ORF/3'-UTR chimeric genes indicate that the poly(A) profile is determined by the 3' UTR. Thus, 3' UTRs are self-contained modular entities sufficient to determine poly(A) profiles and relative 3'-isoform half-lives. In the one atypical instance, ORF deletion causes an upstream shift of poly(A) sites, likely because juxtaposition of an unusually high AT-rich stretch directs polyadenylation closely downstream. This suggests that long AT-rich stretches, which are not encountered until after coding regions, are important for restricting polyadenylation to 3' UTRs.

An atlas of plant full-length RNA reveals tissue-specific and monocots-dicots conserved regulation of poly(A) tail length.

Poly(A) tail is a hallmark of eukaryotic messenger RNA and its length plays an essential role in regulating mRNA metabolism. However, a comprehensive resource for plant poly(A) tail length has yet to be established. Here, we applied a poly(A)-enrichment-free, nanopore-based method to profile full-length RNA with poly(A) tail information in plants. Our atlas contains over 120 million polyadenylated mRNA molecules from seven different tissues of Arabidopsis, as well as the shoot tissue of maize, soybean and rice. In most tissues, the size of plant poly(A) tails shows peaks at approximately 20 and 45 nucleotides, while the poly(A) tails in pollen exhibit a distinct pattern with strong peaks centred at 55 and 80 nucleotides. Moreover, poly(A) tail length is regulated in a gene-specific manner-mRNAs with short half-lives in general have long poly(A) tails, while mRNAs with long half-lives are featured with relatively short poly(A) tails that peak at ~45 nucleotides. Across species, poly(A) tails in the nucleus are almost twice as long as in the cytoplasm. Our comprehensive dataset lays the groundwork for future functional and evolutionary studies on poly(A) tail length regulation in plants.

Review: Mechanisms underlying alternative polyadenylation in plants - looking in the right places.

Recent years have seen an explosion of interest in the subject of alternative polyadenylation in plants. Connections between the polyadenylation complex and numerous developmental and stress responses are well-established. However, those that link stimuli with the functioning of the polyadenylation complex are less well understood. To this end, it is imperative to clearly delineate the roles of the polyadenylation complex in both plant growth AND alternative polyadenylation. It is also necessary to understand the ways by which other molecular processes may contribute to alternative polyadenylation. This review discusses these issues, with a focus on instances that reveal mechanisms by which mRNA polyadenylation may be regulated. Insights from from characterizations of mutants affected in the polyadenylation complex are discussed, as are the limitations of such characterizations when it comes to teasing out cause and effect. These limitations encourage explorations to other processes that are beyond the core polyadenylation complex. Two such processes that sculpt the plant transcriptome - transcription termination and the epigenetic control of transposon activity - also contribute to regulated poly(A) site choice. These subjects define "the right places" - molecular mechanisms that contribute to the wide-ranging control of gene expression via mRNA polyadenylation.

Novel Method of Full-Length RNA-seq That Expands the Identification of Non-Polyadenylated RNAs Using Nanopore Sequencing.

The occurrence of diseases displayed transcriptome alteration, including both coding and non-coding transcripts. The third-generation sequencing (TGS) technologies allow for intensive and comprehensive research of the transcriptome. However, the present standard TGS RNA sequencing method is unable to detect many of the non-polyadenylated [non-poly(A)] RNAs. To obtain more complete transcriptome information, we presented a new comprehensive sequencing approach by performing conventional poly(A) RNA-sequencing combined with the sequencing of non-poly(A) RNA fraction which was tailed by poly(U) on HepG2 and HL-7702 cell lines, enabling the detection of multiple categories of non-poly(A) RNAs excluded by the existing standard approach. Moreover, the length distribution of the full-splice match transcripts was longer than that assembled by short-reads, which contributed to characterizing alternative splicing events and provided a comprehensive portrait of transcriptional complexity. Besides the detection of genes with differential expression patterns in the poly(A) library between HepG2 and HL-7702, we also found a cancer-related non-coding gene in the poly(U) data, that is, growth arrest special 5 (GAS5). Collectively, our results suggested that the novel method effectively captured both poly(A) and non-poly(A) transcripts in the tested cell lines and allowed a deeper exploration of the transcriptome.

Poly(a) selection introduces bias and undue noise in direct RNA-sequencing.

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.

PCDetection: PolyA-CRISPR/Cas12a-based miRNA detection without PAM restriction.

MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally regulate gene expression. The aberrant expression of miRNAs is related to many diseases. MiRNAs can serve as potential biomarkers for the prognosis and diagnosis of cancers and other human diseases. However, the short sequence and high sequence similarity of miRNAs impede detection. Herein, we propose a method to integrate polyA-tailing and CRISPR/Cas12a to amplify and detect all miRNAs with high specificity and sensitivity. PolyA-tailing enables efficient amplification of RNA and introduces a universal PAM sequence for Cas12a to unlock its PAM restriction. The CRISPR-Cas system guarantees the specific recognition of nucleic acid sequences with a single base mismatch. A limit of detection (LOD) as low as 50 fM was achieved. The practical application ability of polyA-CRISPR/Cas12a-based miRNA detection was validated by miRNA analyses in multiple cancer cell samples. With the increasing stability of RNA samples, low cost, excellent specificity, and sensitivity, this method demonstrates great potential to scale up to parallel diagnostic sets for miRNA-related disease.

Transcriptome-wide measurement of poly(A) tail length and composition at subnanogram total RNA sensitivity by PAIso-seq.

Poly(A) tails are added to the 3' ends of most mRNAs in a non-templated manner and play essential roles in post-transcriptional regulation, including mRNA export, stability and translation. Measuring poly(A) tails is critical for understanding their regulatory roles in almost every aspect of biological and medical studies. Previous methods for analyzing poly(A) tails require large amounts of input RNA (microgram-level total RNA), which limits their application. We recently developed a poly(A) inclusive full-length RNA isoform-sequencing method (PAIso-seq) at single-oocyte-level sensitivity (a single mammalian oocyte contains ~0.5 ng of total RNA) based on PacBio sequencing that enabled accurate measurement of the poly(A) tail length and non-A residues within the body of poly(A) tails along with the full-length cDNA, providing the opportunity to study precious in vivo samples with very limited input material. Here, we describe a detailed protocol for PAIso-seq library preparation from single mouse oocytes or bulk oocyte samples. In addition, we provide a complete bioinformatic pipeline to perform the analysis from the raw data to downstream analysis. The minimum time required is ~14.5 h for PAIso-seq double-stranded cDNA preparation, 2 d for PacBio sequencing in HiFi mode and 8 h for the initial data analysis.

Poly(m6A) tails stabilize transcripts.

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.

PolyAtailor: measuring poly(A) tail length from short-read and long-read sequencing data.

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.