Takeshi Chujo

The TDRD9-MIWI2 Complex Is Essential for piRNA-Mediated Retrotransposon Silencing in the Mouse Male Germline

Host-defense mechanisms against transposable elements are critical to protect the genome information. Here we show that tudor-domain containing 9 (Tdrd9) is essential for silencing Line-1 retrotransposon in the mouse male germline. Tdrd9 encodes an ATPase/DExH-type helicase, and its mutation causes male sterility showing meiotic failure. In Tdrd9 mutants, Line-1 was highly activated and piwi-interacting small RNAs (piRNAs) corresponding to Line-1 were increased, suggesting that feedforward amplification operates in the mutant. In fetal testes, Tdrd9 mutation causes Line-1 desilencing and an aberrant piRNA profile in prospermatogonia, followed by cognate DNA demethylation. TDRD9 complexes with MIWI2 with distinct compartmentalization in processing bodies, and this TDRD9-MIWI2 localization is regulated by MILI and TDRD1 residing at intermitochondrial cement. Our results identify TDRD9 as a functional partner of MIWI2 and indicate that the tudor-piwi association is a conserved feature, while two separate axes, TDRD9-MIWI2 and TDRD1-MILI, cooperate nonredundantly in the piwi-small RNA pathway in the mouse male germline.

Architectural RNAs (arcRNAs): A class of long noncoding RNAs that function as the scaffold of nuclear bodies ☆

Mammalian transcriptome analyses elucidated the presence of thousands of unannotated long noncoding RNAs (lncRNAs) with distinct transcriptional units. Molecular characterization and functional classification of these lncRNAs are important challenges in the next decade. A subset of these lncRNAs is the core of nuclear bodies, which are the sites of the biogenesis, maturation, storage, and sequestration of specific RNAs, proteins, and ribonucleoprotein complexes. Here, we define a class of lncRNAs termed architectural RNAs (arcRNAs) that function as the essential scaffold or platform of nuclear bodies. Presently, five lncRNAs from mammals, insects, and yeast are classified as arcRNAs. These arcRNAs are temporarily upregulated upon specific cellular stresses, in developmental stages, or in various disease conditions, and sequestrate specific regulatory proteins, thereby changing gene expression patterns. In this review, we introduce common aspects of these arcRNAs and discuss why RNA is used as the architectural component of nuclear bodies. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa.

Structural, super-resolution microscopy analysis of paraspeckle nuclear body organization

Paraspeckles are nuclear bodies built on the long noncoding RNA Neat1. Using structural illumination microscopy, West et al. analyze the organization of paraspeckles at the submicron scale and show that paraspeckle proteins are arranged around bundles of Neat1, forming core-shell spheroidal structures dependent on the RNA binding protein Fus.

Trmt61B is a methyltransferase responsible for 1-methyladenosine at position 58 of human mitochondrial tRNAs.

In human mitochondria, 1-methyladenosine (m¹A) occurs at position 58 of tRNA(Leu(UUR)). In addition, partial m¹A58 modifications have been found in human mitochondrial tRNA(Lys) and tRNA(Ser(UCN)). We identified human Trmt61B, which encodes a mitochondria-specific tRNA methyltransferase responsible for m¹A58 in these three tRNAs. Trmt61B is dominantly localized to the mitochondria. m¹A58 formation in human mitochondrial tRNA(Leu(UUR)) could be reconstituted in vitro using recombinant Trmt61B in the presence of Ado-Met as a methyl donor. Unlike the cytoplasmic tRNA m¹A58 methyltransferase that consists of an α2β2 heterotetramer formed by Trmt61A and Trmt6, Trmt61B formed a homo-oligomer (presumably a homotetramer) that resembled the bacterial homotetrameric m¹A58 methyltransferase. The bacterial origin of Trmt61B is supported by the results of the phylogenetic analysis.

Mitochondrial 16S rRNA Is Methylated by tRNA Methyltransferase TRMT61B in All Vertebrates.

The mitochondrial ribosome, which translates all mitochondrial DNA (mtDNA)-encoded proteins, should be tightly regulated pre- and post-transcriptionally. Recently, we found RNA-DNA differences (RDDs) at human mitochondrial 16S (large) rRNA position 947 that were indicative of post-transcriptional modification. Here, we show that these 16S rRNA RDDs result from a 1-methyladenosine (m1A) modification introduced by TRMT61B, thus being the first vertebrate methyltransferase that modifies both tRNA and rRNAs. m1A947 is conserved in humans and all vertebrates having adenine at the corresponding mtDNA position (90% of vertebrates). However, this mtDNA base is a thymine in 10% of the vertebrates and a guanine in the 23S rRNA of 95% of bacteria, suggesting alternative evolutionary solutions. m1A, uridine, or guanine may stabilize the local structure of mitochondrial and bacterial ribosomes. Experimental assessment of genome-edited Escherichia coli showed that unmodified adenine caused impaired protein synthesis and growth. Our findings revealed a conserved mechanism of rRNA modification that has been selected instead of DNA mutations to enable proper mitochondrial ribosome function.

Unusual semi‐extractability as a hallmark of nuclear body‐associated architectural noncoding RNAs

NEAT1_2 long noncoding RNA (lncRNA) is the molecular scaffold of paraspeckle nuclear bodies. Here, we report an improved RNA extraction method: extensive needle shearing or heating of cell lysate in RNA extraction reagent improved NEAT1_2 extraction by 20‐fold (a property we term “semi‐extractability”), whereas using a conventional method NEAT1_2 was trapped in the protein phase. The improved extraction method enabled us to estimate that approximately 50 NEAT1_2 molecules are present in a single paraspeckle. Another architectural lncRNA, IGS16, also exhibited similar semi‐extractability. A comparison of RNA‐seq data from needle‐sheared and control samples revealed the existence of multiple semi‐extractable RNAs, many of which were localized in subnuclear granule‐like structures. The semi‐extractability of NEAT1_2 correlated with its association with paraspeckle proteins and required the prion‐like domain of the RNA‐binding protein FUS. This observation suggests that tenacious RNA–protein and protein–protein interactions, which drive nuclear body formation, are responsible for semi‐extractability. Our findings provide a foundation for the discovery of the architectural RNAs that constitute nuclear bodies.

Correction: Mitochondrial 16S rRNA Is Methylated by tRNA Methyltransferase TRMT61B in All Vertebrates

[This corrects the article DOI: 10.1371/journal.pbio.1002557.].

Long Noncoding RNAs as Structural and Functional Components of Nuclear Bodies

The mammalian cell nucleus harbors various membraneless suborganelles named nuclear bodies that are characterized by distinct sets of resident proteins. Nuclear bodies are thought to serve as sites for the biogenesis, assembly, and storage of specific proteins and RNAs. In the last decade, multiple nuclear bodies were found to contain long noncoding RNAs (lncRNAs), and the physiological and molecular functions of these lncRNAs have been elucidated. Numerous lncRNAs are induced in response to cellular stresses, presumably as a mechanism to cope with environmental changes. Some lncRNAs play architectural or structural roles to construct and sustain nuclear bodies; these lncRNAs exert their physiological functions by sequestering specific regulatory proteins in nuclear bodies. Other lncRNAs do not contribute to the integrity of the nuclear body structure but play significant roles in the transcriptional and post-transcriptional regulation of genes by modulating the function and localization of related regulatory proteins. In this review, we focus on the recently unveiled roles of lncRNAs that act as structural and/or functional components of nuclear bodies.