Benchmarking sequencing methods and tools that facilitate the study of alternative polyadenylation

Background Alternative cleavage and polyadenylation (APA), an RNA processing event, occurs in over 70% of human protein-coding genes. APA results in mRNA transcripts with distinct 3′ ends. Most APA occurs within 3′ UTRs, which harbor regulatory elements that can impact mRNA stability, translation, and localization. Results APA can be profiled using a number of established computational tools that infer polyadenylation sites from standard, short-read RNA-seq datasets. Here, we benchmarked a number of such tools—TAPAS, QAPA, DaPars2, GETUTR, and APATrap— against 3′-Seq, a specialized RNA-seq protocol that enriches for reads at the 3′ ends of genes, and Iso-Seq, a Pacific Biosciences (PacBio) single-molecule full-length RNA-seq method in their ability to identify polyadenylation sites and quantify polyadenylation site usage. We demonstrate that 3′-Seq and Iso-Seq are able to identify and quantify the usage of polyadenylation sites more reliably than computational tools that take short-read RNA-seq as input. However, we find that running one such tool, QAPA, with a set of polyadenylation site annotations derived from small quantities of 3′-Seq or Iso-Seq can reliably quantify variation in APA across conditions, such asacross genotypes, as demonstrated by the successful mapping of alternative polyadenylation quantitative trait loci (apaQTL). Conclusions We envisage that our analyses will shed light on the advantages of studying APA with more specialized sequencing protocols, such as 3′-Seq or Iso-Seq, and the limitations of studying APA with short-read RNA-seq. We provide a computational pipeline to aid in the identification of polyadenylation sites and quantification of polyadenylation site usages using Iso-Seq data as input.

Poly(A) tail length is a major regulator of maternal gene expression during the mammalian oocyte-to-embryo transition

Transcription is silent during the mammalian oocyte-to-embryo transition (OET) until zygotic genome activation (ZGA). Therefore, the OET relies on post-transcriptional regulation of maternal mRNA, among which poly(A) tail lengths have been found to regulate translation for a small number of genes1–3. However, transcriptome-wide poly(A) tail length dynamics and their role in gene expression during the mammalian OET remain unknown. Here, we quantified transcriptome-wide mRNA poly(A) tail length dynamics during the mammalian OET using PAIso-seq1 and PAIso-seq24,5, two methods with different underlying principles that preserve the poly(A) tail information. We revealed that poly(A) tail length was highly dynamic during the mouse OET, and Btg4 is responsible for global maternal mRNA deadenylation. We found that the poly(A) tail length positively associated with translational efficiency transcriptome-wide in mouse oocytes. In addition, genes with different alternative polyadenylation isoforms show longer poly(A) tails for isoforms with distal polyadenylation sites compared to those with proximal polyadenylation sites in mouse, rat, pig and human oocytes after meiotic resumption, which is not seen in cultured cell lines. Surprisingly, mammalian embryos, namely mouse, rat, pig, and human embryos, all experience highly conserved global mRNA re-polyadenylation after fertilization, providing molecular evidence that the early embryo development before ZGA is driven by re-polyadenylated maternal mRNAs rather than newly transcribed mRNAs. Together, our study reveals the conserved mRNA poly(A) tail length landscape. This resource can be used for exploring spatiotemporal post-transcriptional regulation throughout the mammalian OET.

Interactions between RNA m6A modification, alternative splicing, and poly(A) tail revealed by MePAIso-seq2

RNA post-transcriptional regulation involves 5-end capping, 3-poly(A) tailing (including polyadenylation sites, tail length, and non-A residues), alternative splicing, and chemical modifications including N6-methyladenosine (m6A). Studying the interplay of m6A, alternative splicing, alternative polyadenylation sites, poly(A) tail length, and non-A residues in poly(A) tails requires monitoring them simultaneously on one transcript, however strategies to achieve this are lacking. Therefore, we developed a new method, combining m6A-specific methylated RNA immunoprecipitation and the PacBio-based, tail-included, full-length RNA sequencing approach PAIso-seq2, which we have named m6A and poly(A) inclusive RNA isoform sequencing 2 (MePAIso-seq2). Using MePAIso-seq2, we revealed that m6A promotes and inhibits a similar number of alternative splicing events in mouse cell lines, showing that m6A does affect alternative splicing. In contrast, no correlation was detected between m6A and alternative polyadenylation sites choice. Surprisingly, we found that m6A-modified RNAs possess longer poly(A) tails and a lower proportion of poly(A) tails containing non-A residues, especially in mouse embryonic stem cells. Together, we developed a new method to detect full-length m6A-modified RNAs to comprehensively study the relationships between m6A, alternative splicing, and poly(A) tailing, laying a foundation for further exploration of the functional coordination of different RNA post-transcriptional modifications.

SCAPTURE: a deep learning-embedded pipeline that captures polyadenylation information from 3′ tag-based RNA-seq of single cells

Single-cell RNA-seq (scRNA-seq) profiles gene expression with high resolution. Here, we develop a stepwise computational method-called SCAPTURE to identify, evaluate, and quantify cleavage and polyadenylation sites (PASs) from 3′ tag-based scRNA-seq. SCAPTURE detects PASs de novo in single cells with high sensitivity and accuracy, enabling detection of previously unannotated PASs. Quantified alternative PAS transcripts refine cell identity analysis beyond gene expression, enriching information extracted from scRNA-seq data. Using SCAPTURE, we show changes of PAS usage in PBMCs from infected versus healthy individuals at single-cell resolution.

ELAV/Hu RNA binding proteins determine multiple programs of neural alternative splicing

ELAV/Hu factors are conserved RNA binding proteins (RBPs) that play diverse roles in mRNA processing and regulation. The founding member,DrosophilaElav, was recognized as a vital neural factor 35 years ago. Nevertheless, little was known about its impacts on the transcriptome, and potential functional overlap with its paralogs. Building on our recent findings that neural-specific lengthened 3’ UTR isoforms are co-determined by ELAV/Hu factors, we address their impacts on splicing. While only a few splicing targets ofDrosophilaare known, ectopic expression of each of the three family members (Elav, Fne and Rbp9) alters hundreds of cassette exon and alternative last exon (ALE) splicing choices. Reciprocally, double mutants ofelav/fne, but notelavalone, exhibit opposite effects on both classes of regulated mRNA processing events in larval CNS. While manipulation ofDrosophilaELAV/Hu RBPs induces both exon skipping and inclusion, characteristic ELAV/Hu motifs are enriched only within introns flanking exons that are suppressed by ELAV/Hu factors. Moreover, the roles of ELAV/Hu factors in global promotion of distal ALE splicing are mechanistically linked to terminal 3’ UTR extensions in neurons, since both processes involve bypass of proximal polyadenylation signals linked to ELAV/Hu motifs downstream of cleavage sites. We corroborate the direct action of Elav in diverse modes of mRNA processing using RRM-dependent Elav-CLIP data from S2 cells. Finally, we provide evidence for conservation in mammalian neurons, which undergo broad programs of distal ALE and APA lengthening, linked to ELAV/Hu motifs downstream of regulated polyadenylation sites. Overall, ELAV/Hu RBPs orchestrate multiple broad programs of neuronal mRNA processing and isoform diversification inDrosophilaand mammalian neurons.

Integrative Transcriptome Sequencing Reveals the Molecular Difference of Maturation Process of Ovary and Testis in Mud Crab Scylla paramamosain

The mud crab Scylla paramamosain is a species with significant sexual dimorphism in growth rate and body size, of which the females are of higher economic and nutritional values than the males. Accordingly, there is an urgent need to explore the molecular mechanism underlying sex determination and gonadal differentiation. The single-molecule long-read technology combining with RNA sequencing was employed to construct a full-length transcriptome for gonads of S. paramamosain. In total, 1,562,819 FLNC reads were obtained from 1,813,758 reads of inserts (ROIs). Among them, the 10,739 fusion isoforms corresponded to 23,634 reads and were involved in 5,369 genes in the reference annotation. According to the criteria for new transcripts, a total of 213,809 isoforms were recognized as novel transcripts and then matched against Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), NR, Swissprot, and KOG databases. We also identified 22,313 SSRs, 169,559 lncRNAs, and 25,451 SNPs. Additionally, 349,854 alternative splicing (AS) events from 8,430 gene models were detected, and 5,129 polyadenylation sites were profiled from 3,090 genes. GO and KEGG annotation indicated that AS and APA probably play important roles in the gonadal development and maturation. Besides, the DEGs associated with gonadal development and maturation were identified and analyzed based on the RNA-Seq data.

SCAPTURE: a deep learning-embedded pipeline that captures polyadenylation information from 3' tag-based RNA-seq of single cells

Single-cell RNA-seq (scRNA-seq) profiles gene expression with a resolution that empowers depiction of cell atlas in complex systems. Here, we developed a stepwise computational pipeline SCAPTURE to identify, evaluate, and quantify cleavage and polyadenylation sites (PASs) from 3' tag-based scRNA-seq. SCAPTURE detects PASs de novo in single cells with high sensitivity and accuracy, enabling detection of previously unannotated PASs. Quantified alternative PAS transcripts refine cell identities, enriching information extracted from scRNA-seq.

Aptardi predicts polyadenylation sites in sample-specific transcriptomes using high-throughput RNA sequencing and DNA sequence

Annotation of polyadenylation sites from short-read RNA sequencing alone is a challenging computational task. Other algorithms rooted in DNA sequence predict potential polyadenylation sites; however, in vivo expression of a particular site varies based on a myriad of conditions. Here, we introduce aptardi (alternative polyadenylation transcriptome analysis from RNA-Seq data and DNA sequence information), which leverages both DNA sequence and RNA sequencing in a machine learning paradigm to predict expressed polyadenylation sites. Specifically, as input aptardi takes DNA nucleotide sequence, genome-aligned RNA-Seq data, and an initial transcriptome. The program evaluates these initial transcripts to identify expressed polyadenylation sites in the biological sample and refines transcript 3′-ends accordingly. The average precision of the aptardi model is twice that of a standard transcriptome assembler. In particular, the recall of the aptardi model (the proportion of true polyadenylation sites detected by the algorithm) is improved by over three-fold. Also, the model—trained using the Human Brain Reference RNA commercial standard—performs well when applied to RNA-sequencing samples from different tissues and different mammalian species. Finally, aptardi’s input is simple to compile and its output is easily amenable to downstream analyses such as quantitation and differential expression.

SRSF3 and SRSF7 modulate 3′UTR length through suppression or activation of proximal polyadenylation sites and regulation of CFIm levels

Background Alternative polyadenylation (APA) refers to the regulated selection of polyadenylation sites (PASs) in transcripts, which determines the length of their 3′ untranslated regions (3′UTRs). We have recently shown that SRSF3 and SRSF7, two closely related SR proteins, connect APA with mRNA export. The mechanism underlying APA regulation by SRSF3 and SRSF7 remained unknown. Results Here we combine iCLIP and 3′-end sequencing and find that SRSF3 and SRSF7 bind upstream of proximal PASs (pPASs), but they exert opposite effects on 3′UTR length. SRSF7 enhances pPAS usage in a concentration-dependent but splicing-independent manner by recruiting the cleavage factor FIP1, generating short 3′UTRs. Protein domains unique to SRSF7, which are absent from SRSF3, contribute to FIP1 recruitment. In contrast, SRSF3 promotes distal PAS (dPAS) usage and hence long 3′UTRs directly by counteracting SRSF7, but also indirectly by maintaining high levels of cleavage factor Im (CFIm) via alternative splicing. Upon SRSF3 depletion, CFIm levels decrease and 3′UTRs are shortened. The indirect SRSF3 targets are particularly sensitive to low CFIm levels, because here CFIm serves a dual function; it enhances dPAS and inhibits pPAS usage by binding immediately downstream and assembling unproductive cleavage complexes, which together promotes long 3′UTRs. Conclusions We demonstrate that SRSF3 and SRSF7 are direct modulators of pPAS usage and show how small differences in the domain architecture of SR proteins can confer opposite effects on pPAS regulation.