Bamba C et al. (2002) Curr Biol "The GTPase Ran regulates chromosome positioning and nuclear envelope assembly in vivo."

Nishida E, Bamba C, Bobinnec Y, Fukuda M

[Curr Biol, 2002]

The GTPase Ran is known to regulate transport of proteins across the nuclear envelope. Recently, Ran has been shown to promote microtubule polymerization and spindle assembly around chromatin in Xenopus mitotic extracts and to stimulate nuclear envelope assembly in Xenopus or HeLa cell extracts. However, these in vitro findings have not been tested in living cells and do not necessarily describe the generalized model of Ran functions. Here we present several lines of evidence that Ran is indispensable for correct chromosome positioning and nuclear envelope assembly in C. elegans. Embryos deprived of Ran by RNAi showed metaphase chromosome misalignment and aberrant chromosome segregation, while astral microtubules seemed unaffected. Depletion of RCC1 or RanGAP by RNAi resulted in essentially the same defects. The immunofluorescent staining showed that Ran localizes to kinetochore regions of metaphase and anaphase chromosomes, suggesting the role of Ran in linking chromosomes to kinetochore microtubules. Ran was shown to localize to the nuclear envelope at telophase and during interphase in early embryos, and the depletion of Ran resulted in failure of nuclear envelope assembly. Thus, Ran is crucially involved in chromosome positioning and nuclear envelope assembly in C. elegans.

Quimby BB et al. (2000) J Biol Chem "The mechanism of ran import into the nucleus by nuclear transport factor 2."

Lamitina T, Quimby BB, Corbett AH, L'Hernault SW

[J Biol Chem, 2000]

The small GTPase Ran is essential for virtually all nucleocytoplasmic transport events. It is hypothesized that Ran drives vectorial transport of macromolecules into and out of the nucleus via the establishment of a Ran gradient between the cytoplasm and nucleoplasm. Although Ran shuttles between the nucleus and cytoplasm, it is concentrated in the nucleus at steady state. We show that nuclear transport factor 2 (NTF2) is required to concentrate Ran in the nucleus in the budding yeast, Saccharomyces cerevisiae. To analyze the mechanism of Ran import into the nucleus by NTF2, we use mutants in a variety of nuclear transport factors along with biochemical analyses of NTF2 complexes. We find that Ran remains concentrated in the nucleus when importin-mediated protein import is disrupted and demonstrate that NTF2 does not form a stable complex with the transport receptor, importin-beta. Consistent with a critical role for NTF2 in establishing and maintaining the Ran gradient, we show that NTF2 is required for early embryogenesis in Caenorhabditis elegans. Our data distinguish between two possible mechanisms for Ran import by NTF2 and demonstrate that Ran import is independent from importin-beta-mediated protein import.

Peter Askjaer et al. (2001) International C. elegans Meeting "Investigation of the function of Ran by RNAi"

Iain W Mattaj, Eva Hannak, Anthony A Hyman, Peter Askjaer

[International C. elegans Meeting, 2001]

Ran is a small GTPase implicated in multiple cellular processes throughout the cell cycle. During interphase Ran was first shown to regulate transport of macromolecules in and out of the cell nucleus. Ran probably does so by serving as a molecular switch that signals the subcellular localization to transport receptors. Later Ran was also demonstrated to have a strong effect on microtubule dynamics in mitotic cell extracts where it is required for formation of a bipolar spindle. Closing the cycle, Ran is finally required for formation of a nuclear envelope around chromatin in Xenopus extracts, most likely reflecting a role for Ran in regeneration of the nuclei after cell division. Strikingly, Ran presumably carries out all three functions by creating protein gradients around chromatin. While the function of Ran in nucleocytoplasmic trafficking is well-characterized from numerous biochemical and in vivo studies it remains to be precisely determined if and how Ran regulates the mitotic spindle apparatus and nuclear envelope assembly in living cells. To address these important issues we have investigated the effects of RNAi-mediated suppression of Ran expression in several transgenic C. elegans lines. We have generated C. elegans lines that express various GFP-tagged cellular reporter proteins in the germline enabling us to make detailed time lapse microscopy recordings of early embryos. From these studies we can now evaluate the effects of Ran depletion on the dynamics of chromatin, microtubules and the nuclear envelope during early embryogenesis. Embryos in which Ran expression is inhibited show strong abnormalities in pronuclear and nuclear appearance and in severe cases no nuclei can be detected. This strongly supports an essential role for Ran in generation of a closed nuclear envelope. Secondly, targeting Ran by RNAi prevents formation of a mitotic spindle while astral microtubules are unaffected, which again provides evidence in favor of recent in vitro observations. We are continuously generating suitable markers to study the effects of Ran on the cell cycle and are currently also disrupting the expression of a broad range of C. elegans genes whose homologues in other systems are known to interact with Ran.

Rudich, Paige et al. (2017) International Worm Meeting "Novel translational products encoded by disease-associated GC-rich repeat expansions cause toxicity in C. elegans."

Rudich, Paige, Lamitina, Todd, Snoznik, Carley

[International Worm Meeting, 2017]

Expanded GC-rich repeats cause many age-onset neurodegenerative diseases. An emerging mechanism that may underlie disease pathology is an unusual type of protein translation called Repeat Associated non-ATG (RAN) translation, which specifically targets these GC-rich repeats. RAN translation requires extended GC-rich repeats and occurs independent of a canonical start codon, allowing translation in all three reading frames. Antisense RNA from these GC-rich repeats also gives rise to RAN protein products causing up to six distinct protein products from one repeat expansion. Most of these RAN proteins have never been studied in any biological system. Defining the role of RAN proteins in neurodegenerative diseases and their potential mechanisms of toxicity represents an exciting new translational research opportunity. We developed RAN protein models in C. elegans for two GC-rich expansion repeat diseases - C9orf72-associated Amyotrophic Lateral Sclerosis (ALS) and Huntington's Disease (HD). To separate RAN protein toxicity from RNA-derived toxicity, we generated 'pure' protein models by synthesizing codon-varied constructs that preserve RAN protein sequence but eliminate repeat-derived RNA structures. We used canonical ATG-dependent protein translation to bypass RAN translation and a C-terminal GFP or RFP tag to examine the subcellular localization. In our C. elegans ALS model, the arginine-containing dipeptides Pro-Arg (PR) and Gly-Arg (GR) exhibited age-onset toxicity when expressed in multiple cell types, including motor neurons. PR and GR exhibited nuclear localization that was necessary for toxicity. PR and GR did not form protein aggregates. Age-onset toxicity of PR, but not GR, was influenced by mutations that alter the rate of ageing, suggesting differences in the toxicity mechanisms of these related RAN peptides. In our HD model, initial studies suggest that HD RAN proteins exhibit distinct cell biological properties from the canonical polyGln (Q) HD protein. Currently, we are comparing the toxicity of these new HD RAN products to the well-described toxicity of polyQ. Understanding how different RAN peptides contribute to HD and to ALS is vital for developing appropriate treatments for these diseases. Moreover, comparing RAN proteins within a single experimental paradigm may reveal common themes that facilitate RAN protein toxicity and generate a more integrated understanding of the role of RAN-derived proteins in neurodegenerative diseases.

Rudich P et al. (2020) J Vis Exp "Measuring RAN Peptide Toxicity in C. elegans."

Lamitina T, Puleo N, Snoznik C, Rudich P

[J Vis Exp, 2020]

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington's disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Lamitina, Todd (2022) MicroPubl Biol

Lamitina, Todd

[MicroPubl Biol, 2022]

GC-rich repeat expansion mutations are implicated in several neurodegenerative diseases and can lead to repeat associated non-AUG-dependent (RAN) translation and concentrations of nuclear RNA foci. To model C9orf72 ALS/FTD, we engineered C. elegans to express pure GGGGCC (G 4 C 2 ) repeats of varying lengths and observed RAN translation and nuclear RNA foci. RNA foci were observed in animals expressing ≥20 G 4 C 2 repeats while RAN translation occured in animals expressing ≥33 G 4 C 2 repeats. These findings show that in C. elegans , RAN translation can occur even in the absence of C9orf72 intronic sequence normally surrounding the repeat. Given that the currently accepted repeat threshold for C9 disease is >30 repeats, our data are consistent with a model in which RAN peptides are key drivers of C9orf72 disease pathology.

Peter Askjaer et al. (2002) European Worm Meeting "The Ran GTPase cycle is essential for spindle formation and nuclear envelope assembly in living Caenorhabditis elegans embryos"

Anthony A Hyman, Peter Askjaer, Eva Hannak, Iain W Mattaj

[European Worm Meeting, 2002]

The small GTPase Ran has been found to play pivotal roles in several aspects of the cell cycle. We have investigated in real time the role of the Ran GTPase cycle in spindle formation and nuclear envelope regeneration in dividing Caenorhabditis elegans embryos. We found that Ran and its co-factors RanBP2, RanGAP and RCC1 are all essential for reformation of the nuclear envelope after division. Knocking down the expression of any of these component of the Ran GTPase cycle by RNAi leads to strong extranuclear clustering of integral nuclear envelope proteins and nucleoporins. Ran, RanBP2, and RanGAP are also required for building a mitotic spindle, whereas astral microtubules are normal in the absence of these proteins. RCC1(RNAi) embryos have similar abnormalities in the initial phase of spindle formation, however, a bipolar spindle eventually forms. Irregular chromatin structures and chromatin bridges due to spindle failure and DNA condensation defects were frequently observed in embryos where the Ran cycle is perturbed. Finally, we have demonstrated that IMA-2, which is a homologue of vertebrate importin , is essential for spindle formation in early embryos, presumably acting downstream of Ran.

Rudich P et al. (2020) PLoS One "PolyQ-independent toxicity associated with novel translational products from CAG repeat expansions."

Rudich P, Lamitina T, Watkins S

[PLoS One, 2020]

Expanded CAG nucleotide repeats are the underlying genetic cause of at least 14 incurable diseases, including Huntington's disease (HD). The toxicity associated with many CAG repeat expansions is thought to be due to the translation of the CAG repeat to create a polyQ protein, which forms toxic oligomers and aggregates. However, recent studies show that HD CAG repeats undergo a non-canonical form of translation called Repeat-associated non-AUG dependent (RAN) translation. RAN translation of the CAG sense and CUG anti-sense RNAs produces six distinct repeat peptides: polyalanine (polyAla, from both CAG and CUG repeats), polyserine (polySer), polyleucine (polyLeu), polycysteine (polyCys), and polyglutamine (polyGln). The toxic potential of individual CAG-derived RAN polypeptides is not well understood. We developed pure C. elegans protein models for each CAG RAN polypeptide using codon-varied expression constructs that preserve RAN protein sequence but eliminate repetitive CAG/CUG RNA. While all RAN polypeptides formed aggregates, only polyLeu was consistently toxic across multiple cell types. In GABAergic neurons, which exhibit significant neurodegeneration in HD patients, codon-varied (Leu)38, but not (Gln)38, caused substantial neurodegeneration and motility defects. Our studies provide the first in vivo evaluation of CAG-derived RAN polypeptides in a multicellular model organism and suggest that polyQ-independent mechanisms, such as RAN-translated polyLeu peptides, may have a significant pathological role in CAG repeat expansion disorders.

Askjaer P et al. (2002) Mol Biol Cell "Ran GTPase cycle and importins alpha and beta are essential for spindle formation and nuclear envelope assembly in living Caenorhabditis elegans embryos."

Askjaer P, Mattaj IW, Hannak E, Galy V

[Mol Biol Cell, 2002]

The small GTPase Ran has been found to play pivotal roles in several aspects of cell function. We have investigated the role of the Ran GTPase cycle in spindle formation and nuclear envelope assembly in dividing Caenorhabditis elegans embryos in real time. We found that Ran and its cofactors RanBP2, RanGAP, and RCC1 are all essential for reformation of the nuclear envelope after cell division. Reducing the expression of any of these components of the Ran GTPase cycle by RNAi leads to strong extranuclear clustering of integral nuclear envelope proteins and nucleoporins. Ran, RanBP2, and RanGAP are also required for building a mitotic spindle, whereas astral microtubules are normal in the absence of these proteins. RCC1(RNAi) embryos have similar abnormalities in the initial phase of spindle formation but eventually recover to form a bipolar spindle. Irregular chromatin structures and chromatin bridges due to spindle failure were frequently observed in embryos where the Ran cycle was perturbed. In addition, connection between the centrosomes and the male pronucleus, and thus centrosome positioning, depends upon the Ran cycle components. Finally, we have demonstrated that both IMA-2 and IMB-1, the homologues of vertebrate importin alpha and beta, are essential for both spindle assembly and nuclear formation in early embryos.

Rudich, P. et al. (2019) International Worm Meeting "PolyQ Independent Toxicity Occurs in C. elegans models of Novel Translational Products from CAG Repeat Expansions."

Rudich, P., Lamitina, S.T.

[International Worm Meeting, 2019]

Expanded CAG nucleotide repeats are the underlying genetic cause of at least 13 incurable diseases, including Huntington's disease (HD). In HD, >37 (CAG) repeats in exon 1 of the Huntington gene (HTT) leads to the development of disease, which includes the death of neurons in several brain regions, such as the striatum and basal ganglia. The major molecular cause for the loss of these neurons is thought to be the translation of the expanded (CAG) repeats into a poly-glutamine (polyGln/polyQ) expanded HTT protein. However, several brain regions that degenerate in HD do not express polyGln aggregates, suggesting that additional polyGln-independent pathways play a significant role in HD. Recent studies discovered HD CAG repeats undergo a novel type of translation called Repeat Associated Non-AUG-dependent (RAN) Translation. RAN translation of the CAG and CUG containing sense and anti-sense RNAs produces five distinct repeat peptides: polyalanine (polyAla), polyserine (polySer), polyleucine (polyLeu), polycysteine (polyCys), and polyglutamine (polyGln). RAN polypeptides are present in HD patients and are particularly prevalent in degenerating regions that lack polyQ, suggesting RAN products are major contributors to HD pathophysiology. We developed C. elegans models for each CAG RAN peptide. To separate RAN protein toxicity from RNA-derived toxicity, we generated 'pure' protein models by synthesizing codon-varied constructs that preserve RAN protein sequence but eliminate repetitive RNA. The RAN CAG peptides were expressed individually in muscle cells or GABAergic neurons. Every RAN peptide formed protein aggregates at 90 repeats, and every RAN peptide except for polyLeu formed aggregates at a disease relevant length of 38 repeats. Surprisingly, (Leu)38 was highly toxic in multiple cell types. In GABAergic neurons, which exhibit significant neurodegeneration in HD patients, codon varied (Leu)38, but not (Gln)38, caused substantial neurodegeneration and motility defects. We performed a forward mutagenesis screen to identify polyLeu toxicity mechanisms and discovered 26 suppressors. Bioinformatic analysis of genome resequencing data from the polyLeu suppressors has identified several candidate genes. RNAi knockdown of some candidates phenocopies the mutant and suppresses polyLeu toxicity. Characterizing the various HD RAN polypeptides and determining the cellular pathways required for their toxicity is vital to fully understanding HD and developing new biomarkers and therapies.