Torres-Ulloa, L. | bioRxiv Science

bioRxiv2024

Raver1 links Ripk1 RNA splicing to caspase-8-mediated pyroptotic cell death, inflammation, and pathogen resistance

Multiple cell death and inflammatory signaling pathways converge on two critical factors: receptor interacting serine/threonine kinase 1 (RIPK1) and caspase-8. Careful regulation of these molecules is critical to control apoptosis, pyroptosis and inflammation. Here we discovered a pivotal role of Raver1 as an essential regulator of Ripk1 pre-mRNA splicing, expression, and functionality, and the subsequent caspase-8-dependent inflammatory cell death. Macrophages from Raver1-deficient mice exhibit altered splicing of Ripk1, accompanied by diminished cell death and reduced activation of caspase-8, Gasdermin D and E, caspase-1, as well as decreased interleukin-18 (IL-18) and IL-1{beta} production. These effects were triggered by Yersinia bacteria, or by restraining TAK1 or IKK{beta} in the presence of LPS, TNF family members, or IFN{gamma}. Consequently, animals lacking Raver1 showed heightened susceptibility to Yersinia infection. Raver1 and RIPK1 also controlled the expression and function of the C-type lectin receptor Mincle. Our study underscores the critical regulatory role of Raver1 in modulating innate immune responses and highlights its significance in directing in vivo and in vitro inflammatory processes. SignificanceCaspase-8 and the kinase RIPK1 are at focal points of several inflammation and cell death pathways. Thus, a careful regulation of their actions is needed. Our work identifies the RNA splicing factor Raver1 as a critical factor directing the splicing of Ripk1 in order to modulate RIPK1/caspase-8-driven pyroptosis, apoptosis and inflammation. Raver1 is central for macrophage responses to Yersinia bacteria, initiated after blockade of kinases TAK1 and IKK, measured as activation of RIPK1, caspase-8, Gasdermin D, caspase-3, IL-1{beta} and IL-18. Importantly, Raver1 is necessary for host resistance to Yersinia infection in vivo. We propose that Raver1 is key for correct tuning of RIPK1-caspase-8 dependent processes.

immunology↗

bioRxiv2023

Genome-wide kinetic profiling of pre-mRNA 3' end cleavage

Cleavage and polyadenylation is necessary for the formation of mature mRNA molecules. The rate at which this process occurs can determine the temporal availability of mRNA for subsequent function throughout the cell and is likely tightly regulated. Despite advances in high-throughput approaches for global kinetic profiling of RNA maturation, genome-wide 3 end cleavage rates have never been measured. Here, we describe a novel approach to estimate the rates of cleavage, using metabolic labeling of nascent RNA, high-throughput sequencing, and mathematical modeling. Using in-silico simulations of nascent RNA-seq data, we show that our approach can accurately and precisely estimate cleavage half-lives for both constitutive and alternative sites. We find that 3 end cleavage is fast on average, with half-lives under a minute, but highly variable across individual sites. Rapid cleavage is promoted by the presence of canonical sequence elements and an increased density of polyadenylation signals near a cleavage site. Finally, we find that cleavage rates are associated with the localization of RNA Polymerase II at the end of a gene and faster cleavage leads to quicker degradation of downstream read-through RNA. Our findings shed light on the features important for efficient 3 end cleavage and the regulation of transcription termination.

genomics↗