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EGFP insertional mutagenesis reveals multiple FXR2P fibrillar states with differing ribosome association in neurons.

ABSTRACT: RNA-binding proteins (RBPs) function in higher-order assemblages such as RNA granules to regulate RNA localization and translation. The Fragile X homolog FXR2P is an RBP essential for formation of neuronal Fragile X granules that associate with axonal mRNA and ribosomes in the intact brain. However, the FXR2P domains important for assemblage formation in a cellular system are unknown. Here we used an EGFP insertional mutagenesis approach to probe for FXR2P intrinsic features that influence its structural states. We tested 18 different in-frame FXR2P^EGFP fusions in neurons and found that the majority did not impact assemblage formation. However, EGFP insertion within a 23 amino acid region of the low complexity (LC) domain induced FXR2P^EGFP assembly into two distinct fibril states that were observed in isolation or in highly-ordered bundles. FXR2P^EGFP fibrils exhibited different developmental timelines, ultrastructures and ribosome associations. Formation of both fibril types was dependent on an intact RNA-binding domain. These results suggest that restricted regions of the LC domain, together with the RNA-binding domain, may be important for FXR2P structural state organization in neurons.

SUBMITTER: Stackpole EE

PROVIDER: S-EPMC6737979 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Insertional mutagenesis was applied to Cryptococcus neoformans to identify genes associated with virulence attributes. Using biolistic transformation, we generated 4,300 nourseothricin (NAT)-resistant strains, of which 590 exhibited stable resistance. We focused on mutants with defects in established virulence factors and identified two with reduced growth at 37 degrees C, four with reduced production of the antioxidant pigment melanin, and two with an increased sensitivity to nitric oxide (NO). The NAT insertion and mutant phenotypes were genetically linked in five of eight mutants, and the DNA flanking the insertions was characterized. For the strains with altered growth at 37 degrees C and altered melanin production, mutations were in previously uncharacterized genes, while the two NO-sensitive strains bore insertions in the flavohemoglobin gene FHB1, whose product counters NO stress. Because of the frequent instability of nourseothricin resistance associated with biolistic transformation, Agrobacterium-mediated transformation was tested. This transkingdom DNA delivery approach produced 100% stable nourseothricin-resistant transformants, and three melanin-defective strains were identified from 576 transformants, of which 2 were linked to NAT in segregation analysis. One of these mutants contained a T-DNA insertion in the promoter of the LAC1 (laccase) gene, which encodes a key enzyme required for melanin production, while the second contained an insertion in the promoter of the CLC1 gene, encoding a voltage-gated chloride channel. Clc1 and its homologs are required for ion homeostasis, and in their absence Cu+ transport into the secretory pathway is compromised, depriving laccase and other Cu(+)-dependent proteins of their essential cofactor. The NAT resistance cassette was optimized for cryptococcal codon usage and GC content and was then used to disrupt a mitogen-activated protein kinase gene, a predicted gene, and two putative chloride channel genes to analyze their contributions to fungal physiology. Our findings demonstrate that both insertional mutagenesis methods can be applied to gene identification, but Agrobacterium-mediated transformation is more efficient and generates exclusively stable insertion mutations.

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EGFP insertional mutagenesis reveals multiple FXR2P fibrillar states with differing ribosome association in neurons.

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