Isabella Pirozzolo

Seminal fluid RNA is essential for embryonic development, but the mechanism by which extracellular RNA is transferred from seminal fluid to sperm remains unclear. Here, we identify a role for functional amyloid fibrils in seminal fluid that bind and traffic RNA to mammalian sperm and, upon fertilization, the zygote. We show that highly cationic peptides in seminal fluid phase separate with RNA to form amyloid fibrils. Cryo-electron tomography reveals that these amyloid fibrils are internalized by sperm. Furthermore, biophysical and in vitro fertilization experiments show that amyloid-bound RNA reaches the zygote where molecular chaperones control release of RNA for translation. Our work uncovers a mechanism for RNA trafficking from seminal fluid to the zygote, establishing amyloid fibrils as potential tools for RNA delivery during fertilization.

Ultra-long-range genomic contacts, which are key components of neuronal genome architecture^1,2,3, constitute a biochemical enigma. This is because regulatory DNA elements make selective and stable contacts with DNA sequences located hundreds of kilobases away, instead of interacting with proximal sequences occupied by the exact same transcription factors^1,4. This is exemplified in olfactory sensory neurons (OSNs), in which only a fraction of LHX2-, EBF1- and LDB1-bound sites interact with each other, converging into highly selective multi-chromosomal enhancer hubs⁵. To obtain biochemical insight into this process, here we assembled olfactory receptor (OR) enhancer hubs in vitro with recombinant proteins and enhancer DNA. Cell-free reconstitution of enhancer hubs revealed that OR enhancers form nucleoprotein condensates with unusual, solid-like characteristics. Assembly of these solid condensates is orchestrated by specific DNA motifs enriched in OR enhancers, which are likely to confer distinct homotypic properties on their resident LHX2–EBF1–LDB1 complexes. Single-molecule tracking and pulse-chase experiments in vivo confirmed that LHX2 and EBF1 assemble OR-transcription-competent condensates with solid properties in OSN nuclei, under physiological concentrations of protein. Thus, homophilic nucleoprotein interactions that are influenced by DNA sequence generate new types of biomolecular condensate, which might provide a generalizable explanation for the stability and specificity of long-range genomic contacts across cell types.

Ultra long-range genomic contacts, which emerge as prominent components of genome architecture, constitute a biochemical paradox. This is because regulatory DNA elements make selective and stable contacts with DNA sequences located megabases away, instead of interacting with proximal sequences occupied by the same exact transcription factors (TF). This is exemplified in olfactory sensory neurons (OSNs), where only a fraction of Lhx2/Ebf1/Ldb1-bound sites interact with each other, converging into highly selective multi-chromosomal enhancer hubs. In vitro hub reconstitution reveals that TF motif variations impose distinct homotypic properties to their resident Lhx2/Ebf1/Ldb1 complexes, enabling formation of nucleoprotein condensates with solid phase characteristics. Live imaging and single molecule tracking of Lhx2/Ebf1 proteins in cultured OSNs confirm that assembly of transcription-competent solid condensates occurs in vivo under physiological protein concentrations. Thus, DNA sequence-induced homophilic nucleoprotein interactions provide a generalizable explanation for the stability and specificity of long-range genomic contacts that control cellular identity and function.

Purpose of review: The microbiota in mammalian hosts can affect maturation and function of the immune system and has been associated with health and disease. We will review new findings on how this dynamic environmental factor impacts alloimmunity and therapy in transplant hosts. Recent findings: The microbiota changes after transplantation and immunosuppressive therapy. New data indicate that different microbial community structures have distinct impact on graft outcome, from promoting, to inhibiting or being neutral to transplant survival. In addition, we will address reciprocal interactions between the microbiota and immunosuppressive drugs, as well as the suitability of the microbiota as a predictive biomarker and its utility as adjunct therapy in transplantation. Summary: Advances in microbiome sequencing and wider availability of gnotobiotic facilities are enabling mechanistic investigations into the commensal communities and pathways that modulate allograft outcome, responsiveness to immunosuppression and side effects of drugs. A better understanding of the functions of the microbiota may help mitigate drug toxicity, predict drug dosage and dampen alloimmunity in transplant patients.

Solid organ transplantation can treat end-stage organ failure, but the half-life of transplanted organs colonized with commensals is much shorter than that of sterile organs. Whether organ colonization plays a role in this shorter half-life is not known. We have previously shown that an intact whole-body microbiota can accelerate the kinetics of solid organ allograft rejection in untreated colonized mice, when compared with germ-free (GF) or with antibiotic-pretreated colonized mice, by enhancing the capacity of antigen-presenting cells (APCs) to activate graft-reactive T cells. However, the contribution of intestinal versus skin microbiota to these effects was unknown. Here, we demonstrate that colonizing the skin of GF mice with a single commensal, Staphylococcus epidermidis , while preventing intestinal colonization with oral vancomycin, was sufficient to accelerate skin graft rejection. Notably, unlike the mechanism by which whole-body microbiota accelerates skin graft rejection, cutaneous S . epidermidis did not enhance the priming of alloreactive T cells in the skin-draining lymph nodes. Rather, cutaneous S . epidermidis augmented the ability of skin APCs to drive the differentiation of alloreactive T cells. This study reveals that the extraintestinal donor microbiota can affect transplant outcome and may contribute to the shorter half-life of colonized organs.