Biology subjects

Lu, F.

Publications and source records attributed to Lu, F..

At least 55 records · Page 3Linked to original sources

bioRxiv2022

Complexity of enhancer networks predicts cell identity and disease genes revealed by single-cell multi-omics analysis

Many enhancers exist as clusters in the genome and control cell identity and disease genes; however, the underlying mechanism remains largely unknown. Here, we introduce an algorithm, eNet, to build enhancer networks by integrating single-cell chromatin accessibility and gene expression profiles. Enhancer network is a gene regulation model we proposed that not only delineates the mapping between enhancers and target genes, but also quantifies the underlying regulatory relationships among enhancers. The complexity of enhancer networks is assessed by two metrics: the number of enhancers and the frequency of predicted enhancer interactions (PEIs) based on chromatin co-accessibility. We apply eNet algorithm to a human blood dataset and find cell identity and disease genes tend to be regulated by complex enhancer networks. The network hub enhancers (enhancers with frequent PEIs) are the most functionally important in enhancer networks. Compared with super-enhancers, enhancer networks show better performance in predicting cell identity and disease genes. The establishment of enhancer networks drives gene expression during lineage commitment. Applying eNet in various datasets in human or mouse tissues across different single-cell platforms, we demonstrate eNet is robust and widely applicable. Thus, we propose a model of enhancer networks containing three modes: Simple, Multiple and Complex, which are distinguished by their complexity in regulating gene expression. Taken together, our work provides an unsupervised approach to simultaneously identify key cell identity and disease genes and explore the underlying regulatory relationships among enhancers in single cells, without requiring the cell type identity in advance. HighlightsO_LIeNet, a computational method to build enhancer network based on scATAC-seq and scRNA-seq data C_LIO_LICell identity and disease genes tend to be regulated by complex enhancer networks, where network hub enhancers are functionally important C_LIO_LIEnhancer network outperforms the existing models in predicting cell identity and disease genes, such as super-enhancer and enhancer cluster C_LIO_LIWe propose a model of enhancer networks in gene regulation containing three modes: Simple, Multiple and Complex C_LI

bioinformatics↗

bioRxiv2022

Population genomics unravels the Holocene history of Triticum-Aegilops species

Deep knowledge of crop biodiversity is essential to improve global food security. Despite bread wheat serving as a keystone crop worldwide, the population history of bread wheat and its wild relatives (a.k.a. wheats) remains elusive. By analyzing whole-genome sequences of 795 wheats, we found that bread wheat originated southwest of the Caspian Sea [~]11,700 years ago and underwent a slow speciation process, lasting [~]3,300 years due to persistent gene flow from wild relatives. Soon after, bread wheat spread across Eurasia and reached Europe, South Asia, and East Asia [~]7,000 to [~]5,000 years ago, shaping a diversified but occasionally convergent adaptive landscape of bread wheat in novel environments. Opposite to cultivated wheat, wild wheat populations have declined by [~]82% in the past [~]2,000 years due to the food choice shift of humans, and likely continue to drop because of the changing climate. These findings will guide future efforts in protecting and utilizing wheat biodiversity to improve global food security.

genetics↗

bioRxiv2022

Emodin Combined with Multiple Low-frequency Low-intensity Ultrasound to Relieve Osteomyelitis Through Sonoantimicrobial Chemotherapy

Treatment of osteomyelitis is still challenging as conventional antibiotic therapy is limited by the emergence of resistant strains and the formation of biofilms. Sonoantimicrobial chemotherapy (SACT) is a novel therapy of low-frequency and low-intensity ultrasound (LFLIU) combined with sonosensitizer. Therefore, in our study, a sonosensitizer named emodin (EM) was proposed to be combined with LFLIU to relieve acute osteomyelitis caused by Methicillin-resistant Staphylococcus aureus (MRSA) through synergistic antibacterial and anti-biofilm effects. The efficiency of different intensities of ultrasound, single (S-LFLIU, 15 min) and multiple ultrasound (M-LFLIU, 5 min every 4 h, three times) against bacteria and biofilm was compared, contributing to develop the best treatment regimen. Our results demonstrated that EM plus S-LFLIU or M-LFLIU (EM+S-LFLIU or EM+M-LFLIU) have significant synergetic bactericidal and anti-biofilm effects and EM+M-LFLIU exhibits superior performance in anti-biofilm. Furthermore, it was suggested that EM+M-LFLIU could produce a large amount of reactive oxygen species (ROS), destroy the integrity of bacterial membrane and wall, down-regulate the expression of oxidative stress, membrane wall synthesis, bacterial virulence and other related genes (agrB, PBP3, sgtB, GMK, zwf, msrA). In vivo study, micro-CT, H&E staining, ELISA assay and bacterial quantification of bone tissue indicated that EM+M-LFLIU could also relieve osteomyelitis of MRSA infection. Our work proffers an original treating bacterial osteomyelitis approach that weakens drug-resistant bacterial and suppresses biofilm formation through SACT, which may provide new prospects for clinical treatment.

microbiology↗

bioRxiv2022

Epigenetic Landscape of HIV Infection in Primary Human Macrophage

HIV-infected macrophages are long-lived cells that sustain persistent virus expression, which is both a barrier to viral eradication and contributor to neurological complications in patients despite antiretroviral therapy (ART). To better understand the regulation of HIV in macrophages, we compared HIV infected primary human monocyte derived macrophages (MDM) to acutely infected primary CD4 T cells and Jurkat cells latently infected with HIV (JLAT 8.4). HIV genomes in MDM were actively transcribed despite enrichment with heterochromatin-associated H3K9me3 across the complete HIV genome in combination with elevated activation marks of H3K9ac and H3K27ac at the LTR. Macrophage patterns contrasted with JLAT cells, which showed conventional bivalent H3K4me3/H3K27me3, and acutely infected CD4 T cells, which showed an intermediate epigenotype. 5 -methylcytosine (5mC) was enriched across the HIV genome in latently infected JLAT cells, while 5 -hydroxymethylcytosine (5hmc) was enriched in CD4 and MDM. HIV infection induced multinucleation of MDMs along with DNA damage associate p53 phosphorylation, as well as loss of TET2 and the nuclear redistribution of 5-hydoxymethylation. Taken together, our findings suggest that HIV induces a unique macrophage nuclear and transcriptional profile, and viral genomes are maintained in a non-canonical bivalent epigenetic state. ImportanceMacrophages serve as a reservoir for long-term persistence and chronic production of HIV. We found an atypical epigenetic control of HIV in macrophages marked by heterochromatic H3K9me3 despite active viral transcription. HIV infection induced changes in macrophage nuclear morphology and epigenetic regulatory factors. These findings may identify new mechanisms to control chronic HIV expression in infected macrophage.

microbiology↗

bioRxiv2021

Increased RNA Polymerase Activity and Pausing at CTCF binding sites on the Epstein Barr Virus Genome During Reactivation from Latency

The ability of Epstein-Barr Virus (EBV) to switch between latent and lytic infection is key to its long-term persistence, yet the molecular mechanisms behind this switch remain unclear. To investigate transcriptional events during the latent to lytic switch we utilized Precision nuclear Run On followed by deep Sequencing (PRO-Seq) to map cellular RNA polymerase (Pol) activity to single-nucleotide resolution on the host and EBV genome in three different models of EBV latency and reactivation. In latently infected Mutu I Burkitt Lymphoma (BL) cells, Pol activity was enriched at the Qp promoter, the EBER region and the BHLF1/LF3 transcripts. Upon reactivation with phorbol ester and sodium butyrate, early phase Pol activity occurred bidirectionally at CTCF sites within the LMP-2A, EBER-1 and RPMS1 loci. PRO-Seq analysis of Akata cells reactivated from latency with anti-IgG and a lymphoblastoid cell-line (LCL) reactivated with small molecule C60 showed a similar pattern of early bidirectional transcription initiating around CTCF binding sites, although the specific CTCF sites and viral genes were different for each latency model. The functional importance of CTCF binding, transcription and reactivation was confirmed using an EBV mutant lacking the LMP-2A CTCF binding site. This virus was unable to reactivate and had disrupted Pol activity at multiple CTCF binding sites relative to WT virus. Overall, these data suggest that CTCF regulates the viral early transcripts during reactivation from latency. These activities likely help maintain the accessibility of the viral genome to initiate productive replication. Author summaryThe ability of EBV to switch between latent and lytic infection is key to its long-term persistence in memory B-cells and its ability to persist in proliferating cells is strongly linked to oncogenesis. During latency, most viral genes are epigenetically silenced, and the virus must overcome this repression to reactivate lytic replication. Reactivation occurs once the immediate early (IE) EBV lytic genes are expressed. However, the molecular mechanisms behind the switch from the latent transcriptional program to begin transcription of the IE genes remain unknown. In this study, we mapped RNA polymerase (Pol) positioning and activity during latency and reactivation. Unexpectedly, Pol activity was not enriched at the IE genes immediately after reactivation but accumulated at distinct regions characteristic of transcription initiation on the EBV genome previously shown to be associated with CTCF. We propose that CTCF binding at these regions retains Pol to maintain a stable latent chromosome conformation and a rapid response to various reactivation signals.

microbiology↗

bioRxiv2021

Genotyping of structural variation using PacBio high-fidelity sequencing

BackgroundStructural variations (SVs) pervade the genome and contribute substantially to the phenotypic diversity of species. However, most SVs were ineffectively assayed because of the complexity of plant genomes and the limitations of sequencing technologies. Recent advancement of third-generation sequencing technologies, particularly the PacBio high-fidelity (HiFi) sequencing, which generates both long and highly accurate reads, offers an unprecedented opportunity to characterize SVs and reveal their functionality. Since HiFi sequencing is new, it is crucial to evaluate HiFi reads in SV detection before applying the technology at scale. ResultsWe sequenced wheat genomes using HiFi, then conducted a comprehensive evaluation of SV detection using mainstream long-read aligners and SV callers. The results showed the accuracy of SV discovery depends more on aligners rather than callers. For aligners, pbmm2 and NGMLR provided the most accurate results while detecting deletion and insertion, respectively. Likewise, cuteSV and SVIM achieved the best performance across all SV callers. We demonstrated that the combination of the aligners and callers mentioned above is optimal for SV detection. Furthermore, we evaluated the impact of sequencing depth on the accuracy of SV detection. The results showed that low-coverage HiFi sequencing is capable of generating high-quality SV genotyping. ConclusionsThis study provides a robust benchmark of SV discovery with HiFi reads, showing the remarkable potential of long-read sequencing to investigate structural variations in plant genomes. The high accuracy SV discovery from low-coverage HiFi sequencing indicates that skim HiFi sequencing is an ideal approach to study structural variations at the population level.

bioinformatics↗

bioRxiv2021

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.

developmental biology↗

bioRxiv2021

Conservation and divergence of poly(A) tail regulation during the mammalian oocyte-to-embryo transition

Poly(A) tail length and non-A residues are vital for oocyte-to-embryo transition (OET) in mice and humans1-5. However, the role of poly(A) tail length and non-A residues during OET in other commonly used mammalian animal models for human diseases remains unexplored. In addition, the degree of conservation in maternal mRNA poly(A) tail dynamics during OET across different mammal species is unknown. Here, we conduct a comparative analysis of the poly(A) tails during OET across four species: mice, rats, pigs, and humans. Dynamics during OET found to be conserved across all four species include: maternal mRNA deadenylation during oocyte maturation and re-polyadenylation after fertilization; a fall-rise trend in poly(A) tail length distribution; a rise-fall trend in the ratio of poly(A) tails with non-A residues; higher abundance of non-A residues in poly(A) tails of maternal mRNA than in zygotic genome activation (ZGA) mRNA; maternal mRNA with U residues degrades faster than those without U residues at the stage when ZGA takes place. While in mice and rats maternal mRNA deadenylation is impaired in parthenogenetic embryos and ZGA inhibition leads to blocked maternal mRNA deadenylation in mice and humans. In contrast, the length of consecutive U residues and the duration time of U residues in poly(A) tail diverges across the four species. Together, these findings reveal that the poly(A) tail mediated maternal mRNA post-transcriptional regulation is highly conserved in mammals with unique divergences in the length and life-span of U residues, providing new insights for the further understanding of OET across different mammals.

developmental biology↗

bioRxiv2021

Dynamics of mitochondrial mRNA abundance and poly(A) tail during the mammalian oocyte-to-embryo transition

Mitochondria are responsible for producing a cells energy and play a central role in many metabolic tasks, as well as signaling transduction and cell death1. Mitochondria dysfunctions cause several human diseases and aging processes2-8. Mammalian oocytes contain far more mitochondria than somatic cells. The nuclear localization of mitochondrial tricarboxylic acid cycle (TCA) cycle enzymes, which normally localize in the mitochondria, is critical for zygotic genome activation (ZGA) and the oocyte-to-embryo transition (OET) in mice9. However, during the mammalian OET, the abundance and post-transcriptional regulation of mitochondrial mRNA (MT-mRNA), particularly the poly(A) tail, has never been studied. Here, we used two independent sequencing methods (PAIso-seq1 and PAIso-seq2) to describe the features of MT-mRNA in mouse cell lines, thirteen mouse tissues and during the OET in mouse, rat, pig, and humans. These features included expression abundance, poly(A) tail length, and non-A residues in poly(A) tails. Unlike nuclear mRNA, we discovered that MT-mRNA has a stable distribution pattern of poly(A) tail length in different cell lines, across tissues, and during mammalian OET. MT-mRNA possesses non-A residues in the poly(A) tail (non-A residues hereafter), which change slightly across tissues and during the OET. We also found that the abundance of MT-mRNA varies substantially across tissues, increases during the OET, and increases along major ZGA in mice, rats, pigs, and humans. These findings provide insights into changes in MT-mRNA abundance and poly(A) tail during the mammalian OET and provide a resource for future studies on the posttranscriptional regulation of mitochondrial transcripts.

developmental biology↗

bioRxiv2021

Dynamics of poly(A) tail length and non-A residues during the human oocyte-to-embryo transition

Poly(A) tail-mediated post-transcriptional regulation of maternal mRNA has been shown to be vital in the oocyte-to-embryo transition (OET) in flies, fish, frogs, and mice1-8. However, nothing is known about poly(A) tail dynamics for even a single gene during the human OET, because of the limited availability of human oocytes and embryos in combination with the low sensitivity of previous methods. Here, we systematically profiled the transcriptome-wide mRNA poly(A) tails in human oocytes at the germ-vesicle (GV), metaphase I (MI), and metaphase II (MII) stages, as well as pre-implantation embryos at the 1-cell (1C), 2-cell (2C), 4-cell (4C), 8-cell (8C), morula (MO), and blastocyst (BL) stages using single-oocyte/embryo PAIso-seq1 and PAIso-seq2 methods. We show that poly(A) tail length is highly dynamic during the OET, with BTG4 responsible for global deadenylation. Moreover, we reveal that non-A residues occur primarily in poly(A) tails of maternal RNA, which begin to increase at the MI stage, become highly abundant after fertilization (with U residues occurring in about two thirds, G residues in about one third, and C residues in about one fifth of mRNAs), and decline at the 8C stage. Importantly, we reveal that TUT4/7 can add U residues to deadenylated mRNA, which can then be re- polyadenylated to produce 5'-end and internal U residues. In addition, the re- polyadenylated mRNA can be stabilized through the addition of G residues by TENT4A/B. Finally, we demonstrate that U residues in poly(A) tails mark the maternal transcripts for quicker degradation in 8C human embryos compared to those without U residues. Together, our results not only reveal the dynamics of poly(A) tail length and non-A residues, but also provide mechanistic insights into the regulation of the length and the role of non-A residues during human OET. These findings further scientific understanding and open a new door for studying the human OET.

developmental biology↗

bioRxiv2021

Abundant non-A residues in the poly(A) tail orchestrate the mouse oocyte-to-embryo transition

Non-A (U, G, and C) residues can be added to the 5-end, internal, and 3-end positions of poly(A) tails of RNA transcripts1-3, and some of these have been shown to regulate mRNA stability4, 5. The mammalian oocyte-to-embryo transition (OET) relies on post-transcriptional regulation of maternal RNA, because transcription is silent during this process until the point of zygotic genome activation (ZGA)6-9. Although the regulation of mRNA translation by poly(A) tail length plays an important role in the mammalian OET, the dynamics and functions of non-A residues in poly(A) tails are completely unknown. In this study, we profiled the genome-wide presence, abundance, and roles of non-A residues during the OET in mice using PAIso-seq1 and PAIso-seq22, 10, two complementary methods of poly(A) tail analysis. We found that non-A residues are highly dynamic in maternal mRNA, following a general pattern of beginning to increase at the MII stage, becoming highly abundant after fertilization with U residues in about half of poly(A) tails in 1-cell embryos, and declining in 2-cell embryos. We revealed that Btg4-mediated global maternal mRNA deadenylation created the substrates for U residue addition by Tut4/7 at their 3-ends and further re-polyadenylation. In addition, G residues can be added by Tent4a/b. Finally, we demonstrate that G residues stabilize the modified mRNA, while the U residues mark maternal RNA for faster degradation in 2-cell mouse embryos. Taken together, these findings demonstrate that non-A residues are abundant and re-sculpt the maternal transcriptome to initiate zygotic development, which reveals the functional importance of the post-transcriptional regulation mediated by non-A residues in mRNA poly(A) tails.

developmental biology↗

bioRxiv2021

Enhancement of synthetic mRNA translation efficiency through engineered poly(A) tails

In vitro transcribed (IVT) mRNA represents a new class of drug in both therapeutics and vaccines. Improving the translation efficiency of IVT mRNA remains a core challenge for mRNA-based applications. Here, using IVT mRNAs with poly(A) tails containing non-A residues which were recently revealed to be widespread in RNA poly(A) tails1,2, we unexpectedly find that non-A residues can effectively promote the mRNA translation. To further support our finding, we provide evidence that non-A residues associated with enhanced mRNA translation efficiency transcriptome-wide in mouse and human cells. Together, our study provides a novel approach to enhance mRNA translation efficiency by inclusion of non-A residues in the mRNA poly(A) tails, holding great potential to promote mRNA-based therapeutics and vaccines.

molecular biology↗

bioRxiv2021

Degradation intermediates as an indicator of mRNA and cell metabolism

Traditional mRNA degradation rate measurements involves complex experimental design with RNA labeling or transcription blocking together with sampling at multiple timepoints1,2. These experimental requirements limit the application of transcriptome-wide mRNA degradation rate analysis mainly in cultured cells, but rarely in in vivo samples. Therefore, a direct and simple strategy needs to be developed to study mRNA degradation rate. Here, we defined mRNA degradation intermediates as transcripts where decay is about to occur or has partially occurred in the 3-untranslated regions after poly(A) tail deadenylation, and found that the proportion of mRNA degradation intermediates is a very simple and convenient indicator for evaluating the degradation rate of mRNA in mouse and human cell lines. In addition, we showed that a higher proportion of mRNA degradation intermediates is correlated with faster cell cycle and higher turnover rate of mouse tissues. Further, we validated that in mouse maturing oocytes where transcription is silent3,4, the proportion of mRNA degradation intermediates is positively correlated with the mRNA degradation rate. Together, these results demonstrate that degradation intermediates can function as a good indicator of mRNA, cell, and tissue metabolism, and can be easily assayed by total RNA 3-end sequencing from a single bulk cell sample without the need for drug treatment or multi-timepoint sampling. This finding is of great potential for studies on mRNA degradation rate at the molecular, cellular, or organic level, including samples or systems that cannot be assayed with previous methods. In addition, further application of the findings into single cells will likely greatly aid the identification and study of rare cells with unique cellular metabolism dynamics such as tissue stem cells and tumor cells.

molecular biology↗

bioRxiv2021

Maternal mRNA deadenylation is defective in in vitro matured mouse and human oocytes

Oocyte in vitro maturation is a technique of assisted reproductive technology that was first introduced in patients with polycystic ovarian syndrome and is now used in most fertility clinics. Thousands of genes show abnormally high expression in in vitro maturated metaphase II (in vitro MII) oocytes compared with in vivo maturated metaphase II (in vivo MII) oocytes in bovines, mice, and humans1-3. However, the underlying mechanisms of this abnormal expression are still poorly understood. In this study, we use PAIso-seq1 to reveal a transcriptome-wide expression profile of full-length transcripts containing entire poly(A) tails in in vivo and in vitro matured mouse and human oocytes. Our results indicate that more genes are up-regulated than down-regulated in in vitro MII oocytes in both mice and humans. Furthermore, we demonstrate that the observed increase in maternal mRNA abundance is caused by impaired deadenylation in in vitro MII oocytes in both mice and humans. We also found that the cytoplasmic polyadenylation of dormant Btg4 and Cnot7 mRNAs, which encode key components of deadenylation machinery, is impaired in in vitro MII oocytes in mice and humans respectively, likely contributing to reduced translation and impaired global maternal mRNA deadenylation. Our findings highlight that impaired maternal mRNA deadenylation is a definite molecular defect in in vitro MII oocytes in both mice and humans. The findings here offer a new criterion for evaluating the quality of in vitro MII oocytes and a potential direction for improving in vitro maturation by fixing the dysregulated maternal mRNA deadenylation.

developmental biology↗

bioRxiv2021

Mitochondrial mRNA is a stable and convenient reference for the normalization of RNA-seq data

The normalization of high-throughput RNA sequencing (RNA-seq) data is needed to accurately analyze gene expression levels. Traditional normalization methods can either correct the differences in sequencing depth, or correct both the sequencing depth and other unwanted variations introduced during sequencing library preparation through exogenous spike-ins1-4. However, the exogenous spike-ins are prone to variation5,6. Therefore, a better normalization approach with a more appropriate reference is an ongoing demand. In this study, we demonstrated that mitochondrial mRNA (mRNA encoded by mitochondria genome) can serve as a steady endogenous reference for RNA-seq data analysis, and performs better than exogenous spike-ins. We also found that using mitochondrial mRNA as a reference can reduce batch effects for RNA-seq data. These results provide a simple and practical normalization strategy for RNA-seq data, which will serve as a valuable tool widely applicable to transcriptomic studies.

molecular biology↗

bioRxiv2021

Comprehensive analysis of mRNA poly(A) tail reveals complex and conserved regulation

Non-templated poly(A) tails are added to the 3'-end of most mRNAs, which have important roles in post-transcriptional regulation1-3. Recent studies have revealed that poly(A) tails are not composed purely of A residues, but also contain U, C and G residues internally and at their 3'-ends4-6, revealing new levels of complexity. However, no method is able to analyze these internal and terminal non-A residues simultaneously. Here, we developed a new method called unbiased capture of 3-terminal followed by full-length RNA sequencing (UCTF-seq) which captures RNA 3'-ends by direct 3' adaptor ligation and rRNA removal by CRISPR/Cas9. This method allows simultaneous evaluation of the poly(A) tail length and 5'-end, internal, and 3'-end non- A residues together with the full-length cDNA for a transcript. Applying this method, we achieved the first complete transcriptome-wide 3' tail map of mRNA within the nuclear and cytoplasmic compartments of mammalian cells, uncovering differences in poly(A) tail length and non-A residues between these two mRNA populations. A survey of diverse eukaryotic species revealed the conservation of a subset of poly(A) tails containing consecutive U residues in the internal positions, whereas those with consecutive C or G residues were of much lower abundance. Together, we established the first method to be able to comprehensively analyze poly(A) tail 5'-end, internal and 3'-end non-A residues in addition to the length simultaneously, and reveal the first complete mRNA 3' tail map, providing rich insights into the regulatory roles of poly(A) tails.

molecular biology↗

bioRxiv2021

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 residues1, 2), alternative splicing, and chemical modifications including N6-methyladenosine (m6A)3. 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 weve 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.

molecular biology↗

bioRxiv2021

Re-polyadenylation occurs predominantly on maternal mRNA degradation intermediates during mammalian oocyte-to-embryo transition

The nascent mRNA transcribed in the nucleus is cleaved and polyadenylated before it is transported to the cytoplasm for translation1. Polyadenylation can also occur in the cytoplasm for post-transcriptional regulation, especially in neurons, oocytes and early embryos1,2. Recently, we revealed transcriptome-wide maternal mRNA cytoplasmic re-polyadenylation during the mammalian oocyte-to-embryo transition (OET)3-6. However, the mechanism of re-polyadenylation during mammalian OET, including the sites to be re-polyadenylated and the enzymes involved, is still poorly understood. Here, by analyzing the PAIso-seq1 and PAIso-seq2 poly(A) inclusive transcriptome data during OET in mice, rats, pigs, and humans, we reveal conserved re-polyadenylation of mRNA degradation intermediates. These re-polyadenylated mRNA degradation intermediates account for over half of the polyadenylated mRNA during OET in all four species. We find that mRNA degradation intermediates for re-polyadenylation are generated through Btg4-mediated deadenylation in both mouse and human. Interestingly, the poly(A) tails on the re-polyadenylated mRNA degradation intermediates are of different lengths and contain different levels of non-A residues compared to regular polyadenylation sites, suggesting specific regulation and function of these poly(A) tails in mammalian OET. Together, our findings reveal the maternal mRNA degradation intermediates as substrates for conserved cytoplasmic dominant re-polyadenylation during mammalian OET, and uncover the mechanism of production of these mRNA degradation intermediates. These findings provide new insights into mRNA post-transcriptional regulation, and a new direction for the study of mammalian OET.

developmental biology↗