Transcriptomic analysis reveals differential gene expression, alternative splicing, and novel exons during mouse trophoblast stem cell differentiation

Ullah, Rahim; Naz, Arab; Akram, Hafiza Sara; Ullah, Zakir; Tariq, Muhammad; Mithani, Aziz; Faisal, Amir

doi:10.1186/s13287-020-01848-8

Cited by 16 publications

(10 citation statements)

References 91 publications

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“…Together, this evidence suggests that these distinct mechanisms of transcriptome regulation (transcription and splicing) are intricately related. While it is believed that this intricate relationship is regulated by tissue-specific regulatory factors, DGE and DAS have either been investigated together in a single biological system [ 89 , 90 ] or separately across multiple biological systems [ 91 , 92 ]. Therefore, there have been no comprehensive investigations of DGE and DAS together across multiple biological systems until now.…”

Section: Discussionmentioning

confidence: 99%

Alternative splicing diversifies the skeletal muscle transcriptome during prolonged spaceflight

Henrich

Wang

et al. 2022

Skeletal Muscle

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Background As the interest in manned spaceflight increases, so does the requirement to understand the transcriptomic mechanisms that underlay the detrimental physiological adaptations of skeletal muscle to microgravity. While microgravity-induced differential gene expression (DGE) has been extensively investigated, the contribution of differential alternative splicing (DAS) to the plasticity and functional status of the skeletal muscle transcriptome has not been studied in an animal model. Therefore, by evaluating both DGE and DAS across spaceflight, we set out to provide the first comprehensive characterization of the transcriptomic landscape of skeletal muscle during exposure to microgravity. Methods RNA-sequencing, immunohistochemistry, and morphological analyses were conducted utilizing total RNA and tissue sections isolated from the gastrocnemius and quadriceps muscles of 30-week-old female BALB/c mice exposed to microgravity or ground control conditions for 9 weeks. Results In response to microgravity, the skeletal muscle transcriptome was remodeled via both DGE and DAS. Importantly, while DGE showed variable gene network enrichment, DAS was enriched in structural and functional gene networks of skeletal muscle, resulting in the expression of alternatively spliced transcript isoforms that have been associated with the physiological changes to skeletal muscle in microgravity, including muscle atrophy and altered fiber type function. Finally, RNA-binding proteins, which are required for regulation of pre-mRNA splicing, were themselves differentially spliced but not differentially expressed, an upstream event that is speculated to account for the downstream splicing changes identified in target skeletal muscle genes. Conclusions Our work serves as the first investigation of coordinate changes in DGE and DAS in large limb muscles across spaceflight. It opens up a new opportunity to understand (i) the molecular mechanisms by which splice variants of skeletal muscle genes regulate the physiological adaptations of skeletal muscle to microgravity and (ii) how small molecule splicing regulator therapies might thwart muscle atrophy and alterations to fiber type function during prolonged spaceflight.

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Section: Discussionmentioning

confidence: 99%

Alternative splicing diversifies the skeletal muscle transcriptome during prolonged spaceflight

Henrich

Wang

et al. 2022

Skeletal Muscle

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“…PE is associated with profound cellular dysfunctions including reduced differentiation and fusion ability [62], and p21 is of crucial importance for differentiation [24]. Indeed, p21 protein levels increased during spontaneous differentiation and fusion of term CTBs [63] as well as CDKN1A during differentiation of mouse trophoblast stem cells and fusion of BeWo cells upon forskolin treatment [64], whereas p53 levels were reduced during BeWo cell differentiation [65]. We report here that p21 levels were significantly decreased in fCTBs, a special portion of CTBs ongoing to fuse to the STB, in early-onset PE samples, suggestive of its involvement in CTB differentiation and fusion.…”

Section: Discussionmentioning

confidence: 99%

Functional Analysis of p21Cip1/CDKN1A and Its Family Members in Trophoblastic Cells of the Placenta and Its Roles in Preeclampsia

Kreis

Friemel

Jennewein

et al. 2021

Cells

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Preeclampsia (PE), a gestational hypertensive disease originating from the placenta, is characterized by an imbalance of various cellular processes. The cell cycle regulator p21Cip1/CDKN1A (p21) and its family members p27 and p57 regulate signaling pathways fundamental to placental development. The aim of the present study was to enlighten the individual roles of these cell cycle regulators in placental development and their molecular involvement in the pathogenesis of PE. The expression and localization of p21, phospho-p21 (Thr-145), p27, and p57 was immunohistochemically analyzed in placental tissues from patients with early-onset PE, early-onset PE complicated by the HELLP (hemolysis, elevated liver enzymes and low platelet count) syndrome as well as late-onset PE compared to their corresponding control tissues from well-matched women undergoing caesarean sections. The gene level was evaluated using real-time quantitative PCR. We demonstrate that the delivery mode strongly influenced placental gene expression, especially for CDKN1A (p21) and CDKN1B (p27), which were significantly upregulated in response to labor. Cell cycle regulators were highly expressed in first trimester placentas and impacted by hypoxic conditions. In support of these observations, p21 protein was abundant in trophoblast organoids and hypoxia reduced its gene expression. Microarray analysis of the trophoblastic BeWo cell line depleted of p21 revealed various interesting candidate genes and signaling pathways for the fusion process. The level of p21 was reduced in fusing cytotrophoblasts in early-onset PE placentas and depletion of p21 led to reduced expression of fusion-related genes such as syncytin-2 and human chorionic gonadotropin (β-hCG), which adversely affected the fusion capability of trophoblastic cells. These data highlight that cell cycle regulators are important for the development of the placenta. Interfering with p21 influences multiple pathways related to the pathogenesis of PE.

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“…While it is believed that this intricate relationship is regulated by tissue-specific regulatory factors, DGE and AS have either been investigated together in a single biological system (cell type, tissue type, disease state, etc.) (Ullah et al, 2020; Brinegar et al, 2017) or separately across multiple biological systems (Shen-Orr et al, 2010; Yeo et al, 2004), without any comprehensive investigations of DGE and AS together across multiple biological systems. As such, the experiments here, documenting an equal but opposite propensity for the quadriceps and gastrocnemius to adapt their transcriptomes to microgravity via DGE or AS, respectively (see Figure 2A, 2B), provides the first evidence of a possible reciprocal, muscle-type-specific relationship between DGE and AS, such that each muscle preferentially employs one mechanism of transcriptome regulation at the other mechanism’s expense.…”

Section: Discussionmentioning

confidence: 99%

Alternative splicing regulates the physiological adaptation of the mouse hind limb postural and phasic muscles to microgravity

Henrich¹,

P²,

Js³

et al. 2021

Preprint

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Muscle atrophy and fiber type alterations are well-characterized physiological adaptations to microgravity with both understood to be primarily regulated by differential gene expression (DGE). While microgravity-induced DGE has been extensively investigated, adaptations to microgravity due to alternative splicing (AS) have not been studied in a mammalian model. We sought to comprehensively elucidate the transcriptomic underpinnings of microgravity-induced muscle phenotypes in mice by evaluating both DGE and changes in AS due to extended spaceflight. Tissue sections and total RNA were isolated from the gastrocnemius and quadriceps, postural and phasic muscles of the hind limb, respectively, of 32-week-old female BALB/c mice exposed to microgravity or ground control conditions for nine weeks. Immunohistochemistry disclosed muscle type-specific physiological adaptations to microgravity that included i) a pronounced reduction in muscle fiber cross-sectional area in both muscles and ii) a prominent slow-to-fast fiber type transition in the gastrocnemius. RNA sequencing revealed that DGE and AS varied across postural and phasic muscle types with preferential employment of DGE in the gastrocnemius and AS in the quadriceps. Gene ontology analysis indicated that DGE and AS regulate distinct molecular processes. Various non-differentially expressed transcripts encoding musculoskeletal proteins (Tnnt3, Tnnt1, Neb, Ryr1, and Ttn) and muscle-specific RNA binding splicing regulators (Mbnl1 and Rbfox1) were found to have significant changes in AS that altered critical functional domains of their protein products. In striking contrast, microgravity-induced differentially expressed genes were associated with lipid metabolism and mitochondrial function. Our work serves as the first comprehensive investigation of coordinate changes in DGE and AS in large limb muscles across spaceflight. We propose that substantial remodeling of pre-mRNA by AS is a major component of transcriptomic adaptation of skeletal muscle to microgravity. The alternatively spliced genes identified here could be targeted by small molecule splicing regulator therapies to address microgravity-induced changes in muscle during spaceflight.

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Transcriptomic analysis reveals differential gene expression, alternative splicing, and novel exons during mouse trophoblast stem cell differentiation

Cited by 16 publications

References 91 publications

Alternative splicing diversifies the skeletal muscle transcriptome during prolonged spaceflight

Alternative splicing diversifies the skeletal muscle transcriptome during prolonged spaceflight

Functional Analysis of p21Cip1/CDKN1A and Its Family Members in Trophoblastic Cells of the Placenta and Its Roles in Preeclampsia

Alternative splicing regulates the physiological adaptation of the mouse hind limb postural and phasic muscles to microgravity

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