DNA contains all the genetic information necessary to build and maintain an organism. Inside the cell nucleus, DNA is wrapped around histone proteins to form chromatin fibers. Depending on the degree of compaction, the chromatin is classified as euchromatin (less condensed, gene-rich, and more accessible to transcription machinery) or heterochromatin (highly condensed, gene-poor, and transcriptionally silent; Huisinga et al., 2006; Figure 1A).
Heterochromatin had long been considered as genetically inert. However, it turned out to be crucial to the biology of cells and contributes, for example, to chromosome segregation during cell division. It is also important for silencing mobile genetic elements – segments of DNA that can move within the genome or jump into the genome of other cells. Mobile genetic elements can pose a real threat to the integrity of the genome, and consequently, several defence mechanisms have evolved to identify them and prevent them from multiplying.
Some heterochromatic loci, formed of remnants of mobile genetic elements, are at the center of these defence mechanisms. These parts of the genome, also known as piRNA clusters, are often compared to a ‘memory system’. Despite being heterochromatic and usually silent, they are transcribed and processed into small non-coding RNAs. These RNAs are called PIWI-interacting RNAs, or piRNAs for short, because they are loaded by proteins of the PIWI family (Figure 1B). They use sequence complementarity to recognize mobile genetic elements residing in other parts of the genome, and then silence them (Brennecke et al., 2007).
In many eukaryotes, the molecular feature defining heterochromatin is the enrichment of a specific histone mark called H3K9me3. This mark is recognized by proteins belonging to the HP1 (Heterochromatin Protein 1) family, which help to pack DNA into its condense structure (Bannister et al., 2001; Lachner et al., 2001). A member of this family, known as Rhino, facilitates the transcription of piRNA clusters in the germline of fruit flies. Although Rhino displays specific affinity for H3K9me3, it only binds to a subset of heterochromatin regions that contain this histone mark. So far, it was unclear how Rhino is guided to these specific parts of the genome (Mohn et al., 2014). Now, in eLife, Julius Brennecke and colleagues – including Lisa Baumgartner as first author – report the results of experiments that clarify this process (Baumgartner et al., 2022).
The team (who are based at the Vienna BioCenter and the Institute of Molecular Biotechnology of the Austrian Academy of Sciences) used a combination of genetic, genomic and imaging approaches to study Rhino in the germline of female fruit flies. The experiments revealed that Rhino interacts with a DNA-binding protein, which Baumgartner et al. named Kipferl. Indeed, a depletion of this protein in the ovaries of fruit flies leads to a broad redistribution and concentration of Rhino at the nuclear periphery in a form that evokes the shape of a Kipferl, an Austrian croissant.
They found that both Kipferl and Rhino are bound at many piRNA source loci, suggesting that Kipferl may act to identify the heterochromatin regions to which Rhino must bind. In the absence of Kipferl, Rhino is sequestered to another part of the genome and its binding to some piRNA clusters is lost, despite the presence of H3K9me3 marks. As a result, piRNA production in ovaries lacking Kipferl is reduced, several mobile elements are reactivated, and the females are less fertile than flies expressing Kipferl.
Kipferl is a DNA-binding protein that specifically binds to DNA sequences that are rich in the guanine nucleotide. This study suggests for the first time that such DNA sequences could help to attract Kipferl, which then recruits Rhino participate in defining piRNA clusters. However, while Kipferl guides and stabilizes Rhino to some chromatin domains enriched in H3K9me3 to convert them into piRNA clusters, some Rhino-dependent piRNA clusters do not need Kipferl (Figure 1B).
The study of Baumgartner et al. also suggests that additional factors help guide Rhino to piRNA clusters during early oogenesis, as Kipferl is not expressed during this developmental stage. Furthermore, piRNAs provided by the mothers may also help to recruit Rhino to specific heterochromatin regions in the embryo (Akkouche et al., 2017; Le Thomas et al., 2014). However, the relative contribution of maternally deposited piRNAs and Kipferl in recruiting Rhino to specify piRNA clusters during embryogenesis requires future investigations.
The study of Baumgartner et al. shows that the relationship between Rhino, Kipferl and DNA is complex, and their elegant dissection of the role of Kipferl provides substantial new insight into how piRNA clusters are defined in the genome.
Alternative polyadenylation yields many mRNA isoforms whose 3’ termini occur disproportionately in clusters within 3’ untranslated regions. Previously, we showed that profiles of poly(A) site usage are regulated by the rate of transcriptional elongation by RNA polymerase (Pol) II (Geisberg et al., 2020). Pol II derivatives with slow elongation rates confer an upstream-shifted poly(A) profile, whereas fast Pol II strains confer a downstream-shifted poly(A) profile. Within yeast isoform clusters, these shifts occur steadily from one isoform to the next across nucleotide distances. In contrast, the shift between clusters – from the last isoform of one cluster to the first isoform of the next – is much less pronounced, even over large distances. GC content in a region 13–30 nt downstream from isoform clusters correlates with their sensitivity to Pol II elongation rate. In human cells, the upstream shift caused by a slow Pol II mutant also occurs continuously at single nucleotide resolution within clusters but not between them. Pol II occupancy increases just downstream of poly(A) sites, suggesting a linkage between reduced elongation rate and cluster formation. These observations suggest that (1) Pol II elongation speed affects the nucleotide-level dwell time allowing polyadenylation to occur, (2) poly(A) site clusters are linked to the local elongation rate, and hence do not arise simply by intrinsically imprecise cleavage and polyadenylation of the RNA substrate, (3) DNA sequence elements can affect Pol II elongation and poly(A) profiles, and (4) the cleavage/polyadenylation and Pol II elongation complexes are spatially, and perhaps physically, coupled so that polyadenylation occurs rapidly upon emergence of the nascent RNA from the Pol II elongation complex.
To mount a protective response to infection while preventing hyperinflammation, gene expression in innate immune cells must be tightly regulated. Despite the importance of pre-mRNA splicing in shaping the proteome, its role in balancing immune outcomes remains understudied. Transcriptomic analysis of murine macrophage cell lines identified Serine/Arginine Rich Splicing factor 6 (SRSF6) as a gatekeeper of mitochondrial homeostasis. SRSF6-dependent orchestration of mitochondrial health is directed in large part by alternative splicing of the pro-apoptosis pore-forming protein BAX. Loss of SRSF6 promotes accumulation of BAX-κ, a variant that sensitizes macrophages to undergo cell death and triggers upregulation of interferon stimulated genes through cGAS sensing of cytosolic mitochondrial DNA. Upon pathogen sensing, macrophages regulate SRSF6 expression to control the liberation of immunogenic mtDNA and adjust the threshold for entry into programmed cell death. This work defines BAX alternative splicing by SRSF6 as a critical node not only in mitochondrial homeostasis but also in the macrophage’s response to pathogens.