Isaacs, T. | bioRxiv Science

bioRxiv2023

Characterization of the RBM15 protein binding with XCI-escaping long noncoding RNAs

The RNA binding motif 15 protein (RBM15) plays a critical role in post-transcriptional regulation. Its role in facilitating N6-methyladenosine (m6A) modification, specifically through guiding the writer complex (WTAP METTL13 METTL14) to DRACH sequence motifs, has been demonstrated for several classes of RNA, including long noncoding RNAs (lncRNAs). The structural mechanism that underlies how RBM15 interacts with RNA has yet to be elucidated. In this study, we mined and bioinformatically assessed publicly available genome-wide RNA 2D structural probing and RBP cross-linking and immunoprecipitation data to investigate how RBM15 interacts with RNA, with a focus on lncRNA transcripts. RBM15, which possesses three RNA recognition motifs (RRMs), primarily interacts with stem-loop structured RNA motifs. Structural modeling reveals RRMs 2 and 3 are coaxially stacked in solution; these two RRMs are responsible for driving RBM15s interaction with RNA. We further demonstrate this experimentally with two RNA hairpins, revealing low micromolar binding affinities. Altogether, this work provides insight into the structural mechanism by which RBM15 interacts with RNAs to govern biological function.

biochemistry↗

bioRxiv2021

A potent myeloid response is rapidly activated in the lungs of premature Rhesus macaques exposed to intra-uterine inflammation

Intrauterine inflammation/infection (IUI), which is present in up to 40% of premature births, leads to elevated levels of pro-inflammatory mediators and microbial products within the amniotic fluid, which come in close contact to fetal mucosae. Yet, knowledge on the fetal mucosal responses to IUI exposure remains limited. To address these questions, we used a non-human primate model of IUI, in which pregnant Rhesus macaques received intra-amniotic (IA) LPS, compared with IA saline. We found that IA LPS exposure induced a robust and rapid inflammation of the fetal lung, but not the intestine. This inflammatory response was characterized by high levels of pro-inflammatory cytokines in the lung and the alveolar wash, and a potent myeloid cell response, dominated by neutrophils and monocytes/macrophages. scRNAseq analyses of fetal lungs showed that the infiltrating (neutrophils and inflammatory monocytes) and the resident (alveolar and interstitial macrophages) myeloid cells exhibited transcriptional profiles consistent with exposure to TLR ligands, as well as to cytokines, notably IL-1 and TNF. However, blocking IL-1 signaling or TNF, alone or simultaneously by administering inhibitors intra-amniotically and subcutaneously to the dam only partially blunted fetal lung inflammation. Together, our novel data indicate that the fetal innate immune system can mount a rapid multi-factorial mucosal innate response to IUI, responding both to direct signaling by bacterial products and to indirect cytokine-mediated pathways of activation. These data thus provide more mechanistic insights into the association between IUI exposure and the post-natal lung morbidities of the premature infant.

immunology↗