Biology subjects

Sossin, W. S.

Publications and source records attributed to Sossin, W. S..

8 recordsLinked to original sources

bioRxiv2026

Non-canonical ribosome organization enables translation stalling in neuronal RNA granules.

Neuronal outgrowth to support cognitive functions relies on a timely supply of proteins to distal sites in the neuron. To meet this demand, neurons transport mRNAs and ribosomes in large assemblies called "neuronal RNA granules". Ribosomes that integrate into these granules initiate protein synthesis in the soma, stall the process, and then become packaged into clusters that are transported to distal sites, where protein synthesis can be reactivated. The exact mechanisms and structure of these ribosome clusters remain unknown. We combined cryo-electron microscopy and cryo-electron tomography to examine these dense ribosome clusters purified from rat brains, which are included in neuronal RNA granules. We found that these clusters contain ribosomes stalled at various stages of elongation and that the mRNA sequences where the ribosomes stall facilitate stalling. The ribosome clusters contain multiple polysomal units, and ribosomes from nearby polysomes interact through rRNA expansion segments, only seen before in disomes of inactive or hibernating ribosomes in neurons. The ribosomes in the clusters form unique three-dimensional arrays that evade detection by the ribosome-associated quality-control and NO-GO decay responses, which normally lead to recycling of 40S and 60S subunits, and degradation of the mRNA and arrested nascent proteins. Overall, this study helps uncover specialized mechanisms in neurons that enable local protein translation at distal sites, supporting proper protein homeostasis, both crucial for neurodevelopment and optimal neuronal function. KEY POINTSO_LIRibosomes in neurons organize into unique clusters that are incorporated into "neuronal RNA granules". C_LIO_LIApproximately half of the ribosomes in these clusters are stalled in elongation. C_LIO_LIThe mRNA sequences read by these ribosomes facilitate stalling. C_LIO_LIRibosomes within the clusters interact through rRNA expansion segments. C_LIO_LIRibosomes in the clusters form unique 3D structures that are not detected by the ribosome-associated quality-control response. C_LI

neuroscience↗

bioRxiv2025

FMRP Regulates Neuronal RNA Granules Containing Stalled Ribosomes, Not Where Ribosomes Stall

Local protein synthesis is a crucial process that maintains local proteostasis in neurons. A large percentage of mRNAs translated in developing neurons are associated with stalled ribosomes. FMRP, the protein lost in Fragile X syndrome, is highly enriched in RNA granules that contain stalled ribosomes. Previous examination of ribosome protected fragments (RPFs) from stalled neuronal ribosomes identified sequences that match those found in mRNAs associated with FMRP, as identified by FMRP cross-linking immunoprecipitation (CLIP) (Anadolu et al, 2023, Journal of Neuroscience doi: 10.1523/JNEUROSCI.1002-22.2023). To investigate whether FMRP recognition of these sequences is important for determining where ribosomes stall on mRNAs, we examined RPFs isolated from P5 mice of both sexes that lack the FMRP protein. We found that the loss of FMRP had no significant effect on the proteins associated with neuronal stalled ribosomes, on ribosome structure, or the stalling sites (locations where RPFs accumulated). However, we observed a small, but significant decrease in the RPF levels from mRNAs previously shown to be associated with FMRP by CLIP in stalled ribosomes. Additionally, the number of neuronal RNA granules containing stalled ribosomes, as assayed by ribopuromycylation, decreased. Unlike neuronal RNA granules in WT neurons, the remaining neuronal RNA granules were resistant to reactivation. These results suggest a role of FMRP in neuronal RNA granules that contain stalled ribosomes, though loss of FMRP does not influence where ribosomes are stalled or the formation of stalled ribosomes.

neuroscience↗

bioRxiv2023

Puromycin reveals a distinct conformation of neuronal ribosomes

Puromycin is covalently added to the nascent chain of proteins by the peptidyl transferase activity of the ribosome and the dissociation of the puromycylated peptide typically follows this event. It was postulated that blocking the translocation of the ribosome with emetine could retain the puromycylated peptide on the ribosome, but evidence against this has recently been published (Hobson et al., 2020 https://doi.org/10.7554/eLife.60048; Enam et al., 2020 https://doi.org/10.7554/eLife.60303). In neurons, puromycylated nascent chains remain in the ribosome even in the absence of emetine, yet direct evidence for this has been lacking. Using biochemistry and cryo-electron microscopy, we show that the puromycylated peptides remain in the ribosome exit channel in the large subunit in a subset of neuronal ribosomes stalled in the hybrid state. These results validate previous experiments to localize stalled polysomes in neurons and provide insight into how neuronal ribosomes are stalled. Moreover, in these hybrid-state neuronal ribosomes, anisomycin, which usually blocks puromycylation, competes poorly with puromycin in the puromycylation reaction, allowing a simple assay to determine the proportion of nascent chains that are stalled in this state. In early hippocampal neuronal cultures, over 50% of all nascent peptides are found in these stalled polysomes. These results provide new insights into the stalling mechanisms of neuronal ribosomes and suggest that puromycylated peptides can be used to reveal subcellular sites of hybrid-state stalled ribosomes in neurons. Significance StatementPuromycin can be covalently linked to the nascent polypeptide chain on ribosomes, followed by dissociation of the puromycylated polypeptide. Here, we conclusively show that in stalled ribosomes isolated from neuronal RNA granules, the puromycylated peptide remains in the polypeptide exit tunnel of the ribosome. This validates previous data using this technique to localize stalled ribosomes in neurons and suggests a unique ribosomal conformation in these cells. Further evidence for the unique state of these ribosomes is the resistance of puromycylation to the inhibitor anisomycin, which prevents puromycylation in all other cellular contexts. These results provide insight into the mechanism underlying neuronal ribosome stalling and resolve a controversy about using puromycin to localize ribosome stalling in neurons.

neuroscience↗

bioRxiv2021

PITA, a pre-bilaterian p75NTR, is the evolutionary ancestor of TNF receptors

Tumor necrosis factor receptors (TNFRs) regulate a diverse array of biological functions, including adaptive immunity, neurodevelopment, and many others. Although TNFRs are expressed in all metazoan phyla, a coherent model of the molecular origins of mammalian TNFRs--and how they relate to TNFRs in other phyla--has remained elusive. To address this, we executed a large-scale, systematic Basic Local Alignment Search Tool (BLAST)-based approach to trace the evolutionary ancestry of all 29 human TNFRs. We discovered that all human TNFRs are descendants of a single pre-bilaterian TNFR with strong sequence similarity to the p75 neurotrophin receptor (p75NTR), which we designate as PITA for p75NTR is the TNFR Ancestor. A distinct subset of human TNFRs--including EDAR, XEDAR and TROY--share a unique history as descendants of EDAR-XEDAR-TROY (EXT), which diverged from PITA in a bilaterian ancestor. Most PITA descendants possess a death domain (DD) within their intracellular domain (ICD) but EXTs do not. PITA descendants are expressed in all bilaterian phyla and Cnidaria, but not in non-planulozoan ParaHoxozoa, suggesting that PITA originated in an ancestral planulozoan. Drosophila melanogaster TNFRs (Wengen (Wgn) and Grindelwald (Grnd)) were identified as divergent PITA descendants, providing the first evolutionary link between this model TNFR system and the mammalian TNFR superfamily. This study reveals PITA as the ancestor to human and Drosophila TNFR systems and describes an evolutionary model that will facilitate deciphering TNF-TNFR functions in health and disease.

evolutionary biology↗

bioRxiv2021

Excitatory postsynaptic calcium transients at Aplysia sensory-motor neuron synapses allow for quantal examination of synaptic strength over multiple days in culture

The ability to monitor changes in strength at individual synaptic contacts is required to test the hypothesis that specialized synapses maintain changes in synaptic strength that underlie memory. Measuring excitatory post-synaptic calcium transients through calcium permeable AMPA receptors is one way to monitor synaptic strength at individual synaptic contacts. Using a membrane targeted genetic calcium sensor, we demonstrate that one can measure synaptic events at individual synaptic contacts in Aplysia sensory-motor neuron synapses. These results show that synaptic strength is not evenly distributed between all contacts in these cultures, but dominated by multiquantal sites of synaptic contact. The probability, quantal size and quantal content can be measured over days at individual synaptic contacts using this technique. Surprisingly, most synaptic contacts were not found opposite presynaptic varicosities, but instead at areas of pre- and post-synaptic contact with no visible thickening of membranes. This technique shows promise in being able to address whether specialized synapses maintain synaptic strength underlying memory.

neuroscience↗

bioRxiv2021

Ribosomes in RNA granules are stalled on mRNA sequences that are consensus sites for FMRP association.

Local translation in neurons is mediated in part by the reactivation of stalled polysomes. Stalled polysomes may be enriched within the pellet of sucrose gradients used to separate polysomes from monosomes. We find that this fraction, isolated from P5 rat brains of both sexes, is enriched in proteins implicated in stalled polysome function, such as the fragile X mental retardation protein (FMRP) and Up-frameshift mutation 1 homolog (UPF1). Cryo-EM analysis of ribosomes in this fraction indicates they are stalled, mainly in the hybrid state. Ribosome profiling of this fraction showed an abundance of footprint reads derived from mRNAs of cytoskeletal proteins implicated in neuronal development and an enrichment of footprint reads on RNA binding proteins. Compared to those usually found in ribosome profiling studies, the footprint reads were more extended on their 3end and were found in reproducible peaks in the mRNAs. These peaks were enriched in motifs previously associated with mRNAs cross-linked to FMRP in vivo, independently linking the ribosomes in the sedimented pellet to the ribosomes associated with FMRP in the cell. The data supports a model in which specific sequences in mRNAs act to stall translation elongation in neurons.

neuroscience↗

bioRxiv2019

Disruption of Capn15 in mice leads to brain and eye deficits

The Small Optic Lobe (SOL) family of calpains are intracellular cysteine proteases that are expressed in the nervous system and play an important role in neuronal development in both Drosophila, where loss of this calpain leads to the eponymous small optic lobes, and in mouse and human, where loss of this calpain leads to eye anomalies. Some human individuals with biallelic variants in CAPN15 also have developmental delay and autism. However, neither the specific effect of the loss of the Capn15 protein on brain development nor the brain regions where this calpain is expressed in the adult is known. Here we show using small animal MRI that mice with the complete loss of Capn15 have smaller brains overall with larger decreases in the thalamus and subregions of the hippocampus. These losses are not seen in Capn15 conditional KO mice where Capn15 is knocked out only in excitatory neurons in the adult. Based on {beta}-galactosidase expression in an insert strain where lacZ is expressed under the control of the Capn15 promoter, we show that Capn15 is expressed in adult mice, particularly in neurons involved in plasticity such as the hippocampus, lateral amygdala and Purkinje neurons, and partially in other non-characterized cell types. The regions of the brain in the adult where Capn15 is expressed do not correspond well to the regions of the brain most affected by the complete knockout suggesting distinct roles of Capn15 in brain development and adult brain function.

neuroscience↗

bioRxiv2019

Isoform specificity of PKMs during long-term facilitation in Aplysia is mediated through stabilization by KIBRA

Persistent activity of protein kinase M (PKM), the truncated form of protein kinase C (PKC), can maintain long-term changes in synaptic strength in many systems including the hermaphrodite marine mollusk, Aplysia californica. Moreover, different types of long-term facilitation (LTF) in cultured Aplysia sensorimotor synapses rely on the activities of different PKM isoforms in the presynaptic sensory neuron and postsynaptic motor neuron. When the atypical PKM isoform was required, the kidney and brain expressed adaptor protein (KIBRA) was also required. Here, we explore how this isoform specificity is established. We find that PKM overexpression in the motor neuron, but not the sensory neuron, is sufficient to increase synaptic strength and that this activity is not isoform specific. KIBRA is not the rate-limiting step in facilitation since overexpression of KIBRA is neither sufficient to increase synaptic strength, nor to prolong a form of PKM-dependent intermediate synaptic facilitation. However, the isoform specificity of dominant negative (DN)-PKMs to erase LTF is correlated with isoform specific competition for stabilization by KIBRA. We identify a new conserved region of KIBRA. Different splice isoforms in this region stabilize different PKMs based on the isoform-specific sequence of an alpha-helix handle in the PKMs. Thus, specific stabilization of distinct PKMs by different isoforms of KIBRA can explain the isoform specificity of PKMs during LTF in Aplysia.\n\nSignificance StatementLong lasting changes in synaptic plasticity associated with memory formation are maintained by persistent protein kinases. We have previously shown in the Aplysia sensorimotor model that distinct isoforms of persistently active protein kinase Cs (PKMs) maintain distinct forms of long-lasting synaptic changes, even when both forms are expressed in the same motor neuron. Here, we show that, while the effects of overexpression of PKMs in not isoform specific, isoform specificity is defined by a handle helix in PKMs that confers stabilization by distinct splice forms in a previously undefined domain of the adaptor protein KIBRA. Thus, we define new regions in both KIBRA and PKMs that define the isoform specificity for maintaining synaptic strength in distinct facilitation paradigms.

neuroscience↗