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The "Repeatome"
The "Repeatome"
Over half of the human genome is made up of repetitive elements, including over 2 million tandem microsatellites. To date, expansions in ~50 of these repeats have been identified as causes of disease, with more found every year. Despite this, we know very little about the normal roles of repetitive elements within the genome and we are likely missing many contributions that such elements make to human disease. Our lab has a long standing interest in repeats and how they specifically function in the nervous system and how their dysfunctions lead to human disease.
Over half of the human genome is made up of repetitive elements, including over 2 million tandem microsatellites. To date, expansions in ~50 of these repeats have been identified as causes of disease, with more found every year. Despite this, we know very little about the normal roles of repetitive elements within the genome and we are likely missing many contributions that such elements make to human disease. Our lab has a long standing interest in repeats and how they specifically function in the nervous system and how their dysfunctions lead to human disease.
The roles of Repeats in Neurological Disease
The roles of Repeats in Neurological Disease
Over the past three decades, studies of how nucleotide repeat expansions cause human disease have revealed significant insights into both the biology of neurodegeneration and into many basic cellular processes, including the biology of RNA in neuronal plasticity and cellular homeostasis and the regulation of translation under both normal conditions and in response to stress.
Over the past three decades, studies of how nucleotide repeat expansions cause human disease have revealed significant insights into both the biology of neurodegeneration and into many basic cellular processes, including the biology of RNA in neuronal plasticity and cellular homeostasis and the regulation of translation under both normal conditions and in response to stress.
Our own research to date has focuses on three repeat expansions that cause neurological diseases.
Our own research to date has focuses on three repeat expansions that cause neurological diseases.
Fragile X-Spectrum Disorders:
Fragile X-Spectrum Disorders:
- X-associated Tremor Ataxia Syndrome (FXTAS), a progressive degenerative illness that causes problems with walking, tremors and dementia.
- Fragile X-syndrome, the most common monogenic cause of autism and intellectual disability.
- Fragile X-associated Premature Ovarian Insufficiency (FXPOI): the most common monogenic cause of early menopause.
Amyotrophic Lateral Sclerosis and Frontotemporal Dementia:
Amyotrophic Lateral Sclerosis and Frontotemporal Dementia:
- We study an intronic GGGGCC repeat in C9orf72 that is the most common known cause of both of these neurodegenerative disorders.
CANVAS (Cerebellar Ataxia with Neuropathy and Vestibular Areflexia Syndrome):
CANVAS (Cerebellar Ataxia with Neuropathy and Vestibular Areflexia Syndrome):
- A common cause of autosomal recessive ataxia and peripheral sensory neuropathy, it results from a homozygous intronic CCTTT repeat expansion in the RFC1 gene.
Fragile X-Spectrum Disorders: A CGG repeat in the 5' leader of the fragile X Gene, FMR1, is present in everyone. This repeat is included in FMR mRNA but not in the mature Fragile X protein, FMRP. THis repeat can become unstable and expand over generations. At intermediate sizes, the repeat expansion leads to enhanced repeat transcription, repeat RNA-protein foci, inefficient FMRP translation, and RAN translation of homopolymeric proteins. Larger (>200) repeats, trigger transcriptional silencing and DNA methylation, leading to a loss of FMR1 mRNA and FMRP and clinically resulting in Fragile X Syndrome.
Fragile X-Spectrum Disorders: A CGG repeat in the 5' leader of the fragile X Gene, FMR1, is present in everyone. This repeat is included in FMR mRNA but not in the mature Fragile X protein, FMRP. THis repeat can become unstable and expand over generations. At intermediate sizes, the repeat expansion leads to enhanced repeat transcription, repeat RNA-protein foci, inefficient FMRP translation, and RAN translation of homopolymeric proteins. Larger (>200) repeats, trigger transcriptional silencing and DNA methylation, leading to a loss of FMR1 mRNA and FMRP and clinically resulting in Fragile X Syndrome.
All of these disorders result from nucleotide repeat expansions in putative non-coding regions of messenger RNAs. The repeat expansions in these disorders are thought to elicit a dominant RNA gain-of-function toxicity by binding to and sequestering specific proteins, thus preventing them from performing their normal functions. However, our group and others have recently discovered that these repeats also support protein translation in the absence of an AUG start codon. Utilizing disease models in Drosophila, mice and cell culture, including neurons derived from human induced pluripotent stem cells, and in collaboration with a number of laboratories here on the UM campus and beyond, we are exploring the mechanisms underlying these disorders with a long term goal of identifying therapeutic targets.
All of these disorders result from nucleotide repeat expansions in putative non-coding regions of messenger RNAs. The repeat expansions in these disorders are thought to elicit a dominant RNA gain-of-function toxicity by binding to and sequestering specific proteins, thus preventing them from performing their normal functions. However, our group and others have recently discovered that these repeats also support protein translation in the absence of an AUG start codon. Utilizing disease models in Drosophila, mice and cell culture, including neurons derived from human induced pluripotent stem cells, and in collaboration with a number of laboratories here on the UM campus and beyond, we are exploring the mechanisms underlying these disorders with a long term goal of identifying therapeutic targets.
RAN translation at nucleotide repeats
RAN translation at nucleotide repeats
Our group has recently identified a role for Repeat-associated non-AUG initiated (RAN) translation in FXTAS pathogenesis (See figure below). This unusual process leads to production of a toxic protein, FMRpolyG, that may contribute to disease pathogenesis. Ongoing work is targeted at understanding how this protein is made, what role it plays in disease pathogenesis, and trying to identify ways that its production can be halted.
Our group has recently identified a role for Repeat-associated non-AUG initiated (RAN) translation in FXTAS pathogenesis (See figure below). This unusual process leads to production of a toxic protein, FMRpolyG, that may contribute to disease pathogenesis. Ongoing work is targeted at understanding how this protein is made, what role it plays in disease pathogenesis, and trying to identify ways that its production can be halted.
RAN translation in fragile X-associated tremor ataxia syndrome.
RAN translation in fragile X-associated tremor ataxia syndrome.
Ribosomes assemble on the 5’ end of the FMR1 message and scan the RNA for an appropriate initiation sequence. Near the CGG repeat hairpin, the 43S preinitiaton complex (red) stalls, triggering RAN translational initiation. Once translation initiates, the ribosome reads through the repeat to produce a polyglycine-containing protein. Normally, this peptide is readily cleared from cells, but with larger repeats the resultant expanded polyglycine protein accumulates in inclusions. The downstream AUG start site for FMRP is not in frame with the polyglycine protein, thus no N-terminal addition onto FMRP occurs with this CGG RAN translation. A product is also generated in the Alanine frame in cellular and in vitro models.
Ribosomes assemble on the 5’ end of the FMR1 message and scan the RNA for an appropriate initiation sequence. Near the CGG repeat hairpin, the 43S preinitiaton complex (red) stalls, triggering RAN translational initiation. Once translation initiates, the ribosome reads through the repeat to produce a polyglycine-containing protein. Normally, this peptide is readily cleared from cells, but with larger repeats the resultant expanded polyglycine protein accumulates in inclusions. The downstream AUG start site for FMRP is not in frame with the polyglycine protein, thus no N-terminal addition onto FMRP occurs with this CGG RAN translation. A product is also generated in the Alanine frame in cellular and in vitro models.
CGG repeat elicited Neuronal dysfunction
CGG repeat elicited Neuronal dysfunction
We are also interested in how CGG repeats impact expression of FMRP. We have discovered that the repeat blocks activity dependent synaptic synthesis of FMRP and that this blockade has a deliterious impact on neuronal plasticity. We are now exploring how this blockade occurs and how its impact might be modulated for therapeutic benefit.
We are also interested in how CGG repeats impact expression of FMRP. We have discovered that the repeat blocks activity dependent synaptic synthesis of FMRP and that this blockade has a deliterious impact on neuronal plasticity. We are now exploring how this blockade occurs and how its impact might be modulated for therapeutic benefit.
A Working Model of mGluR-LTD in CGG KI mice. Group I mGluR receptors modulate synaptic overactivity. Normally, FMRP bound transcripts, including fmr1 mRNA, exist in stalled ribosomal complexes at synapses. Activation of group I mGluRs triggers internalization of AMPA receptors and the dissociation/clearance of FMRP from target mRNAs, allowing for rapid translation of proteins required for LTD. In parallel, FMRP is itself synthesized at synapses and this new FMRP acts as a brake on further translation of mRNA targets. In Fragile X CGG repeat model mice, there is adequate basal expression of FMRP to allow for localization of FMRP with associated transcripts at synapses. i) mGluR activation triggers dissociation of FMRP from these transcripts normally. ii) However, the CGG repeat expansion blocks rapid FMRP synthesis. Without this new FMRP, there is no brake to prevent ongoing synthesis of FMRP target transcripts. iii) The result is over-production of LTD effector proteins and enhanced mGluR-LTD. In contrast to FXS model mice, synaptic protein translation in CGG repeat model mice remains coupled to mGluR activation and the mGluR-LTD is thus dependent on new protein synthesis. (Adapted from Iliff et al, 2012)
A Working Model of mGluR-LTD in CGG KI mice. Group I mGluR receptors modulate synaptic overactivity. Normally, FMRP bound transcripts, including fmr1 mRNA, exist in stalled ribosomal complexes at synapses. Activation of group I mGluRs triggers internalization of AMPA receptors and the dissociation/clearance of FMRP from target mRNAs, allowing for rapid translation of proteins required for LTD. In parallel, FMRP is itself synthesized at synapses and this new FMRP acts as a brake on further translation of mRNA targets. In Fragile X CGG repeat model mice, there is adequate basal expression of FMRP to allow for localization of FMRP with associated transcripts at synapses. i) mGluR activation triggers dissociation of FMRP from these transcripts normally. ii) However, the CGG repeat expansion blocks rapid FMRP synthesis. Without this new FMRP, there is no brake to prevent ongoing synthesis of FMRP target transcripts. iii) The result is over-production of LTD effector proteins and enhanced mGluR-LTD. In contrast to FXS model mice, synaptic protein translation in CGG repeat model mice remains coupled to mGluR activation and the mGluR-LTD is thus dependent on new protein synthesis. (Adapted from Iliff et al, 2012)
The sequestration hypothesis of RNA dominant disorders.
The sequestration hypothesis of RNA dominant disorders.
Nucleotide repeat expansions elicit cellular dysfunction in many tissues including the nervous system. A) Under normal conditions, numerous RNA binding proteins are involved in RNA splicing, processing and other cellular functions. B) Nucleotide repeats in RNA induce repetitive secondary structures that bind to and sequester numerous RNA binding proteins. This sequestration prevents the normal splicing and processing of other mRNAs, leading to retention of rare splice isoforms that are either less stable and thus rapidly degraded, or that encode proteins with different functional characteristics. In the central nervous system, splicing errors can also interfere with proper distribution of the mRNA within neurons, especially dendrites. (Adapted from Todd and Paulson, Annals of Neurology 2009).
Nucleotide repeat expansions elicit cellular dysfunction in many tissues including the nervous system. A) Under normal conditions, numerous RNA binding proteins are involved in RNA splicing, processing and other cellular functions. B) Nucleotide repeats in RNA induce repetitive secondary structures that bind to and sequester numerous RNA binding proteins. This sequestration prevents the normal splicing and processing of other mRNAs, leading to retention of rare splice isoforms that are either less stable and thus rapidly degraded, or that encode proteins with different functional characteristics. In the central nervous system, splicing errors can also interfere with proper distribution of the mRNA within neurons, especially dendrites. (Adapted from Todd and Paulson, Annals of Neurology 2009).
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