The Tug1 lncRNA locus is essential for male fertility

Abstract

Background: Several long noncoding RNAs (lncRNAs) have been shown to function as components of molecular machines that play fundamental roles in biology. While the number of annotated lncRNAs in mammalian genomes has greatly expanded, studying lncRNA function has been a challenge due to their diverse biological roles and because lncRNA loci can contain multiple molecular modes that may exert function. Results: We previously generated and characterized a cohort of 20 lncRNA loci knockout mice. Here, we extend this initial study and provide a more detailed analysis of the highly conserved lncRNA locus, taurine-upregulated gene 1 (Tug1). We report that Tug1-knockout male mice are sterile with underlying defects including a low number of sperm and abnormal sperm morphology. Because lncRNA loci can contain multiple modes of action, we wanted to determine which, if any, potential elements contained in the Tug1 genomic region have any activity. Using engineered mouse models and cell-based assays, we provide evidence that the Tug1 locus harbors two distinct noncoding regulatory activities, as a cis-DNA repressor that regulates neighboring genes and as a lncRNA that can regulate genes by a trans-based function. We also show that Tug1 contains an evolutionary conserved open reading frame that when overexpressed produces a stable protein which impacts mitochondrial membrane potential, suggesting a potential third coding function. Conclusions: Our results reveal an essential role for the Tug1 locus in male fertility and uncover evidence for distinct molecular modes in the Tug1 locus, thus highlighting the complexity present at lncRNA loci.

One-shot analysis of translated mammalian lncRNAs with AHARIBO

“…To further validate AHARIBO-rC, we took advantage of a micropeptide (176 aa) originating from an open reading frame of the TUG1 lncRNA, called TUG1-BOAT ( Lewandowski et al, 2020 ). The wild-type (WT) ORF has a non-canonical start codon and a methionine 75 nt upstream of the stop codon.…”

“…We ectopically express the putative protein produced by the open reading frame of TUG1, called TUG1-BOAT (Tug1-Bifunctional ORF and Transcript), in HeLa cells. Briefly, construct generation and transfection was performed as in Lewandowski et al, 2020 with some minor changes to adapt the experimental setup to the AHARIBO method. We synthesized three different constructs for human Tug1 ORF1 (Thermo Scientific):…”

“…The first (called WT TUG1-BOAT ) is the one reported in Lewandowski et al, 2020 . It has a non-canonical start codon and a methionine at 75 nt (25 aa) upstream of the stop codon.…”

A Testis-Specific Long Noncoding RNA, Start, Is a Regulator of Steroidogenesis in Mouse Leydig Cells

“…While the hormonal endocrine system that is responsible for steroidogenesis in the HPG axis has been relatively well characterized ( 20 , 21 ), the molecular mechanism underlying the precise control of spermatogenetic genes is unclear. The findings in previous studies suggest some pivotal roles of a novel regulator in spermatogenesis and androgen production in the testis ( 22 , 23 ).…”

IP3R Channels in Male Reproduction

“…The lncRNA eosinophil granule ontogeny transcript (EGOT) upregulated IP3R1 levels via formation of a pre-ITPR1/EGOT dsRNA that induced accumulation and enhanced alternative splicing of IP3R1 primary RNA [75]. Furthermore, a large number of miRNAs and lncRNAs are exclusively or highly expressed in the testes and related to spermatogenesis and even male infertility [76][77][78][79][80]. However, to the best of our knowledge, whether these lncRNAs take part in IP3Rs-mediated Ca 2+ signal transduction in male reproduction remains undetermined.…”