Skip to main content
eScholarship
Open Access Publications from the University of California

Scholarly Works (9 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2023)

Transcription factors play an essential role in organism development and homeostasis; and loss or impaired function of transcription factors has been implicated in a variety of developmental disorders and chronic diseases. Here, we show the transcription factor Ras Responsive Element Binding Protein 1 (RREB1) is essential for Purkinje cell survival in the mammalian brain. We identified a spontaneous mutation that results in hypomorphic loss of Rreb1 and leads to progressive Purkinje cell degeneration and ataxia in mice. ChIP-seq of RREB1 in wild-type cells and RNA-seq in wild-type and Rreb1-deficient Purkinje cells demonstrated RREB1 directly, positively regulates genes associated with the microtubule network and autophagy.Dysregulation of genes associated with the microtubule network lead to alterations in posttranslational modifications of tubulin and reduced dendritic branching in Purkinje cells. Altered expression of genes associated with autophagy lead to a reduction in autophagosomes and lysosomes, and the accumulation of P62- and Ubiquitin-positive inclusions. Interestingly, while this study demonstrates a role for RREB1 in the microtubule network and proteostasis in neurons, examination of RREB1 in HEK 293-T cells suggests it plays a different role in this cell type. Analysis of RREB1 ChIP-seq data from Purkinje cells and HEK 293-T cells identified an overlap in many target genes. Surprisingly, while RREB1 positively regulates these genes in Purkinje cells, it appears to negatively regulate these genes in HEK 293-T cells. Examination of autophagic flux in Rreb1 knockout cells demonstrated no significant difference between knockout cells and wild-type cells. Together, these studies demonstrate a novel and specific role for RREB1 in maintaining the microtubule network and proteostasis in mammalian neurons.

    • Thesis
    • Peer Reviewed
    UC San Diego Electronic Theses and Dissertations (2021)

    Errors in tRNA aminoacylation can lead to misfolded proteins, which can form protein aggregates. Protein aggregation have been linked to many neurological diseases, thus understanding the mechanisms that can lead to the formation of protein aggregates is critical for researching degenerative illnesses. Aminoacyl tRNA synthetases (AARSs) have been found to have hydrolytic editing functions that can correct tRNA mischarging errors and prevent mistranslation. A mutation, referred to as sticky, has been discovered to cause a dysfunction in the hydrolytic editing function of alanyl-tRNA synthetase (AlaRS), resulting in mischarged Ser-tRNAAla and causes serine to be incorrectly incorporated into newly-formed peptides. However, Ankrd16, a newly discovered protein, has been found to aid AlaRS in editing of mischarged tRNAs by removing serine from the aminoacyl active site of AlaRS. By conditionally deleting Ankrd16 in CaMKIIa-positive neurons in the forebrain of Aars^sti/sti mice, the Ackerman lab was able to induce neuronal death in targeted neurons (Vo et al., 2018). In this study, we attempted to induce motor neuron death by conditionally deleting Ankrd16 in ChAT-positive motor neurons in Aars^sti/sti mice. Although motor neuron death was not observed, other phenotypes, such as decreased weight, kyphosis, and decreased axon diameters were found to be caused by the conditional deletion of Ankrd16. These results suggest that motor neurons may be more resistant to Ankrd16 loss than other neurons, such as Purkinje cells.

      Cover page: Effects of Ankrd16 loss in motor neurons of aminoacyl tRNA synthetase editing-deficient mice
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2021)
      Homozygous mutation of the RNA kinase CLP1 (cleavage factor polyribonucleotide kinase subunit 1) causes pontocerebellar hypoplasia type 10 (PCH10), a pediatric neurodegenerative disease. CLP1 is associated with the transfer RNA (tRNA) splicing endonuclease complex and the cleavage and polyadenylation machinery, but its function remains unclear. We generated two mouse models of PCH10: one homozygous for the disease-associated Clp1 mutation, R140H, and one heterozygous for this mutation and a null allele. Both models exhibit loss of lower motor neurons and neurons of the deep cerebellar nuclei. To explore whether Clp1 mutation impacts tRNA splicing, we profiled the products of intron-containing tRNA genes. While mature tRNAs were expressed at normal levels in mutant mice, numerous other products of intron-containing tRNA genes were dysregulated, with pre-tRNAs, introns, and certain tRNA fragments up-regulated, and other fragments down-regulated. However, the spatiotemporal patterns of dysregulation do not correlate with pathogenicity for most altered tRNA products. To elucidate the effect of Clp1 mutation on precursor messenger RNA (pre-mRNA) cleavage, we analyzed poly(A) site (PAS) usage and gene expression in Clp1R140H/- spinal cord. PAS usage was shifted from proximal to distal sites in the mutant mouse, particularly in short and closely spaced genes. Many such genes were also expressed at lower levels in the Clp1R140H/- mouse, possibly as a result of impaired transcript maturation. These findings are consistent with the hypothesis that select genes are particularly dependent upon CLP1 for proper pre-mRNA cleavage, suggesting that impaired mRNA 3' processing may contribute to pathogenesis in PCH10.
        Cover page: The Clp1 R140H mutation alters tRNA metabolism and mRNA 3′ processing in mouse models of pontocerebellar hypoplasia
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2024)
        Defects in tRNA biogenesis are associated with multiple neurological disorders, yet our understanding of these diseases has been hampered by an inability to determine tRNA expression in individual cell types within a complex tissue. Here, we developed a mouse model in which RNA polymerase III is conditionally epitope tagged in a Cre-dependent manner, allowing us to accurately profile tRNA expression in any cell type in vivo. We investigated tRNA expression in diverse nervous system cell types, revealing dramatic heterogeneity in the expression of tRNA genes between populations. We found that while maintenance of levels of tRNA isoacceptor families is critical for cellular homeostasis, neurons are differentially vulnerable to insults to distinct tRNA isoacceptor families. Cell-type-specific translatome analysis suggests that the balance between tRNA availability and codon demand may underlie such differential resilience. Our work provides a platform for investigating the complexities of mRNA translation and tRNA biology in the brain.
          Cover page: Cell-type-specific expression of tRNAs in the brain regulates cellular homeostasis
          • Article
          • Peer Reviewed
          UC San Diego Previously Published Works (2016)
          Ribosome stalling during translation has recently been shown to cause neurodegeneration, yet the signaling pathways triggered by stalled elongation complexes are unknown. To investigate these pathways we analyzed the brain of C57BL/6J-Gtpbp2(nmf205)(-/-) mice in which neuronal elongation complexes are stalled at AGA codons due to deficiencies in a tRNA(Arg)UCU tRNA and GTPBP2, a mammalian ribosome rescue factor. Increased levels of phosphorylation of eIF2α (Ser51) were detected prior to neurodegeneration in these mice and transcriptome analysis demonstrated activation of ATF4, a key transcription factor in the integrated stress response (ISR) pathway. Genetic experiments showed that this pathway was activated by the eIF2α kinase, GCN2, in an apparent deacylated tRNA-independent fashion. Further we found that the ISR attenuates neurodegeneration in C57BL/6J-Gtpbp2(nmf205)(-/-) mice, underscoring the importance of cellular and stress context on the outcome of activation of this pathway. These results demonstrate the critical interplay between translation elongation and initiation in regulating neuron survival during cellular stress.
            Cover page: Activation of GCN2 kinase by ribosome stalling links translation elongation with translation initiation
            • Article
            • Peer Reviewed
            UC San Diego Previously Published Works (2021)
            Translation-dependent quality control pathways such as no-go decay (NGD), non-stop decay (NSD), and nonsense-mediated decay (NMD) govern protein synthesis and proteostasis by resolving non-translating ribosomes and preventing the production of potentially toxic peptides derived from faulty and aberrant mRNAs. However, how translation is altered and the in vivo defects that arise in the absence of these pathways are poorly understood. Here, we show that the NGD/NSD factors Pelo and Hbs1l are critical in mice for cerebellar neurogenesis but expendable for survival of these neurons after development. Analysis of mutant mouse embryonic fibroblasts revealed translational pauses, alteration of signaling pathways, and translational reprogramming. Similar effects on signaling pathways, including mTOR activation, the translatome and mouse cerebellar development were observed upon deletion of the NMD factor Upf2. Our data reveal that these quality control pathways that function to mitigate errors at distinct steps in translation can evoke similar cellular responses.
              Cover page: Defects in translation-dependent quality control pathways lead to convergent molecular and neurodevelopmental pathology
              • Article
              • Peer Reviewed
              UC San Diego Previously Published Works (2020)
              Ribosome-associated quality control pathways respond to defects in translational elongation to recycle arrested ribosomes and degrade aberrant polypeptides and mRNAs. Loss of a tRNA gene leads to ribosomal pausing that is resolved by the translational GTPase GTPBP2, and in its absence causes neuron death. Here, we show that loss of the homologous protein GTPBP1 during tRNA deficiency in the mouse brain also leads to codon-specific ribosome pausing and neurodegeneration, suggesting that these non-redundant GTPases function in the same pathway to mitigate ribosome pausing. As observed in Gtpbp2-/- mice (Ishimura et al., 2016), GCN2-mediated activation of the integrated stress response (ISR) was apparent in the Gtpbp1-/- brain. We observed decreased mTORC1 signaling which increased neuronal death, whereas ISR activation was neuroprotective. Our data demonstrate that GTPBP1 functions as an important quality control mechanism during translation elongation and suggest that translational signaling pathways intricately interact to regulate neuronal homeostasis during defective elongation.
                Cover page: GTPBP1 resolves paused ribosomes to maintain neuronal homeostasis
                • Article
                • Peer Reviewed
                UC San Diego Previously Published Works (2022)
                SignificanceUnderstanding and treating neurological disorders are global priorities. Some of these diseases are engendered by mutations that cause defects in the cellular synthesis of transfer RNAs (tRNAs), which function as adapter molecules that translate messenger RNAs into proteins. During tRNA biogenesis, ribonuclease P catalyzes removal of the transcribed sequence upstream of the mature tRNA. Here, we focus on a cytoplasmic tRNAArgUCU that is expressed specifically in neurons and, when harboring a particular point mutation, contributes to neurodegeneration in mice. Our results suggest that this mutation favors stable alternative structures that are not cleaved by mouse ribonuclease P and motivate a paradigm that may help to understand the molecular basis for disease-associated mutations in other tRNAs.
                  Cover page: Structural basis for impaired 5′ processing of a mutant tRNA associated with defects in neuronal homeostasis
                  • Article
                  • Peer Reviewed
                  UC San Diego Previously Published Works (2018)
                  Editing domains of aminoacyl tRNA synthetases correct tRNA charging errors to maintain translational fidelity. A mutation in the editing domain of alanyl tRNA synthetase (AlaRS) in Aars sti mutant mice results in an increase in the production of serine-mischarged tRNAAla and the degeneration of cerebellar Purkinje cells. Here, using positional cloning, we identified Ankrd16, a gene that acts epistatically with the Aars sti mutation to attenuate neurodegeneration. ANKRD16, a vertebrate-specific protein that contains ankyrin repeats, binds directly to the catalytic domain of AlaRS. Serine that is misactivated by AlaRS is captured by the lysine side chains of ANKRD16, which prevents the charging of serine adenylates to tRNAAla and precludes serine misincorporation in nascent peptides. The deletion of Ankrd16 in the brains of Aarssti/sti mice causes widespread protein aggregation and neuron loss. These results identify an amino-acid-accepting co-regulator of tRNA synthetase editing as a new layer of the machinery that is essential to the prevention of severe pathologies that arise from defects in editing.
                    Cover page: ANKRD16 prevents neuron loss caused by an editing-defective tRNA synthetase
                    Top