4.5SH RNA counteracts deleterious exonization of SINE B1 in mice.

4.5SH RNA is a highly abundant, small rodent-specific noncoding RNA that localizes to nuclear speckles enriched in pre-mRNA-splicing regulators. To investigate the physiological functions of 4.5SH RNA, we have created mutant mice that lack the expression of 4.5SH RNA. The mutant mice exhibited embryonic lethality, suggesting that 4.5SH RNA is an essential species-specific noncoding RNA in mice. RNA-sequencing analyses revealed that 4.5SH RNA protects the transcriptome from abnormal exonizations of the antisense insertions of the retrotransposon SINE B1 (asB1), which would otherwise introduce deleterious premature stop codons or frameshift mutations. Mechanistically, 4.5SH RNA base pairs with complementary asB1-containing exons via the target recognition region and recruits effector proteins including Hnrnpm via its 5' stem loop region. The modular organization of 4.5SH RNA allows us to engineer a programmable splicing regulator to induce the skipping of target exons of interest. Our results also suggest the general existence of splicing regulatory noncoding RNAs.

Genomic Profiling by ALaP-Seq Reveals Transcriptional Regulation by PML Bodies through DNMT3A Exclusion.

The promyelocytic leukemia (PML) body is a phase-separated nuclear structure physically associated with chromatin, implying its crucial roles in genome functions. However, its role in transcriptional regulation is largely unknown. We developed APEX-mediated chromatin labeling and purification (ALaP) to identify the genomic regions proximal to PML bodies. We found that PML bodies associate with active regulatory regions across the genome and with ∼300 kb of the short arm of the Y chromosome (YS300) in mouse embryonic stem cells. The PML body association with YS300 is essential for the transcriptional activity of the neighboring Y-linked clustered genes. Mechanistically, PML bodies provide specific nuclear spaces that the de novo DNA methyltransferase DNMT3A cannot access, resulting in the steady maintenance of a hypo-methylated state at Y-linked gene promoters. Our study underscores a new mechanism for gene regulation in the 3D nuclear space and provides insights into the functional properties of nuclear structures for genome function.

The essential role of architectural noncoding RNA Neat1 in cold-induced beige adipocyte differentiation in mice.

Neat1 is an architectural RNA that provides the structural basis for nuclear bodies known as paraspeckles. Although the assembly processes by which Neat1 organizes paraspeckle components are well-documented, the physiological functions of Neat1 are not yet fully understood. This is partly because Neat1 knockout (KO) mice, lacking paraspeckles, do not exhibit overt phenotypes under normal laboratory conditions. During our search for conditions that elicit clear phenotypes in Neat1 KO mice, we discovered that the differentiation of beige adipocytes-inducible thermogenic cells that emerge upon cold exposure-is severely impaired in these mutant mice. Neat1_2, the architectural isoform of Neat1, is transiently upregulated during the early stages of beige adipocyte differentiation, coinciding with increased paraspeckle formation. Genes with altered expression during beige adipocyte differentiation typically cluster at specific chromosomal locations, some of which move closer to paraspeckles upon cold exposure. These observations suggest that paraspeckles might coordinate the regulation of these gene clusters by controlling the activity of certain transcriptional condensates that coregulate multiple genes. We propose that our findings highlight a potential role for Neat1 and paraspeckles in modulating chromosomal organization and gene expression, potentially crucial processes for the differentiation of beige adipocytes.

RNA: The Unsuspected Conductor in the Orchestra of Macromolecular Crowding.

This comprehensive Review delves into the chemical principles governing RNA-mediated crowding events, commonly referred to as granules or biological condensates. We explore the pivotal role played by RNA sequence, structure, and chemical modifications in these processes, uncovering their correlation with crowding phenomena under physiological conditions. Additionally, we investigate instances where crowding deviates from its intended function, leading to pathological consequences. By deepening our understanding of the delicate balance that governs molecular crowding driven by RNA and its implications for cellular homeostasis, we aim to shed light on this intriguing area of research. Our exploration extends to the methodologies employed to decipher the composition and structural intricacies of RNA granules, offering a comprehensive overview of the techniques used to characterize them, including relevant computational approaches. Through two detailed examples highlighting the significance of noncoding RNAs, NEAT1 and XIST, in the formation of phase-separated assemblies and their influence on the cellular landscape, we emphasize their crucial role in cellular organization and function. By elucidating the chemical underpinnings of RNA-mediated molecular crowding, investigating the role of modifications, structures, and composition of RNA granules, and exploring both physiological and aberrant phase separation phenomena, this Review provides a multifaceted understanding of the intriguing world of RNA-mediated biological condensates.

Species-specific formation of paraspeckles in intestinal epithelium revealed by characterization of NEAT1 in naked mole-rat.

Paraspeckles are mammalian-specific nuclear bodies built on the long noncoding RNA NEAT1_2 The molecular mechanisms of paraspeckle formation have been mainly studied using human or mouse cells, and it is not known if the same molecular components are involved in the formation of paraspeckles in other mammalian species. We thus investigated the expression pattern of NEAT1_2 in naked mole-rats (nNEAT1_2), which exhibit extreme longevity and lower susceptibility to cancer. In the intestine, nNEAT1_2 is widely expressed along the entire intestinal epithelium, which is different from the expression of mNeat1_2 that is restricted to the cells of the distal tip in mice. Notably, the expression of FUS, a FET family RNA binding protein, essential for the formation of paraspeckles both in humans and mice, was absent in the distal part of the intestinal epithelium in naked mole-rats. Instead, mRNAs of other FET family proteins EWSR1 and TAF15 were expressed in the distal region. Exogenous expression of these proteins in Fus-deficient murine embryonic fibroblast cells rescued the formation of paraspeckles. These observations suggest that nNEAT1_2 recruits a different set of RNA binding proteins in a cell type-specific manner during the formation of paraspeckles in different organisms.

Evidence for a functional role of Start, a long noncoding RNA, in mouse spermatocytes.

A mouse testis-specific long noncoding RNA (lncRNA), Start, is localized in the cytosol of Leydig cells and in the nucleus of pachytene spermatocytes. We previously showed that Start regulates steroidogenesis through controlling the expression of Star and Hsd3b1 genes in Leydig cells, but its function in germ cells was not known. Here we verified that a spermatocyte-specific protease gene, Prss43/Tessp-3, was downregulated in Start-knockout testes. To investigate the transcriptional regulatory activity of Start in spermatocytes, we first performed a series of reporter gene assays using a thymidine kinase promoter in spermatocyte-derived GC-2spd(ts) cells. A 5.4-kb genome sequence encompassing Start exhibited enhancer activity for this promoter, and the activity was decreased by knockdown of Start. Deletion of the Start promoter and replacement of the Start sequence abolished the enhancer activity and, consistently, the activity was detected in further experiments only when Start was actively transcribed. We then examined whether the Prss43/Tessp-3 gene could be a target of Start. A reporter gene assay demonstrated that the 5.4-kb sequence exhibited enhancer activity for a Prss43/Tessp-3 promoter in GC-2spd(ts) cells and that the activity was significantly decreased by knockdown of Start. These results suggest that Start functions in transcriptional activation of the Prss43/Tessp-3 gene in spermatocytes. Given that Start is presumed to regulate steroidogenic genes at the posttranscriptional level in Leydig cells, the function in spermatocytes is a novel role of Start. These findings provide an insight into multifunctionality of lncRNAs in the testis.

A Long Noncoding RNA, lncRNA-Amhr2, Plays a Role in Amhr2 Gene Activation in Mouse Ovarian Granulosa Cells.

Anti-Müllerian hormone (AMH) is critical to the regression of Müllerian ducts during mammalian male differentiation and targets ovarian granulosa cells and testicular Sertoli and Leydig cells of adults. Specific effects of AMH are exerted via its receptor, AMH type II receptor (Amhr2), but the mechanism by which the Amhr2 gene is specifically activated is not fully understood. To see whether a proximal promoter was sufficient for Amhr2 gene activation, we generated transgenic mice that bore the enhanced green fluorescent protein (EGFP) gene driven by a 500-bp mouse Amhr2 gene promoter. None of the established 10 lines, however, showed appropriate EGFP expression, indicating that the 500-bp promoter was insufficient for Amhr2 gene activation. As a regulatory element, we found a long noncoding RNA, lncRNA-Amhr2, transcribed from upstream of the Amhr2 gene in ovarian granulosa cells and testicular Sertoli cells. In primary granulosa cells, knockdown of lncRNA-Amhr2 resulted in a decrease of Amhr2 messnger RNA level, and a transient reporter gene assay showed that lncRNA-Amhr2 activation increased Amhr2 promoter activity. The activity was correlated with lncRNA-Amhr2 transcription in stably transfected OV3121 cells derived from mouse granulosa cells. Moreover, by the Tet-on system, the induction of lncRNA-Amhr2 transcription dramatically increased Amhr2 promoter activity in OV3121 cells. These results indicate that lncRNA-Amhr2 plays a role in Amhr2 gene activation in ovarian granulosa cells by enhancing promoter activity, providing insight into Amhr2 gene regulation underlying the AMH signaling in the female reproductive system.

A Testis-Specific Long Non-Coding RNA, lncRNA-Tcam1, Regulates Immune-Related Genes in Mouse Male Germ Cells.

Spermatogenesis is precisely controlled by hormones from the hypothalamus-pituitary-gonadal axis and testis-specific genes, but the regulatory mechanism is not fully understood. Recently, a large number of long non-coding RNAs (lncRNAs) are found to be transcribed at each stage of meiosis of male germ cells, and their functions in spermatogenesis have yet to be fully investigated. lncRNA-testicular cell adhesion molecule 1 (lncRNA-Tcam1) is a nuclear lncRNA which is specifically expressed in mouse male germ cells and presumed to play a role in gene regulation during meiosis. Here, we present the identification of potential target genes of lncRNA-Tcam1 using spermatocyte-derived GC-2spd(ts) cells. Initially, 55 target gene candidates were detected by RNA-sequencing of two GC-2spd(ts) cell clones that were stably transfected with transgenes to express lncRNA-Tcam1 at different levels. Expression of 21 genes of the candidates was found to be correlated with lncRNA-Tcam1 at 7-14 postnatal days, when lncRNA-Tcam1 expression was elevated. Subsequently, we examined expression levels of the 21 genes in other two GC-2spd(ts) clones, and 11 genes exhibited the correlation with lncRNA-Tcam1. Induction of lncRNA-Tcam1 transcription using the Tet-off system verified that six genes, Trim30a, Ifit3, Tgtp2, Ifi47, Oas1g, and Gbp3, were upregulated in GC-2spd(ts) cells, indicating that lncRNA-Tcam1 is responsible for the regulation of gene expression of the six genes. In addition, five of the six genes, namely, Ifit3, Tgtp2, Ifi47, Oas1g, and Gbp3, are immune response genes, and Trim30a is a negative regulator of immune response. Altogether, the present study suggests that lncRNA-Tcam1 is responsible for gene regulation for the immune response during spermatogenesis.

Characterization of the human TCAM1P pseudogene and its activation by a potential dual promoter-enhancer: comparison with a protein-coding mouse orthologue.

TCAM1P is a unitary pseudogene, which was disabled since the human-mouse divergence. Here we found that TCAM1P was specifically expressed in the human testis, with different cell type-specificity from mouse Tcam1, and characterized its transcripts. At the mouse locus, a multifunctional dual promoter-enhancer (DPE) controls the expression of Tcam1 and Smarcd2 genes. The corresponding human sequence was found to potentially function as a DPE, although the molecular mechanism was different from mouse. Interestingly, the change in DPE activity occurred before pseudogenization of TCAM1P. These data suggest the presence of a DPE in the human genome for the first time, and provide an important model of evolutionary changes in the regulatory mechanism of a pseudogene.

A long non-coding RNA transcribed from conserved non-coding sequences contributes to the mouse prolyl oligopeptidase gene activation.

Prolyl oligopeptidase (POP) is a multifunctional protease which is involved in many physiological events, but its gene regulatory mechanism is poorly understood. To identify novel regulatory elements of the POP gene, we compared the genomic sequences at the mouse and human POP loci and found six conserved non-coding sequences (CNSs) at adjacent intergenic regions. From these CNSs, four long non-coding RNAs (lncRNAs) were transcribed and the expression pattern of one (lncPrep+96kb) was correlated with that of POP. lncPrep+96kb was transcribed as two forms due to the different transcriptional start sites and was localized at the nucleus and cytoplasm, although more was present at the nucleus. When we knocked down lncPrep+96kb in the primary ovarian granulosa cell and a hepatic cell line, the POP expression was decreased in both cells. In contrast, overexpression of lncPrep+96kb increased the POP expression only in the granulosa cell. Because lncPrep+96kb was upregulated with the same timing as POP in the hormone-treated ovary, this lncRNA could play a role in the POP gene activation in the granulosa cell. Moreover, a downstream region of the human POP gene was also transcribed. We propose a novel mechanism for the POP gene activation.

A conserved noncoding sequence can function as a spermatocyte-specific enhancer and a bidirectional promoter for a ubiquitously expressed gene and a testis-specific long noncoding RNA.

Tissue-specific gene expression is tightly regulated by various elements such as promoters, enhancers, and long noncoding RNAs (lncRNAs). In the present study, we identified a conserved noncoding sequence (CNS1) as a novel enhancer for the spermatocyte-specific mouse testicular cell adhesion molecule 1 (Tcam1) gene. CNS1 was located 3.4kb upstream of the Tcam1 gene and associated with histone H3K4 mono-methylation in testicular germ cells. By the in vitro reporter gene assay, CNS1 could enhance Tcam1 promoter activity only in GC-2spd(ts) cells, which were derived from mouse spermatocytes. When we integrated the 6.9-kb 5'-flanking sequence of Tcam1 with or without a deletion of CNS1 linked to the enhanced green fluorescent protein gene into the chromatin of GC-2spd(ts) cells, CNS1 significantly enhanced Tcam1 promoter activity. These results indicate that CNS1 could function as a spermatocyte-specific enhancer. Interestingly, CNS1 also showed high bidirectional promoter activity in the reporter assay, and consistent with this, the Smarcd2 gene and lncRNA, designated lncRNA-Tcam1, were transcribed from adjacent regions of CNS1. While Smarcd2 was ubiquitously expressed, lncRNA-Tcam1 expression was restricted to testicular germ cells, although this lncRNA did not participate in Tcam1 activation. Ubiquitous Smarcd2 expression was correlated to CpG hypo-methylation of CNS1 and partially controlled by Sp1. However, for lncRNA-Tcam1 transcription, the strong association with histone acetylation and histone H3K4 tri-methylation also appeared to be required. The present data suggest that CNS1 is a spermatocyte-specific enhancer for the Tcam1 gene and a bidirectional promoter of Smarcd2 and lncRNA-Tcam1.