Highlights d R-rich DPRs sequester NPM1 into large, soluble phaseseparation-inhibited complexes d NPM1 sequestration dissolves droplets in vitro and delocalizes nucleolar NPM1 in cells d poly(PR) entraps rRNA in static puncta in vitro and in nucleoli d poly(PR) interactions disrupt nucleolar organization in a length-dependent manner
Axonally synthesized proteins support nerve regeneration through retrograde signaling and local growth mechanisms. RNA binding proteins (RBP) are needed for this and other aspects of post-transcriptional regulation of neuronal mRNAs, but only a limited number of axonal RBPs are known. We used targeted proteomics to profile RBPs in peripheral nerve axons. We detected 76 proteins with reported RNA binding activity in axoplasm, and levels of several change with axon injury and regeneration. RBPs with altered levels include KHSRP that decreases neurite outgrowth in developing CNS neurons. Axonal KHSRP levels rapidly increase after injury remaining elevated up to 28 days post axotomy. Khsrp mRNA localizes into axons and the rapid increase in axonal KHSRP is through local translation of Khsrp mRNA in axons. KHSRP can bind to mRNAs with 3’UTR AU-rich elements and targets those transcripts to the cytoplasmic exosome for degradation. KHSRP knockout mice show increased axonal levels of KHSRP target mRNAs, Gap43, Snap25, and Fubp1, following sciatic nerve injury and these mice show accelerated nerve regeneration in vivo. Together, our data indicate that axonal translation of the RNA binding protein Khsrp mRNA following nerve injury serves to promote decay of other axonal mRNAs and slow axon regeneration.
Public Health Service increased risk donor kidneys are discarded 50% more often than nonincreased risk donor kidneys despite equivalent patient and graft survival outcomes. Patient and provider biases as well as challenges in risk interpretation contribute to the underuse of increased risk donor organs. As the ultimate decision to accept or reject an increased risk donor organ results from the patient–provider conversation, there is an opportunity to improve this dialogue. This report introduces the best-case/worst-case communication guide for structuring high-stake conversations on increased risk kidney offers between transplant providers and their patients. Through best case/worst case, providers focus on eliciting patient values and long-term goals. The patient’s unique context can then inform an individualized discussion of “best,” “worst,” and “most likely” outcomes and support the provider’s ultimate recommendation. Transplant providers are encouraged to adopt this communication strategy to enhance shared decision-making and improve patient outcomes.
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Liquid-liquid phase separation (LLPS) underlies the assembly of biomolecular condensates such as stress granules. Stress granule persistence or disrupted stress granule dynamics is hypothesized to lead to the characteristic protein inclusions that are a hallmark of ALS (amyotrophic lateral sclerosis) and other neurological disorders. We recently demonstrated that Ubiquilin-2 (UBQLN2), an ALS-linked protein critical for maintaining protein quality control, is recruited to stress granules in cells and undergoes LLPS in vitro under physiological conditions. Mutations in the intrinsically-disordered, proline-rich (Pxx) region of UBQLN2 cause ALS and ALS/dementia and are linked to protein inclusions that form in degenerated motor neurons. The molecular mechanisms for how these Pxx mutations cause disease are unknown or poorly understood. We hypothesized that Pxx mutations disrupt UBQLN2 LLPS. Using spectrophotometric assays, light and fluorescence microscopy, we show that a subset of these mutations at positions T487, P497 or P506 significantly increase UBQLN2 LLPS propensity and/or alter material properties of UBQLN2 protein droplets in vitro. Importantly, these UBQLN2 mutants still undergo LLPS reversibly, and are all eliminated by ubiquitin binding. Biophysical characterization reveal that these single point mutations do not alter UBQLN2 structure, but likely promote UBQLN2 self-association and oligomerization, a prerequisite for LLPS. Our preliminary results suggested that increased hydrophobicity of the amino acid promotes UBQLN2 LLPS. We speculate that increased hydrophobicity promotes UBQLN2 oligomerization and self-assembly, which in turn, promotes UBQLN2 phase separation. Overall, our experiments suggest that disease-linked mutations modulate UBQLN2 oligomerization and LLPS, and potentially alter material properties of UBQLN2-containing biomolecular condensates in the cell, promoting disease states.
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