Generation of the floxed allele of the SIP1 (Smad-interacting protein 1) gene for Cre-mediated conditional knockout in the mouse

Abstract Background Our previous study have shown that the PSMD11 protein was an important survival factor for cancer cells except for its key role in regulation of assembly and activity of the 26S proteasome. To further investigate the role of PSMD11 in carcinogenesis, we constructed a conditional exon 5 floxed allele of PSMD11 (PSMD11flx) in mice. Results It was found that homozygous PSMD11 flx/flx mice showed normal and exhibited a normal life span and fertility, and showed roughly equivalent expression of PSMD11 in various tissues, suggesting that the floxed allele maintained the wild-type function. Cre recombinase could induce efficient knockout of the floxed PSMD11 allele both in vitro and in vivo. Mice with constitutive single allele deletion of PSMD11 derived from intercrossing between PSMD11flx/flx and CMV-Cre mice were all viable and fertile, and showed apparent growth retardation, suggesting that PSMD11 played a significant role in the development of mice pre- or postnatally. No whole-body PSMD11 deficient embryos (PSMD11−/−) were identified in E7.5–8.5 embryos in uteros, indicating that double allele knockout of PSMD11 leads to early embryonic lethality. To avoid embryonic lethality produced by whole-body PSMD11 deletion, we further developed conditional PSMD11 global knockout mice with genotype Flp;FSF-R26CAG − CreERT2/+; PSMD11flx/flx, and demonstrated that PSMD11 could be depleted in a temporal and tissue-specific manner. Meanwhile, it was found that depletion of PSMD11 could induce massive apoptosis in MEFs. Conclusions In summary, our data demonstrated that we have successfully generated a conditional knockout allele of PSMD11 in mice, and found that PSMD11 played a key role in early and postnatal development in mice, the PSMD11 flx/flx mice will be an invaluable tool to explore the functions of PSMD11 in development and diseases.

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Binding of LncRNA-DACH1 to dystrophin impairs the membrane trafficking of Nav1.5 protein and increases ventricular arrhythmia susceptibility

10.21203/rs.3.rs-156367/v1 ◽

2021 ◽

Author(s):

Zhenwei Pan ◽

Gen-Long Xue ◽

Yang Zhang ◽

Jiming Yang ◽

Ying Yang ◽

...

Keyword(s):

Ventricular Arrhythmia ◽

Membrane Trafficking ◽

Ex Vivo ◽

Conditional Knockout ◽

Human Homologue ◽

Interacting Protein ◽

Membrane Anchoring ◽

Ventricular Arrhythmogenesis

Abstract Dystrophin is a critical interacting protein of Nav1.5 that determines its membrane anchoring in cardiomyocytes. The study aims to explore whether lncRNA-DACH1(lncDACH1) can regulate the distribution of Nav1.5 by binding to dystrophin and participate in ventricular arrhythmogenesis. LncDACH1 was confirmed to bind to dystrophin. Cardiomyocyte-specific transgenic overexpression of lncDACH1(lncDACH1-TG) reduced the membrane distribution of dystrophin and Nav1.5 in cardiomyocytes. The opposite data were collected from lncDACH1 cardiomyocyte conditional knockout (lncDACH1-CKO) mice. Moreover, increased ventricular arrhythmia susceptibility was observed in lncDACH1-TG mice in vivo and ex vivo. The conservative fragment of lncDACH1 inhibited membrane distribution of dystrophin and Nav1.5 and promoted the inducibility of ventricular arrhythmia. Upregulation of dystrophin in lncDACH1-TG mice rescued the impaired membrane distribution of dystrophin and Nav1.5. The human homologue of lncDACH1 inhibited the membrane distribution of Nav1.5 in human iPS-differentiated cardiomyocytes. Collectively, lncDACH1 regulates Nav1.5 membrane distribution by binding to dystrophin and participates in ventricular arrhythmogenesis.

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A molecular brake that modulates spliceosome pausing at detained introns contributes to neurodegeneration

10.1101/2022.01.11.475943 ◽

2022 ◽

Author(s):

Yichang Jia

Keyword(s):

Rna Binding ◽

Rna Binding Protein ◽

Conditional Knockout ◽

Interacting Protein ◽

U2 Snrna ◽

Mrna Pool ◽

Splicing Efficiency ◽

Underlying Mechanisms ◽

Molecular Brake ◽

Polyadenylated Mrna

Emerging evidence suggests that intron-detaining transcripts (IDTs) are a nucleus-detained and polyadenylated mRNA pool for cell to quickly and effectively respond to environmental stimuli and stress. However, the underlying mechanisms of detained intron (DI) splicing are still largely unknown. Here, we suggest that post-transcriptional DI splicing is paused at Bact 29 state, an active spliceosome but not catalytically primed, which depends on SNIP1 (Smad Nuclear Interacting Protein 1) and RNPS1 (a serine-rich RNA binding protein) interaction. RNPS1 and Bact component preferentially dock at DIs and the RNPS1 docking is sufficient to trigger spliceosome pausing. Haploinsufficiency of Snip1 attenuates neurodegeneration and globally rescues IDT accumulation caused by a previously reported mutant U2 snRNA, a basal spliceosomal component. Snip1 conditional knockout in cerebellum decreases DI splicing efficiency and causes neurodegeneration. Therefore, we suggest that SNIP1 and RNPS1 form a molecular brake for the spliceosome pausing, and that its misregulation contributes to neurodegeneration.

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Early signalling events of autophagy

Essays in Biochemistry ◽

10.1042/bse0550001 ◽

2013 ◽

Vol 55 ◽

pp. 1-15 ◽

Cited By ~ 16

Author(s):

Laura E. Gallagher ◽

Edmond Y.W. Chan

Keyword(s):

Protein Kinase ◽

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Mammalian Cells ◽

Mammalian Target Of Rapamycin ◽

Interacting Protein ◽

The Core ◽

Autophagy Gene ◽

Autophagy Induction ◽

Cellular Pathways

Autophagy is a conserved cellular degradative process important for cellular homoeostasis and survival. An early committal step during the initiation of autophagy requires the actions of a protein kinase called ATG1 (autophagy gene 1). In mammalian cells, ATG1 is represented by ULK1 (uncoordinated-51-like kinase 1), which relies on its essential regulatory cofactors mATG13, FIP200 (focal adhesion kinase family-interacting protein 200 kDa) and ATG101. Much evidence indicates that mTORC1 [mechanistic (also known as mammalian) target of rapamycin complex 1] signals downstream to the ULK1 complex to negatively regulate autophagy. In this chapter, we discuss our understanding on how the mTORC1–ULK1 signalling axis drives the initial steps of autophagy induction. We conclude with a summary of our growing appreciation of the additional cellular pathways that interconnect with the core mTORC1–ULK1 signalling module.

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Functional amyloid: widespread in Nature, diverse in purpose

Essays in Biochemistry ◽

10.1042/bse0560207 ◽

2014 ◽

Vol 56 ◽

pp. 207-219 ◽

Cited By ~ 77

Author(s):

Chi L.L. Pham ◽

Ann H. Kwan ◽

Margaret Sunde

Keyword(s):

Spider Silk ◽

Epigenetic Inheritance ◽

Fibrillar Structure ◽

Cytoplasmic Polyadenylation ◽

Functional Amyloid ◽

Interacting Protein ◽

Diverse Range ◽

Element Binding Protein ◽

Functional Amyloids ◽

Micro Organisms

Amyloids are insoluble fibrillar protein deposits with an underlying cross-β structure initially discovered in the context of human diseases. However, it is now clear that the same fibrillar structure is used by many organisms, from bacteria to humans, in order to achieve a diverse range of biological functions. These functions include structure and protection (e.g. curli and chorion proteins, and insect and spider silk proteins), aiding interface transitions and cell–cell recognition (e.g. chaplins, rodlins and hydrophobins), protein control and storage (e.g. Microcin E492, modulins and PMEL), and epigenetic inheritance and memory [e.g. Sup35, Ure2p, HET-s and CPEB (cytoplasmic polyadenylation element-binding protein)]. As more examples of functional amyloid come to light, the list of roles associated with functional amyloids has continued to expand. More recently, amyloids have also been implicated in signal transduction [e.g. RIP1/RIP3 (receptor-interacting protein)] and perhaps in host defence [e.g. aDrs (anionic dermaseptin) peptide]. The present chapter discusses in detail functional amyloids that are used in Nature by micro-organisms, non-mammalian animals and mammals, including the biological roles that they play, their molecular composition and how they assemble, as well as the coping strategies that organisms have evolved to avoid the potential toxicity of functional amyloid.

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