in-vitro-biochemical-assays-using-biotin-labels-to-study-protein

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...

Nanjing Medical University

Transcription factors of the Sox protein family contain a DNA-binding HMG box and are key regulators of progenitor cell fate. Here, we report that expression of Sox30 is restricted to meiotic spermatocytes and postmeiotic haploids.  mutant males are sterile owing to spermiogenic arrest at the early round spermatid stage. Specifically, in the absence of Sox30, proacrosomic vesicles fail to form a single acrosomal organelle, and spermatids arrest at step 2-3. Although most  mutant spermatocytes progress through meiosis, accumulation of diplotene spermatocytes indicates a delayed or impaired transition from meiotic to postmeiotic stages. Transcriptome analysis of isolated stage-specific spermatogenic cells reveals that Sox30 controls a core postmeiotic gene expression program that initiates as early as the late meiotic cell stage. ChIP-seq analysis shows that Sox30 binds to specific DNA sequences in mouse testes, and its genomic occupancy correlates positively with expression of many postmeiotic genes including , ,  and  These results define Sox30 as a crucial transcription factor that controls the transition from a late meiotic to a postmeiotic gene expression program and subsequent round spermatid development.

Sox30 initiates transcription of haploid genes during late meiosis and spermiogenesis in mouse testes.

Mechanistic target of rapamycin (mTOR) controls cell growth and metabolism in response to environmental and metabolic signals. Rapamycin robustly extends the lifespan in mammals and has clinical relevance in organ transplantation and cancer therapy but side effects include male infertility. Here, we report that chronic rapamycin treatment causes spermatogenic arrest in adult male mice due to defects in sex body formation and meiotic sex chromosome inactivation (MSCI). Many sex chromosome-linked genes were up-regulated in isolated pachytene spermatocytes from rapamycin-treated mice. RNA-Seq analysis also identified mRNAs encoding the core piRNA pathway components were decreased. Furthermore, rapamycin treatment was associated with a drastic reduction in pachytene piRNA populations. The inhibitory effects of rapamycin on spermatogenesis were partially reversible, with restoration of testis mass and sperm motility within 2 months of treatment cessation. Collectively, we have defined an essential role of mTOR in MSCI and identified a novel function as a regulator of small RNA homeostasis in male germ cells.

Rapamycin-mediated mTOR inhibition impairs silencing of sex chromosomes and the pachytene piRNA pathway in the mouse testis.

RNA regulation by RNA-binding proteins (RBPs) involve extremely complicated mechanisms. MOV10 and MOV10L1 are two homologous RNA helicases implicated in distinct intracellular pathways. MOV10L1 participates specifically in Piwi-interacting RNA (piRNA) biogenesis and protects mouse male fertility. In contrast, the functional complexity of MOV10 remains incompletely understood, and its role in the mammalian germline is unknown. Here, we report a study of the biological and molecular functions of the RNA helicase MOV10 in mammalian male germ cells.

Biological and RNA regulatory function of MOV10 in mammalian germ cells.

MOV10L1 and its paralog MOV10 are evolutionally conserved RNA helicases involved in distinct RNA regulatory pathways. The testis-specific MOV10L1 is essential for spermatogenesis and PIWI-interacting RNAs biogenesis, whereas MOV10 is ubiquitous and multifunctional. Although both proteins have been implied to correlate with RNA G-quadruplex (RG4) in vivo, their capabilities in binding and resolving RG4 and their respective biological significance remain unclear. Herein, we comprehensively characterize and compare the activities of these two helicases on various nucleic acid substrates in vitro, with a focus on RG4 structure. We find that both MOV10L1 and MOV10 are able to resolve RG4, with MOV10L1 being more efficient in that. In contrast to MOV10, MOV10L1 prefers to bind to a junction between single-stranded RNA and RG4, which is mediated by both its N and C termini. Furthermore, we show that RG4 unwinding by MOV10L1 facilitates the cleavage of this specific RNA structure by an endonuclease.

Article	Year
Sox30 initiates transcription of haploid genes during late meiosis and spermiogenesis in mouse testes. Development (Cambridge, England)\| PubMed ID: 29866902	2018
Rapamycin-mediated mTOR inhibition impairs silencing of sex chromosomes and the pachytene piRNA pathway in the mouse testis. Aging\| PubMed ID: 30636722	2019
Biological and RNA regulatory function of MOV10 in mammalian germ cells. BMC biology\| PubMed ID: 31088452	2019
MOV10L1 Binds RNA G-Quadruplex in a Structure-Specific Manner and Resolves It More Efficiently Than MOV10. iScience\| PubMed ID: 31252377	2019

Jie Xie

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