Biology subjects

Alvarado, C.

Publications and source records attributed to Alvarado, C..

10 recordsLinked to original sources

bioRxiv2025

RNA regulates repeat-associated non-AUG (RAN) translation initiation in C9orf72 FTD/ALS

Repeat-associated non-AUG (RAN) translation synthesizes protein in the absence of a cognate AUG start codon 1,2In frontotemporal dementia and amyotrophic lateral sclerosis, a GGGGCC (G4C2) repeat expansion in an intron of C9orf72 leads to synthesis of neurotoxic dipeptide-repeat proteins, underscoring the need to understand the mechanism of C9orf72 RAN translation1-5. RNA sequence and structure have been implicated, but how they direct C9orf72 RAN translation, particularly the rate-limiting, multi-step initiation phase, remains unclear6-10. We applied single-molecule biophysics to a reconstituted human translation initiation system and tracked fluorescently labeled ribosomes and initiation factors in real time. We show that RNA G4C2 repeats and sequence context alter initiation factor dynamics after ribosomal scanning, generating a kinetic bottleneck in the commitment to initiate at a near-cognate CUG start codon. Our model of C9orf72 RAN translation provides a mechanistic framework for how repeat expansions change underlying translation dynamics and may be broadly relevant to other disorders that involve RAN translation.

biophysics↗

bioRxiv2025

Neural Synchrony in Parent-Child Dyads: Profiles Associated with Interparental Conflict and Internalizing Symptoms

Interparental conflict and parental stress are well-established risk factors for child psychopathology, including elevated internalizing and externalizing symptoms. From a family systems framework, these stressors may spill over into the parent-child relationship, undermining emotional attachment and co-regulation processes central to childrens mental health. Neural synchrony, defined as the dynamic, mutual alignment of brain activity between a parent and child, offers a biological index of these dyadic processes. Using functional near-infrared spectroscopy, researchers have shown that greater neural synchrony (NS) in prefrontal brain regions is associated with more attuned caregiving and positive child adjustment. Yet, NS is not uniform; it varies across dyads in pattern and regional distribution, potentially reflecting differences in relational dynamics, regulation, or stress exposure. To capture this heterogeneity, we used latent profile analysis to identify distinct synchrony patterns along the right and left ventrolateral and dorsolateral prefrontal cortices during the DB-DOS:Biosynch - a mild stress, three-context task. We further examined whether interparental conflict and perceived parental stress predicted profile membership, and whether childrens internalizing and externalizing behaviors differed by profile. Among 194 dyads, two profiles emerged: lower baseline synchrony (LB; n = 132) and higher baseline synchrony (HB; n = 62). Greater interparental conflict reduced the odds of membership in HB, while parental stress was not predictive of profile membership. Additionally, children in LB exhibited higher levels of internalizing behaviors compared to HB, with no group differences observed for externalizing behaviors. These findings underscore the value of capturing synchrony heterogeneity in understanding family stress and child psychopathology.

developmental biology↗

bioRxiv2025

ARAP2 regulates responses to interferon-gamma by restricting SOCS1

Interferon-gamma (IFN{gamma}) is critical for immunity against intra-macrophagic pathogens, signaling through the JAK-STAT pathway to induce a tyrosine-phosphorylation cascade that ensures a potent immune response. Excessive JAK-STAT signaling can drive hyperinflammation and autoimmunity, and thus signaling is tightly and selectively regulated by the IFN{gamma}-inducible protein, Suppressor of Cytokine Signaling 1 (SOCS1). SOCS1 inhibits signaling by directly blocking JAK kinase activity. Here we identified a SOCS1-interacting partner, ARAP2 that fine-tunes SOCS1 function. We report that tyrosine 415 in ARAP2 binds the SOCS1-Src Homology 2 (SH2) domain and limits the ability of SOCS1 to inhibit IFN{gamma} signaling. Our findings show that ARAP2 promotes the IFN{gamma} response through a phosphorylation dependent interaction with the negative regulator SOCS1.

cell biology↗

bioRxiv2024

Comparative transcriptomics in ferns reveals key innovations and divergent evolution of secondary cell wall

Despite ferns being crucial to understanding plant evolution, their large and complex genomes has kept their genetic landscape largely uncharted, with only a handful of genomes sequenced and sparse transcriptomic data. Addressing this gap, we generated extensive RNA-sequencing data for multiple organs across 22 representative species over the fern phylogeny, assembling high-quality transcriptomes. These data facilitated the construction of a time-calibrated fern phylogeny covering all major clades, revealing numerous whole-genome duplications and highlighting the uniqueness of fern genetics, with half of the uncovered gene families being fern-specific. Our investigation into fern cell walls through biochemical and immunological analyses identified occurrences of the lignin syringyl unit and its independent evolution in ferns. Moreover, the discovery of an unusual sugar in fern cell walls hints at a divergent evolutionary path in cell wall biochemistry, potentially driven by gene duplication and sub-functionalization. We provide an online database preloaded with genomic and transcriptomic data for ferns and other land plants, which we used to identify an independent evolution of lignocellulosic gene modules in ferns. Our data provide a framework for the unique evolutionary path that ferns have navigated since they split from the last common ancestor of euphyllophytes more than 360 million years ago.

plant biology↗

bioRxiv2024

eIF1 and eIF5 dynamically control translation start site fidelity

Translation initiation defines the identity of a synthesized protein through selection of a translation start site on a messenger RNA. This process is essential to well-controlled protein synthesis, modulated by stress responses, and dysregulated in many human diseases. The eukaryotic initiation factors eIF1 and eIF5 interact with the initiator methionyl-tRNAiMet on the 40S ribosomal subunit to coordinate start site selection. Here, using single-molecule analysis of in vitro reconstituted human initiation combined with translation assays in cells, we examine eIF1 and eIF5 function. During translation initiation on a panel of RNAs, we monitored both proteins directly and in real time using single-molecule fluorescence. As expected, eIF1 loaded onto mRNAs as a component of the 43S initiation complex. Rapid ([~] 2 s) eIF1 departure required a translation start site and was delayed by alternative start sites and a longer 5 untranslated region (5UTR). After its initial departure, eIF1 rapidly and transiently sampled initiation complexes, with more prolonged sampling events on alternative start sites. By contrast, eIF5 only transiently bound initiation complexes late in initiation immediately prior to association of eIF5B, which allowed joining of the 60S ribosomal subunit. eIF5 association required the presence of a translation start site and was inhibited and destabilized by alternative start sites. Using both knockdown and overexpression experiments in human cells, we validated that eIF1 and eIF5 have opposing roles during initiation. Collectively, our findings demonstrate how multiple eIF1 and eIF5 binding events control start-site selection fidelity throughout initiation, which is tuned in response to changes in the levels of both proteins.

biophysics↗

bioRxiv2024

Transient inhibition of type I interferon enhances CD8+ T cell stemness and vaccine protection

Developing vaccines that promote CD8+ T cell memory is a challenge for infectious disease and cancer immunotherapy. TCF-1+ stem cell-like memory T (TSCM) cells are important determinants of long-lived memory. Yet, the developmental requirements for TSCM formation are unclear. Here, we identify the temporal window for type I interferon (IFN-I) receptor (IFNAR) blockade to drive TSCM cell generation. TSCM cells were transcriptionally distinct and emerged from a transitional precursor of exhausted (TPEX) cellular state concomitant with viral clearance. TSCM differentiation correlated with T cell retention within the lymph node paracortex, due to increased CXCR3 chemokine abundance which disrupted gradient formation. These affects were due a counterintuitive increase in IFN{psi}, which controlled cell location. Combining IFNAR inhibition with mRNA-LNP vaccination promoted specific TSCM differentiation and enhanced protection against chronic infection. These finding propose a new approach to vaccine design whereby modulation of inflammation promotes memory formation and function. HIGHLIGHTSO_LIEarly, transient inhibition of the type I interferon (IFN) receptor (IFNAR) during acute viral infection promotes stem cell-like memory T (TSCM) cell differentiation without establishing chronic infection. C_LIO_LITSCM and precursor of exhausted (TPEX) cellular states are distinguished transcriptionally and by cell surface markers. C_LIO_LIDevelopmentally, TSCM cell differentiation occurs via a transition from a TPEX state coinciding with viral clearance. C_LIO_LITransient IFNAR blockade increases IFN{psi} production to modulate the ligands of CXCR3 and couple TSCM differentiation to cell retention within the T cell paracortex of the lymph node. C_LIO_LISpecific promotion of TSCM cell differentiation with nucleoside-modified mRNA-LNP vaccination elicits enhanced protection against chronic viral challenge. C_LI

immunology↗

bioRxiv2024

Divergent cytokine and transcriptional signatures control functional T follicular helper cell heterogeneity

Adaptive immune responses protect against multiple classes of pathogens, including viral, bacterial, fungal, and helminth infections. In all these settings, CD4+ T follicular helper (Tfh) cells tailor high-affinity class-switched B cells responses. How Tfh lineage sovereignty is established while allowing for this context-specific functional heterogeneity is unclear. Here, we identify Tfh transcriptional networks in response to diverse infections. While Bcl-6 is the transcriptional linchpin of the core Tfh signature, this is overlayed with pathogen-specific transcriptional modules that shape Tfh function. Cytokine-transcriptional Tfh programing in mouse and human lymphoid tissue demonstrated that type I interferon and TGF{beta} signaling direct individual Tfh subpopulations to instruct B cell output. Here, we provide a transcriptional map and cell surface resource to interrogate Tfh diversity in humans and mice. This resource can be leveraged to further understand the origins of immune flexibility, perform immune monitoring in infection and antibody-mediated diseases and to develop context-specific vaccines. ONE-SENTENCE SUMMARYDalit, Tan and colleagues provide a resource that functionally and transcriptionally profiles T follicular helper cells (Tfh) during diverse pathogen responses to reveal a blueprint for transcriptional flexibility and new tools to interrogate Tfh heterogeneity in mice and humans.

immunology↗

bioRxiv2022

Survey of activation-induced genome architecture reveals a novel enhancer of Myc

The transcription factor Myc is critically important in driving cell proliferation, a function that is frequently dysregulated in cancer. To avoid this dysregulation Myc is tightly controlled by numerous layers of regulation. One such layer is the use of distal regulatory enhancers to drive Myc expression. Here, using chromosome conformation capture to examine B cells of the immune system in the first hours after their activation, we reveal a previously unidentified enhancer of myc. The interactivity of this enhancer coincides with a dramatic, but discrete, spike in Myc expression 3 hours post-activation. However, genetic deletion of this region, has little impact on Myc expression, Myc protein level or in vitro and in vivo cell proliferation. Examination of the enhancer deleted regulatory landscape suggests that enhancer redundancy likely sustains Myc expression. This work highlights not only the importance of temporally examining enhancers, but also the complexity and dynamics of the regulation of critical genes such as Myc.

genetics↗

bioRxiv2021

eIF5B and eIF1A remodel human translation initiation complexes to mediate ribosomal subunit joining

Joining of the ribosomal subunits at a translation start site on a messenger RNA during initiation commits the ribosome to synthesize a protein. Here, we combined single-molecule spectroscopy and structural methods using an in vitro reconstituted system to examine how the human ribosomal subunits join. Single-molecule fluorescence revealed when universally-conserved eukaryotic initiation factors (eIFs) eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we examined initiation complexes that contained both eIF1A and eIF5B using single-particle electron cryo-microscopy. The resulting structure illuminated how eukaryote-specific contacts between eIF1A and eIF5B remodel the initiation complex to orient initiator tRNA in a conformation compatible with ribosomal subunit joining. Collectively, our findings provide a quantitative and architectural framework for the molecular choreography orchestrated by eIF1A and eIF5B during human translation initiation.

biophysics↗

bioRxiv2021

Rapid 40S scanning and its regulation by mRNA structure during eukaryotic translation initiation

How the eukaryotic 43S preinitiation complex scans along the 5' untranslated region (5'UTR) of a capped mRNA to locate the correct start codon remains elusive. Here, we directly track yeast 43S-mRNA binding, scanning, and 60S subunit joining by real-time single-molecule fluorescence spectroscopy. Once engaged with the mRNA, 43S scanning occurs at >100 nucleotides per second, independent of multiple cycles of ATP-hydrolysis by RNA helicases. The scanning ribosomes can proceed through RNA secondary structures, but 5'UTR hairpin sequences near start codons drive scanning ribosomes at start codons back in the 5' direction, requiring rescanning to arrive once more at a start codon. Direct observation of scanning ribosomes provides a mechanistic framework for translational regulation by 5'UTR structures and upstream near-cognate start codons. One Sentence SummaryDirect observation of scanning eukaryotic ribosomes establishes a quantitative framework of scanning and its regulation.

biophysics↗