ArcRNAs and the formation of nuclear bodies.

Long noncoding RNAs (lncRNAs) have long been collectively and passively defined as transcripts that do not encode proteins. However, extensive functional studies performed over the last decade have enabled the classification of lncRNAs into multiple categories according to their functions and/or molecular properties. Architectual RNAs (arcRNAs) are a group of lncRNAs that serve as architectural components of submicron-scale cellular bodies or nonmembranous organelles, which are composed of specific sets of proteins and nucleic acids involved in particular molecular processes. In this review, we focus on arcRNAs that function in the nucleus, which provide a structural basis for the formation of nuclear bodies, nonmembranous organelles in the cell nucleus. We will summarize the current list of arcRNAs and proteins associated with classic and more recently discovered nuclear bodies and discuss general rules that govern the formation of nuclear bodies, emphasizing weak multivalent interactions mediated by innately flexible biomolecules.

C9orf72-derived arginine-rich poly-dipeptides impede phase modifiers.

Nuclear import receptors (NIRs) not only transport RNA-binding proteins (RBPs) but also modify phase transitions of RBPs by recognizing nuclear localization signals (NLSs). Toxic arginine-rich poly-dipeptides from C9orf72 interact with NIRs and cause nucleocytoplasmic transport deficit. However, the molecular basis for the toxicity of arginine-rich poly-dipeptides toward NIRs function as phase modifiers of RBPs remains unidentified. Here we show that arginine-rich poly-dipeptides impede the ability of NIRs to modify phase transitions of RBPs. Isothermal titration calorimetry and size-exclusion chromatography revealed that proline:arginine (PR) poly-dipeptides tightly bind karyopherin-ß2 (Kapß2) at 1:1 ratio. The nuclear magnetic resonances of Kapß2 perturbed by PR poly-dipeptides partially overlapped with those perturbed by the designed NLS peptide, suggesting that PR poly-dipeptides target the NLS binding site of Kapß2. The findings offer mechanistic insights into how phase transitions of RBPs are disabled in C9orf72-related neurodegeneration.

Distinct RNA polymerase transcripts direct the assembly of phase-separated DBC1 nuclear bodies in different cell lines.

The mammalian cell nucleus is a highly organized organelle that contains membrane-less structures referred to as nuclear bodies (NBs). Some NBs carry specific RNA types that play architectural roles in their formation. Here, we show two types of RNase-sensitive DBC1-containing NBs, DBC1 nuclear body (DNB) in HCT116 cells and Sam68 nuclear body (SNB) in HeLa cells, that exhibit phase-separated features and are constructed using RNA polymerase I or II transcripts in a cell type-specific manner. We identified additional protein components present in DNB by immunoprecipitation-mass spectrometry, some of which (DBC1 and heterogeneous nuclear ribonucleoprotein L [HNRNPL]) are required for DNB formation. The rescue experiment using the truncated HNRNPL mutants revealed that two RNA-binding domains and intrinsically disordered regions of HNRNPL play significant roles in DNB formation. All these domains of HNRNPL promote in vitro droplet formation, suggesting the need for multivalent interactions between HNRNPL and RNA as well as proteins in DNB formation.

Mechanism of karyopherin-ß2 binding and nuclear import of ALS variants FUS(P525L) and FUS(R495X).

Mutations in the RNA-binding protein FUS cause familial amyotropic lateral sclerosis (ALS). Several mutations that affect the proline-tyrosine nuclear localization signal (PY-NLS) of FUS cause severe juvenile ALS. FUS also undergoes liquid-liquid phase separation (LLPS) to accumulate in stress granules when cells are stressed. In unstressed cells, wild type FUS resides predominantly in the nucleus as it is imported by the importin Karyopherin-ß2 (Kapß2), which binds with high affinity to the C-terminal PY-NLS of FUS. Here, we analyze the interactions between two ALS-related variants FUS(P525L) and FUS(R495X) with importins, especially Kapß2, since they are still partially localized to the nucleus despite their defective/missing PY-NLSs. The crystal structure of the Kapß2·FUS(P525L)PY-NLS complex shows the mutant peptide making fewer contacts at the mutation site, explaining decreased affinity for Kapß2. Biochemical analysis revealed that the truncated FUS(R495X) protein, although missing the PY-NLS, can still bind Kapß2 and suppresses LLPS. FUS(R495X) uses its C-terminal tandem arginine-glycine-glycine regions, RGG2 and RGG3, to bind the PY-NLS binding site of Kapß2 for nuclear localization in cells when arginine methylation is inhibited. These findings suggest the importance of the C-terminal RGG regions in nuclear import and LLPS regulation of ALS variants of FUS that carry defective PY-NLSs.

Siwi levels reversibly regulate secondary piRISC biogenesis by affecting Ago3 body morphology in Bombyx mori.

Silkworm ovarian germ cells produce the Siwi-piRNA-induced silencing complex (piRISC) through two consecutive mechanisms, the primary pathway and the secondary ping-pong cycle. Primary Siwi-piRISC production occurs on the outer mitochondrial membrane in an Ago3-independent manner, where Tudor domain-containing Papi binds unloaded Siwi via its symmetrical dimethylarginines (sDMAs). Here, we now show that secondary Siwi-piRISC production occurs at the Ago3-positive nuage Ago3 bodies, in an Ago3-dependent manner, where Vreteno (Vret), another Tudor protein, interconnects unloaded Siwi and Ago3-piRISC through their sDMAs. Upon Siwi depletion, Ago3 is phosphorylated and insolubilized in its piRISC form with cleaved RNAs and Vret, suggesting that the complex is stalled in the intermediate state. The Ago3 bodies are also enlarged. The aberrant morphology is restored upon Siwi re-expression without Ago3-piRISC supply. Thus, Siwi depletion aggregates the Ago3 bodies to protect the piRNA intermediates from degradation until the normal cellular environment returns to re-initiate the ping-pong cycle. Overall, these findings reveal a unique regulatory mechanism controlling piRNA biogenesis.

RNase Sensitivity Screening for Nuclear Bodies with RNA Scaffolds in Mammalian Cells.

The mammalian cell nucleus is highly organized and contains membraneless nuclear bodies (NBs) characterized by distinct resident factors. The NBs are thought to serve as sites for biogenesis and storage of certain RNA and protein factors as well as assembly of ribonucleoprotein complexes. Some NBs are formed with architectural RNAs (arcRNAs) as their structural scaffolds and additional NBs likely remain unidentified in mammalian cells. Here, we describe an experimental protocol to search for new NBs built on certain arcRNAs. RNase-sensitive NBs were identified by monitoring nuclear foci visualized by tagging thousands of human cDNA products.

The Sam68 nuclear body is composed of two RNase-sensitive substructures joined by the adaptor HNRNPL.

The mammalian cell nucleus contains membraneless suborganelles referred to as nuclear bodies (NBs). Some NBs are formed with an architectural RNA (arcRNA) as the structural core. Here, we searched for new NBs that are built on unidentified arcRNAs by screening for ribonuclease (RNase)-sensitive NBs using 32,651 fluorescently tagged human cDNA clones. We identified 32 tagged proteins that required RNA for their localization in distinct nuclear foci. Among them, seven RNA-binding proteins commonly localized in the Sam68 nuclear body (SNB), which was disrupted by RNase treatment. Knockdown of each SNB protein revealed that SNBs are composed of two distinct RNase-sensitive substructures. One substructure is present as a distinct NB, termed the DBC1 body, in certain conditions, and the more dynamic substructure including Sam68 joins to form the intact SNB. HNRNPL acts as the adaptor to combine the two substructures and form the intact SNB through the interaction of two sets of RNA recognition motifs with the putative arcRNAs in the respective substructures.

Prion-like domains in RNA binding proteins are essential for building subnuclear paraspeckles.

Prion-like domains (PLDs) are low complexity sequences found in RNA binding proteins associated with the neurodegenerative disorder amyotrophic lateral sclerosis. Recently, PLDs have been implicated in mediating gene regulation via liquid-phase transitions that drive ribonucleoprotein granule assembly. In this paper, we report many PLDs in proteins associated with paraspeckles, subnuclear bodies that form around long noncoding RNA. We mapped the interactome network of paraspeckle proteins, finding enrichment of PLDs. We show that one protein, RBM14, connects key paraspeckle subcomplexes via interactions mediated by its PLD. We further show that the RBM14 PLD, as well as the PLD of another essential paraspeckle protein, FUS, is required to rescue paraspeckle formation in cells in which their endogenous counterpart has been knocked down. Similar to FUS, the RBM14 PLD also forms hydrogels with amyloid-like properties. These results suggest a role for PLD-mediated liquid-phase transitions in paraspeckle formation, highlighting this nuclear body as an excellent model system for understanding the perturbation of such processes in neurodegeneration.

Respective functions of two distinct Siwi complexes assembled during PIWI-interacting RNA biogenesis in Bombyx germ cells.

PIWI-interacting RNA (piRNA) biogenesis consists of two sequential steps: primary piRNA processing and the ping-pong cycle that depends on reciprocal Slicer-mediated RNA cleavage by PIWI proteins. However, the molecular functions of the factors involved remain elusive. Here, we show that RNAs cleaved by a Bombyx mori PIWI, Siwi, remain bound to the protein upon cleavage but are released by a DEAD box protein BmVasa. BmVasa copurifies with Siwi but not another PIWI BmAgo3. A lack of BmVasa does not affect primary piRNA processing but abolishes the ping-pong cycle. Siwi also forms a complex with BmSpn-E and BmQin. This complex is physically separable from the Siwi/BmVasa complex. BmSpn-E, unlike BmVasa, is necessary for primary piRNA production. We propose a model for piRNA biogenesis, where the BmSpn-E/BmQin dimer binds Siwi to function in primary piRNA processing, whereas BmVasa, by associating with Siwi, ensures target RNA release upon cleavage to facilitate the ping-pong cycle.

Rapid and efficient elimination of specific nuclear noncoding RNAs in mammalian cells with antisense oligonucleotides.

A wide variety of noncoding RNAs are thought to play fundamental and regulatory functions in the mammalian cell nucleus, although their primary functions have yet to be elucidated. Here, we describe a method for the rapid and efficient elimination of specific nuclear noncoding RNAs in mammalian cells using chemically modified antisense oligonucleotides, which may facilitate the functional assessment of nuclear ncRNAs.

How does the royal family of Tudor rule the PIWI-interacting RNA pathway?

PIWI (P-element-induced wimpy testis) proteins are a subset of the Argonaute proteins and are expressed predominantly in the germlines of a variety of organisms, including Drosophila and mammals. PIWI proteins associate specifically with PIWI-interacting RNAs (piRNAs), small RNAs that are also expressed predominantly in germlines, and silence transposable DNA elements and other genes showing complementarities to the sequences of associated piRNAs. This mechanism helps to maintain the integrity of the genome and the development of gametes. PIWI proteins have been shown recently to contain symmetrical dimethyl arginines (sDMAs), and this modification is mediated by the methyltransferase PRMT5 (also known as Dart5 or Capsuleen). It was then demonstrated that multiple members of the Tudor (Tud) family of proteins, which are necessary for gametogenesis in both flies and mice, associate with PIWI proteins specifically through sDMAs in various but particular combinations. Although Tud domains in Tud family members are known to be sDMA-binding modules, involvement of the Tudor family at the molecular level in the piRNA pathway has only recently come into focus.

Dss1 associating with the proteasome functions in selective nuclear mRNA export in yeast.

Dss1p is an evolutionarily conserved small protein that interacts with BRCA2, a tumor suppressor protein, in humans. The Schizosaccharomyces pombe strain lacking the dss1(+) gene (Deltadss1) shows a temperature-sensitive growth defect and accumulation of bulk poly(A)(+) RNA in the nucleus at a nonpermissive temperature. In situ hybridization using probes for several specific mRNAs, however, revealed that the analyzed mRNAs were exported normally to the cytoplasm in Deltadss1, suggesting that Dss1p is required for export of some subsets of mRNAs. We identified the pad1(+) gene, which encodes a component of the 26S proteasome, as a suppressor for the ts(-) phenotype of Deltadss1. Unexpectedly, overexpression of Pad1p could suppress neither the defect in nuclear mRNA export nor a defect in proteasome function. In addition, loss of proteasome functions does not cause defective nuclear mRNA export. Dss1p seems to be a multifunctional protein involved in nuclear export of specific sets of mRNAs and the ubiquitin-proteasome pathway in fission yeast.