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In eukaryotes, poly(A) tails are present on almost every mRNA. Early experiments led to the hypothesis that poly(A) tails and the cytoplasmic polyadenylate-binding protein (PABPC) promote translation and prevent mRNA degradation, but the details remained unclear. More recent data suggest that the role of poly(A) tails is much more complex: poly(A)-binding protein can stimulate poly(A) tail removal (deadenylation) and the poly(A) tails of stable, highly translated mRNAs at steady state are much shorter than expected. Furthermore, the rate of translation elongation affects deadenylation. Consequently, the interplay between poly(A) tails, PABPC, translation and mRNA decay has a major role in gene regulation. In this Review, we discuss recent work that is revolutionizing our understanding of the roles of poly(A) tails in the cytoplasm. Specifically, we discuss the roles of poly(A) tails in translation and control of mRNA stability and how poly(A) tails are removed by exonucleases (deadenylases), including CCR4–NOT and PAN2–PAN3. We also discuss how deadenylation rate is determined, the integration of deadenylation with other cellular processes and the function of PABPC. We conclude with an outlook for the future of research in this field.Recent studies have revolutionized our understanding of the interplay between mRNA poly(A) tails and the processes of translation and mRNA decay in the cytoplasm. Poly(A) tails interact with dedicated RNA-binding proteins and deadenylases, which together determine the impact of poly(A) tails on gene expression.

In mammals, translational control plays critical roles during oocyte-to-embryo transition (OET) when transcription ceases. However, the underlying regulatory mechanisms remain challenging to study. Here, using low-input Ribo-seq (Ribo-lite), we investigated translational landscapes during OET using 30–150 mouse oocytes or embryos per stage. Ribo-lite can also accommodate single oocytes. Combining PAIso-seq to interrogate poly(A) tail lengths, we found a global switch of translatome that closely parallels changes of poly(A) tails upon meiotic resumption. Translation activation correlates with polyadenylation and is supported by polyadenylation signal proximal cytoplasmic polyadenylation elements (papCPEs) in 3′ untranslated regions. By contrast, translation repression parallels global de-adenylation. The latter includes transcripts containing no CPEs or non-papCPEs, which encode many transcription regulators that are preferentially re-activated before zygotic genome activation. CCR4-NOT, the major de-adenylation complex, and its key adaptor protein BTG4 regulate translation downregulation often independent of RNA decay. BTG4 is not essential for global de-adenylation but is required for selective gene de-adenylation and production of very short-tailed transcripts. In sum, our data reveal intimate interplays among translation, RNA stability and poly(A) tail length regulation underlying mammalian OET. Using an optimized Ribo-seq protocol that is applicable for low-input samples, Xie, Li and colleagues revealed the translation landscape during oocyte-to-embryo transition and in pre-implantation embryos.

Understanding genome organization and gene regulation requires insight into RNA transcription, processing and modification. We adapted nanopore direct RNA sequencing to examine RNA from a wild-type accession of the model plant Arabidopsis thaliana and a mutant defective in mRNA methylation (m6A). Here we show that m6A can be mapped in full-length mRNAs transcriptome-wide and reveal the combinatorial diversity of cap-associated transcription start sites, splicing events, poly(A) site choice and poly(A) tail length. Loss of m6A from 3’ untranslated regions is associated with decreased relative transcript abundance and defective RNA 3′ end formation. A functional consequence of disrupted m6A is a lengthening of the circadian period. We conclude that nanopore direct RNA sequencing can reveal the complexity of mRNA processing and modification in full-length single molecule reads. These findings can refine Arabidopsis genome annotation. Further, applying this approach to less well-studied species could transform our understanding of what their genomes encode.

RNA N 6 -methyladenosine (m 6 A) modifications are essential in plants. Here, we show that transgenic expression of the human RNA demethylase FTO in rice caused a more than threefold increase in grain yield under greenhouse conditions. In field trials, transgenic expression of FTO in rice and potato caused ~50% increases in yield and biomass. We demonstrate that the presence of FTO stimulates root meristem cell proliferation and tiller bud formation and promotes photosynthetic efficiency and drought tolerance but has no effect on mature cell size, shoot meristem cell proliferation, root diameter, plant height or ploidy. FTO mediates substantial m 6 A demethylation (around 7% of demethylation in poly(A) RNA and around 35% decrease of m 6 A in non-ribosomal nuclear RNA) in plant RNA, inducing chromatin openness and transcriptional activation. Therefore, modulation of plant RNA m 6 A methylation is a promising strategy to dramatically improve plant growth and crop yield. Rice and potato plants are more productive after epitranscriptome engineering.

Most human genes have multiple sites at which RNA 3ʹ end cleavage and polyadenylation can occur, enabling the expression of distinct transcript isoforms under different conditions. Novel methods to sequence RNA 3′ ends have generated comprehensive catalogues of polyadenylation (poly(A)) sites; their analysis using innovative computational methods has revealed how poly(A) site choice is regulated by core RNA 3ʹ end processing factors, such as cleavage factor I and cleavage and polyadenylation specificity factor, as well as by other RNA-binding proteins, particularly splicing factors. Here, we review the experimental and computational methods that have enabled the global mapping of mRNA and of long non-coding RNA 3ʹ ends, quantification of the resulting isoforms and the discovery of regulators of alternative cleavage and polyadenylation (APA). We highlight the different types of APA-derived isoforms and their functional differences, and illustrate how APA contributes to human diseases, including cancer and haematological, immunological and neurological diseases.

Late-onset ataxia is common, often idiopathic, and can result from cerebellar, proprioceptive, or vestibular impairment; when in combination, it is also termed cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS). We used non-parametric linkage analysis and genome sequencing to identify a biallelic intronic AAGGG repeat expansion in the replication factor C subunit 1 ( RFC1 ) gene as the cause of familial CANVAS and a frequent cause of late-onset ataxia, particularly if sensory neuronopathy and bilateral vestibular areflexia coexist. The expansion, which occurs in the poly(A) tail of an AluSx3 element and differs in both size and nucleotide sequence from the reference (AAAAG) 11 allele, does not affect RFC1 expression in patient peripheral and brain tissue, suggesting no overt loss of function. These data, along with an expansion carrier frequency of 0.7% in Europeans, implies that biallelic AAGGG expansion in RFC1 is a frequent cause of late-onset ataxia. Biallelic expansion of an intronic AAGGG repeat in RFC1 is identified here as a common cause of late-onset ataxia. This expansion occurs in the poly(A) tail of an AluSx3 element and is observed at a carrier frequency of 0.7% in populations of European ancestry.

DNA can determine where and when genes are expressed, but the full set of sequence determinants that control gene expression is unknown. Here, we measured the transcriptional activity of DNA sequences that represent an ~100 times larger sequence space than the human genome using massively parallel reporter assays (MPRAs). Machine learning models revealed that transcription factors (TFs) generally act in an additive manner with weak grammar and that most enhancers increase expression from a promoter by a mechanism that does not appear to involve specific TF–TF interactions. The enhancers themselves can be classified into three types: classical, closed chromatin and chromatin dependent. We also show that few TFs are strongly active in a cell, with most activities being similar between cell types. Individual TFs can have multiple gene regulatory activities, including chromatin opening and enhancing, promoting and determining transcription start site (TSS) activity, consistent with the view that the TF binding motif is the key atomic unit of gene expression. Analysis of massively parallel reporter assays measuring the transcriptional activity of DNA sequences indicates that most transcription factor (TF) activity is additive and does not rely on specific TF–TF interactions. Individual TFs can have different gene regulatory activities.

Background N 6 -methyladenosine (m 6 A) mRNA modification is essential for mammalian and plant viability. The U6 m 6 A methyltransferases in other species regulate S-adenosylmethionine (SAM) homeostasis through installing m 6 A in pre-mRNAs of SAM synthetases. However, U6 m 6 A methyltransferase has not been characterized in Arabidopsis and little is known about its role in regulating photomorphogenesis and flowering. Results Here we characterize that FIONA1 is an Arabidopsis U6 m 6 A methyltransferase that installs m 6 A in U6 snRNA and a small subset of poly(A) + RNA. Disruption of FIONA1 leads to phytochrome signaling-dependent hypocotyl elongation and photoperiod-independent early flowering. Distinct from mammalian METTL16 and worm METT-10, FIONA1 neither installs m 6 A in the mRNAs of Arabidopsis SAM synthetases nor affects their transcript expression levels under normal or high SAM conditions. We confirm that FIONA1 can methylate plant mRNA m 6 A motifs in vitro and in vivo. We further show that FIONA1 installs m 6 A in several phenotypic related transcripts, thereby affecting downstream mRNA stability and regulating phytochrome signaling and floral transition. Conclusion FIONA1 is functional as a U6 m 6 A methyltransferase in Arabidopsis, distinct from mammalian METTL16 and worm METT-10. Our results demonstrate that FIONA1-mediated m 6 A post-transcriptional regulation is an autonomous regulator for flowering and phytochrome signaling-dependent photomorphogenesis.

Although messenger RNA (mRNA) has proved effective as a vaccine, its potential as a general therapeutic modality is limited by its instability and low translation capacity. To increase the duration and level of protein expression from mRNA, we designed and synthesized topologically and chemically modified mRNAs with multiple synthetic poly(A) tails. Here we demonstrate that the optimized multitailed mRNA yielded ~4.7–19.5-fold higher luminescence signals than the control mRNA from 24 to 72 h post transfection in cellulo and 14 days detectable signal versus <7 days signal from the control in vivo. We further achieve efficient multiplexed genome editing of the clinically relevant genes Pcsk9 and Angptl3 in mouse liver at a minimal mRNA dosage. Taken together, these results provide a generalizable approach to synthesize capped branched mRNA with markedly enhanced translation capacity. mRNA with engineered poly(A) tails produces prolonged higher levels of protein.

Genome-wide association studies have identified thousands of noncoding variants associated with human traits and diseases. However, the functional interpretation of these variants is a major challenge. Here, we constructed a multi-tissue atlas of human 3′UTR alternative polyadenylation (APA) quantitative trait loci (3′aQTLs), containing approximately 0.4 million common genetic variants associated with the APA of target genes, identified in 46 tissues isolated from 467 individuals (Genotype-Tissue Expression Project). Mechanistically, 3′aQTLs can alter poly(A) motifs, RNA secondary structure and RNA-binding protein–binding sites, leading to thousands of APA changes. Our CRISPR-based experiments indicate that such 3′aQTLs can alter APA regulation. Furthermore, we demonstrate that mapping 3′aQTLs can identify APA regulators, such as La-related protein 4. Finally, 3′aQTLs are colocalized with approximately 16.1% of trait-associated variants and are largely distinct from other QTLs, such as expression QTLs. Together, our findings show that 3′aQTLs contribute substantially to the molecular mechanisms underlying human complex traits and diseases. A multi-tissue atlas of alternative polyadenylation (APA) quantitative trait loci (3′aQTLs) identifies approximately 0.4 million common genetic variants associated with the APA of target genes. Approximately 16% of trait-associated variants colocalize with 3′aQTLs.