Alternative Polyadenylation Sites Research Articles

Sleep and diurnal alternative polyadenylation sites associated with human APA-linked brain disorders

Disruption of sleep and circadian rhythms are a comorbid feature of many pathologies, and can negatively influence many health conditions, including neurodegenerative disease, metabolic illness, cancer, and various neurological disorders. Genetic association studies linking sleep and circadian disturbances with disease susceptibility have mainly focused on changes in gene expression due to mutations, such as single-nucleotide polymorphisms. The interaction between sleep and/or circadian rhythms with the use of Alternative Polyadenylation (APA) has been largely undescribed, particularly in the context of other disorders. APA generates transcript isoforms by utilizing various polyadenylation sites (PASs) from the same gene affecting its mRNA translation, stability, localization, and subsequent function. Here we identified unique APAs expressed in rat brain over time-of-day, immediately following sleep deprivation, and the subsequent recovery period. From these data, we performed a secondary analysis of these sleep- or time-of-day associated PASs with recently described APA-linked human brain disorder susceptibility genes.

SCAPE-APA: a package for estimating alternative polyadenylation events from scRNA-seq data

SCAPE was a package that was previously developed to estimate alternative polyadenylation events from single-cell RNA-seq (scRNA-seq) data. It was composed of ad-hoc python scripts and has speed issues when handling large scRNA-seq data. To suit the needs of analyzing large scRNA-seq datasets, we present SCAPE-APA, which is a reimplementation of SCAPE with substantial changes. We made the following updates to the package: (1) we binned similar reads together to accelerate the estimation; (2) we re-derived the mixture model to tailor it for binned reads; (3) we implemented the inference algorithm using Taichi language for acceleration; (4) we re-implemented the untranslated region (UTR) annotation extraction script using the professional package gffutils for better maintenance; (5) we wrote a script to detect spurious alternative polyadenylation sites generated due to junction reads, and (6) we created a formal Python package and uploaded it to the Python Package Index website (Pypi).

Full-Length Transcriptome Profile of Apis cerana Revealed by Nanopore Sequencing

The Asian honey bee (Apis cerana) plays a crucial role in providing abundant bee products and in maintaining ecological balance. Despite the availability of the genomic sequence of the Asian honey bee, its transcriptomic information remains largely incomplete. To address this issue, here we constructed three pooled RNA samples from the queen, drone, and worker bees of A. cerana and performed full-length RNA sequencing using Nanopore single-molecule sequencing technology. Ultimately, we obtained 160,811 full-length transcript sequences from 19,859 genes, with 141,189 being novel transcripts, of which 130,367 were functionally annotated. We detected 520, 324, and 1823 specifically expressed transcripts in the queen, worker, and drone bees, respectively. Furthermore, we identified 38,799 alternative splicing (AS) events from 5710 genes, 44,243 alternative polyadenylation (APA) sites from 1649 gene loci, 88,187 simple sequence repeats (SSRs), and 17,387 long noncoding RNAs (lncRNAs). Leveraging these transcripts as references, we identified 6672, 7795, and 6804 differentially expressed transcripts (DETs) in comparisons of queen ovaries vs drone testes, worker ovaries vs drone testes, and worker ovaries vs queen ovaries, respectively. Our research results provide a comprehensive set of reference transcript datasets for Apis cerana, offering important sequence information for further exploration of its gene functions.

Regulation of alternative polyadenylation isoforms of Timp2 is an effector event of RAS signaling in cell transformation.

Alternative polyadenylation (APA) generates mRNA isoforms with different lengths of the 3' untranslated region (3' UTR). The tissue inhibitor of metalloproteinase 2 (TIMP2) plays a key role in extracellular matrix remodeling under various developmental and disease conditions. Both human and mouse genes encoding TIMP2 contain two highly conserved 3'UTR APA sites, leading to mRNA isoforms that differ substantially in 3'UTR size. APA of Timp2 is one of the most significantly regulated events in multiple cell differentiation lineages. Here we show that Timp2 APA is highly regulated in transformation of NIH3T3 cells by the oncogene HRAS G12V . Perturbations of isoform expression with long 3'UTR isoform-specific knockdown or genomic removal of the alternative UTR (aUTR) region indicate that the long 3'UTR isoform contributes to the secreted Timp2 protein much more than the short 3'UTR isoform. The short and long 3'UTR isoforms differ in subcellular localization to endoplasmic reticulum (ER). Strikingly, Timp2 aUTR enhances secreted protein expression but no effect on intracellular proteins in reporter assays. Furthermore, downregulation of Timp2 long isoform mitigates gene expression changes elicited by HRAS G12V . Together, our data indicate that regulation of Timp2 protein expression through APA isoform changes is an integral part of RAS-mediated cell transformation and 3'UTR isoforms of Timp2 can have distinct impacts on expression of secreted vs. intracellular proteins.

Large-Scale Alternative Polyadenylation-Wide Association Studies to Identify Putative Cancer Susceptibility Genes.

Alternative polyadenylation (APA) modulates mRNA processing in the 3'-untranslated regions (3' UTR), affecting mRNA stability and translation efficiency. Research into genetically regulated APA has the potential to provide insights into cancer risk. In this study, we conducted large APA-wide association studies to investigate associations between APA levels and cancer risk. Genetic models were built to predict APA levels in multiple tissues using genotype and RNA sequencing data from 1,337 samples from the Genotype-Tissue Expression project. Associations of genetically predicted APA levels with cancer risk were assessed by applying the prediction models to data from large genome-wide association studies of six common cancers among European ancestry populations: breast, ovarian, prostate, colorectal, lung, and pancreatic cancers. A total of 58 risk genes (corresponding to 76 APA sites) were associated with at least one type of cancer, including 25 genes previously not linked to cancer susceptibility. Of the identified risk APAs, 97.4% and 26.3% were supported by 3'-UTR APA quantitative trait loci and colocalization analyses, respectively. Luciferase reporter assays for four selected putative regulatory 3'-UTR variants demonstrated that the risk alleles of 3'-UTR variants, rs324015 (STAT6), rs2280503 (DIP2B), rs1128450 (FBXO38), and rs145220637 (LDHA), significantly increased the posttranscriptional activities of their target genes compared with reference alleles. Furthermore, knockdown of the target genes confirmed their ability to promote proliferation and migration. Overall, this study provides insights into the role of APA in the genetic susceptibility to common cancers. Significance: Systematic evaluation of associations of alternative polyadenylation with cancer risk reveals 58 putative susceptibility genes, highlighting the contribution of genetically regulated alternative polyadenylation of 3'UTRs to genetic susceptibility to cancer.

Mitogenome, Poly(A) Mitotranscriptome, and Molecular Phylogeny of Rasbora rasbora (Family Danionidae; Subfamily Rasborinae)

The subfamily Rasborinae is a species-rich group of freshwater fish related to zebrafish; however, its taxonomy remains unclear. We present the complete mitogenome and corresponding polyadenylated mitotranscriptome of Rasbora rasbora (Hamilton 1822) based on long-read and high-coverage Oxford Nanopore Technology sequencing. The mitogenome size, gene content, and gene organization correspond to the typical vertebrate composition, and the mitogenome generates 10 polyadenylated mRNAs. Two alternative polyadenylation sites of ND5 mRNA were detected, one with a 596 nt 3′untranslated region corresponding to the antisense ND6 gene. Polyadenylation also generates seven of the mRNA UAA stop codons. Complete mitogenome sequences, excluding the control region, were carefully aligned for RNA-coding and protein-coding features using 54 available species of the subfamily Rasborinae. The phylogenetic analyses based on maximum likelihood, Bayesian inference, and neighbor-joining tree building methods confirm the transfer of R. rasbora into the Sumatrana species group. The overall phylogeny of the subfamily Rasborinae supports with high confidence some previously observed changes within this subfamily, as well as contradicts some conclusions set by previous studies.

Sleep and diurnal alternative polyadenylation sites associated with human APA-linked brain disorders.

Disruption of sleep and circadian rhythms are a comorbid feature of many pathologies, and can negatively influence many health conditions, including neurodegenerative disease, metabolic illness, cancer, and various neurological disorders. Genetic association studies linking sleep and circadian disturbances with disease susceptibility have mainly focused on changes in gene expression due to mutations, such as single-nucleotide polymorphisms. The interaction between sleep and/or circadian rhythms with the use of Alternative Polyadenylation (APA) has been largely undescribed, particularly in the context of other disorders. APA is a process that generates various transcript isoforms of the same gene affecting its mRNA translation, stability, localization, and subsequent function. Here we identified unique APAs expressed in rat brain over time-of-day, immediately following sleep deprivation, and the subsequent recovery period. From these data, we performed a secondary analysis of these sleep- or time-of-day associated PASs with recently described APA-linked human brain disorder susceptibility genes.

Comprehensive analysis of single-nucleotide variants and alternative polyadenylation between inbred and outbred pigs

Inbreeding can lead to the accumulation of homozygous single nucleotide polymorphisms (SNPs) in the genome, which can significantly affect gene expression and phenotype. In this study, we examined the impact of homozygous SNPs resulting from inbreeding on alternative polyadenylation (APA) site selection and the underlying genetic mechanisms using inbred Luchuan pigs. Genome resequencing revealed that inbreeding results in a high accumulation of homozygous SNPs within the pig genome. 3′ mRNA-seq on leg muscle, submandibular lymph node, and liver tissues was performed to identify differences in APA events between inbred and outbred Luchuan pigs. We revealed different tissue-specific APA usage caused by inbreeding, which were associated with different biological processes. Furthermore, we explored the role of polyadenylation signal (PAS) SNPs in APA regulation under inbreeding and identified key genes such as PUM1, SCARF1, RIPOR2, C1D, and LRRK2 that are involved in biological processes regulation. This study provides resources and sheds light on the impact of genomic homozygosity on APA regulation, offering insights into genetic characteristics and biological processes associated with inbreeding.

Arabidopsis and maize terminator strength is determined by GC content, polyadenylation motifs and cleavage probability

The 3’ end of a gene, often called a terminator, modulates mRNA stability, localization, translation, and polyadenylation. Here, we adapted Plant STARR-seq, a massively parallel reporter assay, to measure the activity of over 50,000 terminators from the plants Arabidopsis thaliana and Zea mays. We characterize thousands of plant terminators, including many that outperform bacterial terminators commonly used in plants. Terminator activity is species-specific, differing in tobacco leaf and maize protoplast assays. While recapitulating known biology, our results reveal the relative contributions of polyadenylation motifs to terminator strength. We built a computational model to predict terminator strength and used it to conduct in silico evolution that generated optimized synthetic terminators. Additionally, we discover alternative polyadenylation sites across tens of thousands of terminators; however, the strongest terminators tend to have a dominant cleavage site. Our results establish features of plant terminator function and identify strong naturally occurring and synthetic terminators.

#314 Systemic lupus erythematous (SLE) and isolated multiple sclerosis: analysis of BAFF—VAR variant on gene TNSF13B

Abstract Background and Aims We report a case of young a woman (33 years old) affected by SLE since 2007. In this year, a renal biopsy showed class IV lupus nephritis and the patient received corticosteroids and immunosuppressant for 3 years. The patient remained thereafter in disease remission with normal renal function, no proteinuria and normal complement levels. In 2013 the patient performed brain MRI for sudden hearing loss. The MRI showed a pattern compatible with isolated multiple sclerosis (RIS), confirmed on subsequent radiographic checks. In 2023, due to the initial onset of renal failure, a new re-staging biopsy showed microscopic aspects compatible with class I/II lupus nephritis (INS/RPS Classification 2004, 2018); the activity index calculated with SLEDAI—2K was +4. A new brain MRI carried out without onset of new symptoms showed a new hyperintense region at the level of the head of the right caudate nucleus and enlargement of the intrasellar CSF content as per the condition of a “partially empty” sella. A genetic variant of the TNFSF13B gene was therefore suspected, since it is involved in encoding the cytochine and drug target B-cell activating factor (BAFF), associated with multiple sclerosis as well as SLE. We searched for the genetic variant in order to undertake target therapy early in our patient. Method We performed PCR amplification and direct sequencing of all coding exons plus all the intron-exon junctions of the analyzed gene, by automatic sequencer with SeqStudio Genetic Analyzer (analytical sensitivity and specificity >99%). Results We found the heterozygous presence of the BAFF-var variant in the 3' UTR region of the TNFSF13B gene (Heterozygous genotype for BAFF-var). This variant involves an insertion/deletion (GCTGT>A) at the level of the 3' UTR region with consequent production of an alternative polyadenylation site (APA). This APA leads to the formation of a shorter transcript (BAFF-var mRNA) which causes greater production of the encoded protein compared to the wild-type transcript (BAFF-WT mRNA). Conclusion The molecular analysis conducted highlighted the presence of the BAFF-var variant in heterozygosis which leads to increased levels of soluble BAFF in the serum, with consequent up-regulation of humoral immunity. It is reported in the literature1 that this variant is associated with both Multiple Sclerosis and SLE and may represent a valid therapeutic target being treatable with the monoclonal antibody Belimumab.

Colocalization analysis of 3' UTR alternative polyadenylation quantitative trait loci reveals novel mechanisms underlying associations with lung function.

While many disease-associated single nucleotide polymorphisms (SNPs) are expression quantitative trait loci (eQTLs), a large proportion of genome-wide association study (GWAS) variants are of unknown function. Alternative polyadenylation (APA) plays an important role in posttranscriptional regulation by allowing genes to shorten or extend 3' untranslated regions (UTRs). We hypothesized that genetic variants that affect APA in lung tissue may lend insight into the function of respiratory associated GWAS loci. We generated alternative polyadenylation (apa) QTLs using RNA sequencing and whole genome sequencing on 1241 subjects from the Lung Tissue Research Consortium (LTRC) as part of the NHLBI TOPMed project. We identified 56 179 APA sites corresponding to 13 582 unique genes after filtering out APA sites with low usage. We found that a total of 8831 APA sites were associated with at least one SNP with q-value < 0.05. The genomic distribution of lead APA SNPs indicated that the majority are intronic variants (33%), followed by downstream gene variants (26%), 3' UTR variants (17%), and upstream gene variants (within 1kb region upstream of transcriptional start site, 10%). APA sites in 193 genes colocalized with GWAS data for at least one phenotype. Genes containing the top APA sites associated with GWAS variants include membrane associated ring-CH-type finger 2 (MARCHF2), nectin cell adhesion molecule 2 (NECTIN2), and butyrophilin subfamily 3 member A2 (BTN3A2). Overall, these findings suggest that APA may be an important mechanism for genetic variants in lung function and chronic obstructive pulmonary disease (COPD).

A revamped rat reference genome improves the discovery of genetic diversity in laboratory rats

The seventh iteration of the reference genome assembly for Rattus norvegicus-mRatBN7.2-corrects numerous misplaced segments and reduces base-level errors by approximately 9-fold and increases contiguity by 290-fold compared with its predecessor. Gene annotations are now more complete, improving the mapping precision of genomic, transcriptomic, and proteomics datasets. We jointly analyzed 163 short-read whole-genome sequencing datasets representing 120 laboratory rat strains and substrains using mRatBN7.2. We defined ∼20.0 million sequence variations, of which 18,700 are predicted to potentially impact the function of 6,677 genes. We also generated a new rat genetic map from 1,893 heterogeneous stock rats and annotated transcription start sites and alternative polyadenylation sites. The mRatBN7.2 assembly, along with the extensive analysis of genomic variations among rat strains, enhances our understanding of the rat genome, providing researchers with an expanded resource for studies involving rats.

Abstract 5838: Un-methylation of NUDT21 promotes the development of enzalutamide resistance in prostate cancer by regulation of 3’UTR usage switch

Abstract Enzalutamide, a second-generation anti-androgen drug, is a new treatment option for castration resistant prostate cancer (CRPC) patient. However, almost all CRPC patients develop drug resistance for enzalutamide after six to twelve months of treatment. According to our current findings, 3'UTR usage switches of RNA transcripts were observed in the prostate cancer specimen after acquiring enzalutamide resistance and nudix hydrolase 21 (NUDT21), an alternative polyadenylation (APA) regulator, was overexpressed in the enzalutamide resistant (EnzaR) cells, suggesting its potential role involved in regulation of 3'UTR usage switch. However, the underlying mechanism of how NUDT21 regulates 3'UTR usage switch during the development of enzalutamide resistance is still unclear. Interestingly, a potential mono-methylation (MME) is identified in NUDT21 protein by us, and it has never been examined before. Thus, we hypothesize that MME of NUDT21 may regulate the specificity of 3'UTR usage switch and involve in the development of EnzaR. To test the idea, a monoclonal antibody recognized NUDT21 MME was generated by us. Moreover, protein arginine methyltransferase 7 (PRMT7) was identified to be responsible for adding MME to NUDT21. In addition, androgen treatment increased both expression levels of NUDT21 MME and PRMT7 while decreased the expression level of NUDT21 total form in LNCaP cells via androgen receptor (AR) dependent manner. Next, co-treatment of enzalutamide and PRMT7 inhibitor leaded to increase of un-MME form of NUDT21 and it greatly enhanced the enzalutamide resistance in LNCaP cells. Importantly, binding profile of NUDT21 total form indeed had 3'UTR usage switch from distal to proximal APA site in COUP-TFII transcript after acquiring enzalutamide resistance, while its MME form attenuated this phenomenon. In addition, expression levels of NUDT21 MME and its total form were shown to conversely express in prostate cancer specimens. Finally, un-MME form of NUDT21 promoted the cancer growth in vitro and in vivo while its MME form did not. Taken together, our results suggest that down-regulation of PRMT7 by enzalutamide may lead to decrease of NUDT21 MME level and contribute to develop enzalutamide resistance by causing the 3'UTR usage switch of NUDT21 downstream transcript. Citation Format: Shin-Chih (Kenny) Lin, Shih-Chieh Lin. Un-methylation of NUDT21 promotes the development of enzalutamide resistance in prostate cancer by regulation of 3’UTR usage switch [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5838.

Deciphering Estrus Expression in Gilts: The Role of Alternative Polyadenylation and LincRNAs in Reproductive Transcriptomics.

The fertility rate and litter size of female pigs are critically affected by the expression of estrus. The objective of this study was to elucidate the regulatory mechanisms of estrus expression by analyzing the differential expression of genes and long intergenic non-coding RNAs (lincRNA), as well as the utilization of alternative polyadenylation (APA) sites, in the vulva and vagina during the estrus and diestrus stages of Large White and indigenous Chinese Mi gilts. Our study revealed that the number of differentially expressed genes (DEG) in the vulva was less than that in the vagina, and the DEGs in the vulva were enriched in pathways such as "neural" pathways and steroid hormone responses, including the "Calcium signaling pathway" and "Oxytocin signaling pathway". The DEGs in the vagina were enriched in the "Metabolic pathways" and "VEGF signaling pathway". Furthermore, 27 and 21 differentially expressed lincRNAs (DEL), whose target genes were enriched in the "Endocrine resistance" pathway, were identified in the vulva and vagina, respectively. Additionally, we observed that 63 and 618 transcripts of the 3'-untranslated region (3'-UTR) were lengthened during estrus in the vulva and vagina, respectively. Interestingly, the genes undergoing APA events in the vulva exhibited species-specific enrichment in neural or steroid-related pathways, whereas those in the vagina were enriched in apoptosis or autophagy-related pathways. Further bioinformatic analysis of these lengthened 3'-UTRs revealed the presence of multiple miRNAs binding sites and cytoplasmic polyadenylation element (CPE) regulatory aspects. In particular, we identified more than 10 CPEs in the validated lengthened 3'-UTRs of the NFIX, PCNX4, CEP162 and ABHD2 genes using RT-qPCR. These findings demonstrated the involvement of APA and lincRNAs in the regulation of estrus expression in female pigs, providing new insights into the molecular mechanisms underlying estrus expression in pigs.

VizAPA: visualizing dynamics of alternative polyadenylation from bulk and single-cell data.

Alternative polyadenylation (APA) is a widespread post-transcriptional regulatory mechanism across all eukaryotes. With the accumulation of genome-wide APA sites, especially those with single-cell resolution, it is imperative to develop easy-to-use visualization tools to guide APA analysis. We developed an R package called vizAPA for visualizing APA dynamics from bulk and single-cell data. vizAPA implements unified data structures for APA data and genome annotations. vizAPA also enables identification of genes with differential APA usage across biological samples and/or cell types. vizAPA provides four unique modules for extensively visualizing APA dynamics across biological samples and at the single-cell level. vizAPA could serve as a plugin in many routine APA analysis pipelines to augment studies for APA dynamics. https://github.com/BMILAB/vizAPA.

Above and belowground phenotypic response to exogenous auxin across Arabidopsis thaliana mutants and natural accessions varies from seedling to reproductive maturity.

Plant hormones influence phenology, development, and function of above and belowground plant structures. In seedlings, auxin influences the initiation and development of lateral roots and root systems. How auxin-related genes influence root initiation at early life stages has been investigated from numerous perspectives. There is a gap in our understanding of how these genes influence root size through the life cycle and in mature plants. Across development, the influence of a particular gene on plant phenotypes is partly regulated by the addition of a poly-A tail to mRNA transcripts via alternative polyadenylation (APA). Auxin related genes have documented variation in APA, with auxin itself contributing to APA site switches. Studies of the influence of exogenous auxin on natural plant accessions and mutants of auxin pathway gene families exhibiting variation in APA are required for a more complete understanding of genotype by development by hormone interactions in whole plant and fitness traits. We studied Arabidopsis thaliana homozygous mutant lines with inserts in auxin-related genes previously identified to exhibit variation in number of APA sites. Our growth chamber experiment included wildtype Col-0 controls, mutant lines, and natural accession phytometers. We applied exogenous auxin through the life cycle. We quantified belowground and aboveground phenotypes in 14 day old, 21 day old seedlings and plants at reproductive maturity. We contrasted root, rosette and flowering phenotypes across wildtype, auxin mutant, and natural accession lines, APA groups, hormone treatments, and life stages using general linear models. The root systems and rosettes of mutant lines in auxin related genes varied in response to auxin applications across life stages and varied between genotypes within life stages. In seedlings, exposure to auxin decreased size, but increased lateral root density, whereas at reproductive maturity, plants displayed greater aboveground mass and total root length. These differences may in part be due to a shift which delayed the reproductive stage when plants were treated with auxin. Root traits of auxin related mutants depended on the number of APA sites of mutant genes and the plant's developmental stage. Mutants with inserts in genes with many APA sites exhibited lower early seedling belowground biomass than those with few APA sites but only when exposed to exogenous auxin. As we observed different responses to exogenous auxin across the life cycle, we advocate for further studies of belowground traits and hormones at reproductive maturity. Studying phenotypic variation of genotypes across life stages and hormone environments will uncover additional shared patterns across traits, assisting efforts to potentially reach breeding targets and enhance our understanding of variation of genotypes in natural systems.

Multiple strategies, including 6mA methylation, affecting plant alternative splicing in allopolyploid peanut.

Alternative splicing (AS), an important post-transcriptional regulation mechanism in eukaryotes, can significantly increase transcript diversity and contribute to gene expression regulation and many other complicated developmental processes. While plant gene AS events are well described, few studies have investigated the comprehensive regulation machinery of plant AS. Here, we use multi-omics to analyse peanut AS events. Using long-read isoform sequencing, 146 464 full-length non-chimeric transcripts were obtained, resulting in annotation corrections for 1782 genes and the identification of 4653 new loci. Using Iso-Seq RNA sequences, 271 776 unique splice junctions were identified, 82.49% of which were supported by transcriptome data. We characterized 50 977 polyadenylation sites for 23 262 genes, 12 369 of which had alternative polyadenylation sites. AS allows differential regulation of the same gene by miRNAs at the isoform level coupled with polyadenylation. In addition, we identified many long non-coding RNAs and fusion transcripts. There is a suppressed effect of 6mA on AS and gene expression. By analysis of chromatin structures, the genes located in the boundaries of topologically associated domains, proximal chromosomal telomere regions, inter- or intra-chromosomal loops were found to have more unique splice isoforms, higher expression, lower 6mA and more transposable elements (TEs) in their gene bodies than the other genes, indicating that chromatin interaction, 6mA and TEs play important roles in AS and gene expression. These results greatly refine the peanut genome annotation and contribute to the study of gene expression and regulation in peanuts. This work also showed AS is associated with multiple strategies for gene regulation.

Microbiota affects mitochondria and immune cell infiltrations via alternative polyadenylation during postnatal heart development.

There is a growing body of evidence supporting the significant impact of microbiota on heart development. Alternative polyadenylation (APA) is a crucial mechanism for gene expression regulation and has been implicated in postnatal heart development. Nonetheless, whether microbiota can influence postnatal heart development through the regulation of APA remains unclear. Therefore, we conducted APA sequencing on heart tissues collected from specific pathogen-free (SPF) mice and germ-free (GF) mice at three different developmental stages: within the first 24h after birth (P1), 7-day-old SPF mice, and 7-day-old GF mice. This approach allowed us to obtain a comprehensive genome-wide profile of APA sites in the heart tissue samples. In this study, we made a significant observation that GF mice exhibited noticeably longer 3' untranslated region (3' UTR) lengths. Furthermore, we confirmed significant alterations in the 3' UTR lengths of mitochondria-related genes, namely Rala, Timm13, and Uqcc3. Interestingly, the GF condition resulted in a marked decrease in mitochondrial cristae density and a reduction in the level of Tomm20 in postnatal hearts. Moreover, we discovered a connection between Rala and Src, which further implicated their association with other differentially expressed genes (DEGs). Notably, most of the DEGs were significantly downregulated in GF mice, with the exceptions being Thbs1 and Egr1. Importantly, the GF condition demonstrated a correlation with a lower infiltration of immune cells, whereby the levels of resting NK cells, Th17 cells, immature dendritic cells, and plasma cells in GF mice were comparable to those observed in P1 mice. Furthermore, we established significant correlations between these immune cells and Rala as well as the related DEGs. Our findings clearly indicated that microbiota plays a vital role in postnatal heart development by affecting APA switching, mitochondria and immune cell infiltrations.

Dynamics of alternative polyadenylation in single root cells of Arabidopsis thaliana.

Single-cell RNA-seq (scRNA-seq) technologies have been widely used to reveal the diversity and complexity of cells, and pioneering studies on scRNA-seq in plants began to emerge since 2019. However, existing studies on plants utilized scRNA-seq focused only on the gene expression regulation. As an essential post-transcriptional mechanism for regulating gene expression, alternative polyadenylation (APA) generates diverse mRNA isoforms with distinct 3' ends through the selective use of different polyadenylation sites in a gene. APA plays important roles in regulating multiple developmental processes in plants, such as flowering time and stress response. In this study, we developed a pipeline to identify and integrate APA sites from different scRNA-seq data and analyze APA dynamics in single cells. First, high-confidence poly(A) sites in single root cells were identified and quantified. Second, three kinds of APA markers were identified for exploring APA dynamics in single cells, including differentially expressed poly(A) sites based on APA site expression, APA markers based on APA usages, and APA switching genes based on 3' UTR (untranslated region) length change. Moreover, cell type annotations of single root cells were refined by integrating both the APA information and the gene expression profile. We comprehensively compiled a single-cell APA atlas from five scRNA-seq studies, covering over 150,000 cells spanning four major tissue branches, twelve cell types, and three developmental stages. Moreover, we quantified the dynamic APA usages in single cells and identified APA markers across tissues and cell types. Further, we integrated complementary information of gene expression and APA profiles to annotate cell types and reveal subtle differences between cell types. This study reveals that APA provides an additional layer of information for determining cell identity and provides a landscape of APA dynamics during Arabidopsis root development.

Single-Molecule Real-Time Sequencing for Identifying Sexual-Dimorphism-Related Transcriptomes and Genes in the Chinese Soft-Shelled Turtle (Pelodiscus sinensis)

The Chinese soft-shelled turtle (Pelodiscus sinensis), an economically important aquatic species in China, displays considerable sexual dimorphism: the male P. sinensis is larger and, thus, more popular in the market. In this study, we obtained the full-length (FL) transcriptome data of P. sinensis by using Pacific Biosciences (PacBio)’s isoform sequencing and analyzed the transcriptome structure. In total, 1,536,849 high-quality FL transcripts were obtained through single-molecule real-time (SMRT) sequencing, which were then corrected using Illumina sequencing data. Next, 89,666 nonredundant FL transcripts were generated after mapping to the reference genome of P. sinensis; 291 fusion genes and 17,366 novel isoforms were successfully annotated using data from the nonredundant protein sequence database (NR), eukaryotic orthology groups (KOG), the Gene Ontology (GO) project, and the KEGG Orthology (KO) database. Additionally, 19,324 alternative polyadenylation sites, 101,625 alternative splicing events, 12,392 long noncoding RNAs, and 5916 transcription factors were identified. Smad4, Wif1, and 17-β-hsd were identified as female-biased genes, while Nkd2 and Prp18 held a higher expression level in males than females. In summary, we found differences between male and female P. sinensis individuals in AS, lncRNA, genes, and transcripts, which relate to the Wnt pathway, oocyte meiosis, and the TGF-β pathway. Female-biased genes such as Smad4, Wif1, and 17-β-hsd and male-biased genes such as Nkd2 and Prp18 played important roles in the sex determination of P. sinensis. FL transcripts are a precious resource for characterizing the transcriptome of P. sinensis, laying the foundation for further research on the sex-determination mechanisms of P. sinensis.