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Single-cell analysis of isoform switching and transposable element expression during preimplantation embryonic development [1]

['Chaoyang Wang', 'Gmu-Gibh Joint School Of Life Sciences', 'The Fifth Affiliated Hospital Of Guangzhou Medical University', 'Guangzhou Laboratory', 'Guangzhou Medical University', 'Guangzhou', 'The Bioland Laboratory', 'Guangzhou Regenerative Medicine', 'Health Guangdong Laboratory', 'State Key Laboratory Of Ophthalmology']

Date: 2024-02

Alternative splicing is an essential regulatory mechanism for development and pathogenesis. Through alternative splicing one gene can encode multiple isoforms and be translated into proteins with different functions. Therefore, this diversity is an important dimension to understand the molecular mechanism governing embryo development. Isoform expression in preimplantation embryos has been extensively investigated, leading to the discovery of new isoforms. However, the dynamics of isoform switching of different types of transcripts throughout the development remains unexplored. Here, using single-cell direct isoform sequencing in over 100 single blastomeres from the mouse oocyte to blastocyst stage, we quantified isoform expression and found that 3-prime partial transcripts lacking stop codons are highly accumulated in oocytes and zygotes. These transcripts are not transcription by-products and might play a role in maternal to zygote transition (MZT) process. Long-read sequencing also enabled us to determine the expression of transposable elements (TEs) at specific loci. In this way, we identified 3,894 TE loci that exhibited dynamic changes along the preimplantation development, likely regulating the expression of adjacent genes. Our work provides novel insights into the transcriptional regulation of early embryo development.

Funding: This work was supported by grants from the National Key Research and Development Program of China (2020YFA0112201 to XF), the National Natural Science Foundation of China (32071451 to XF), the Guangdong Provincial Pearl River Talents Program (2021QN02Y747 to XF), and the R&D Program of Guangzhou National Laboratory (SRPG21-001 to XF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: All single-cell isoform sequencing data generated in this study are available at NCBI Gene Expression Omnibus ( https://www.ncbi.nlm.nih.gov/geo/ ) under the accession number GSE250381. Code availability The HIT-scISOseq analysis pipeline and source code are available from https://github.com/shizhuoxing/scISA-Tools . Additionally, the source code utilized in this study has been published at: https://zenodo.org/records/10394889 .

In this study, we adapted the HIT-scISOseq method for low-throughput cell analysis and sequenced isoforms in single blastomeres of mouse preimplantation embryos [ 6 ]. We analyzed cell heterogeneity within the same embryos at the same stage, providing insights into the timing of cell fate diversification during preimplantation embryo development. Isoforms of each gene in every single cell were quantified, and different isoform types showed varied proportions across embryonic stages. Notably, a significant number of 3-prime partial transcripts (lacking stop codons and generating proteins lacking C-termini) were observed in oocytes and zygotes, but were quickly degraded at the early 2-cell (E2C) stage. Furthermore, locus-specific TEs were analyzed, revealing dynamic expression changes during embryonic development. These TEs showed high correlation with the expression of adjacent genes, indicating their potential importance in developmental events.

Transposable elements (TEs) account for approximately 46% and approximately 37.5% of the human and mouse genome, respectively [ 20 , 21 ], contributing to evolution and genetic regulation. They can be divided into 2 major classes based on the transposition mode: class I retrotransposon and class II DNA transposon [ 22 , 23 ]. Class I, which makes up about 95% of total TEs, includes long and short interspersed elements (LINEs and SINEs, respectively) and long terminal repeats (LTRs). TEs are known to play a crucial role during embryo development [ 24 ]. For instance, MERVL and MT2_mm (a truncated form of ERVL containing only the LTR domain) can serve as promoters for totipotent gene expression, and their expression has been considered an essential totipotent biomarker [ 18 , 25 , 26 ]. LINEs, particularly LINE1, have been reported to suppress the expression of totipotent genes such as Dux [ 27 , 28 ]. A previous study showed that the hominoid-specific transposon (SINE-VNTR-Alu) acts as an enhancer to promote the ZGA process [ 29 ]. However, due to their highly repetitive nature, it is challenging to determine the activity of TEs at the locus level with limited read length. Analyzing TE expression in specific loci is therefore important for gene transcriptional regulation.

Isoform switch plays an important role in cell fate determination. PBX1, for example, can be transcribed into 3 different isoforms, each with distinct functions. PBX1a maintains the pluripotency of mouse embryonic stem cells (mESCs), while PBX1b promotes differentiation [ 10 ]. Other genes such as Tcf3 and Sall4 have similar regulatory patterns in mESCs [ 11 , 12 ]. The molecular regulation of preimplantation embryo development has been the focus of many studies, particularly maternal to zygote transition (MZT), which is crucial for whole-body development [ 13 – 16 ]. Although hundreds of genes have been identified in zygotic genome activation (ZGA), the functional regulators remain largely unclear, including whether isoform switching participates in the process [ 17 – 19 ].

A gene can be transcribed into various isoforms, which are then translated into different proteins. Isoform compositions differ between cell types and states, making isoform switching a crucial factor in determining cell identity [ 1 , 2 ]. Third-generation sequencing-based single-cell RNA-sequencing methods like SCAN-seq, HIT-scISOseq, and MAS-ISO-seq have been developed to directly sequence gene isoforms [ 1 , 3 – 7 ]. SCAN-seq is known for its high gene detection sensitivity and ability to detect many novel transcripts in rare samples [ 4 ]. However, it fails to quantify the absolute abundance of genes and isoforms due to the high error rate of Nanopore sequencing [ 8 , 9 ]. On the other hand, HIT-scISOseq and MAS-ISO-seq use the PacBio HiFi sequencing platform to quantify isoform abundance in single cells with improved data throughput [ 6 , 7 ].

Results

Isoform diversity decreases along preimplantation embryo development To explore the connection between gene and isoform expression during mouse preimplantation development, we grouped genes into 6 categories based on the number of isoform types they expressed (S2A Fig). While most genes expressed only 1 type of isoform across different stages, more genes expressed multiple types of isoforms in the earlier stages. In mouse oocytes and zygotes, around 60% of genes expressed more than 1 type of isoform, and nearly 20% of genes were found with over 5 types of isoforms. In contrast, approximately 70% of genes in mESCs expressed only 1 type of isoform, and less than 5% of genes expressed more than 5 types of isoforms (S2A Fig). The same isoform expression characteristics were observed in SCAN-seq data (S2B Fig), indicating a diverse range of isoforms in early mouse embryos [4]. To rule out the possibility that this observation was caused by higher mRNA abundance in early embryos, especially in oocytes and zygotes, we performed oocyte splits. The results showed that the ratios of genes containing different numbers of isoform types were almost consistent among intact oocytes, 1/2 oocytes, and 1/4 oocytes (S2C Fig), suggesting that the detected isoform diversity was hardly affected by the amount of mRNAs. Additionally, the genes expressing more types of isoforms were detected with higher expression levels in both our data and SCAN-seq data (S2D and S2E Fig). To validate this hypothesis, we randomly selected 3 highly expressed genes (CPM > 100) and 3 lowly expressed genes (CPM < 10) in mESC to confirm their isoform diversity by reverse transcription and PCR (RT-PCR). Although there were more types of isoforms revealed by RT-PCR than the sequencing results, the highly expressed genes still showed higher isoform diversity (S2F Fig). We then assessed the isoform dominant level in each gene expressing multiple types of isoforms by calculating the ratio of the UMI number of the major isoform to the total UMI number of the corresponding gene. The major isoform ratios increased from early to late embryonic stages, especially after the ZGA process (S2G Fig). In comparison, the major isoforms accounted for 90% of most genes in mESCs, indicating a dominant isoform expression pattern and less isoform diversity in these cells.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002505

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