El silenciamiento génico de Mucor circinelloides regula la cromatina centromérica y la virulencia

Resumen

Since its discovery in the early 1990s, post-transcriptional gene silencing or RNA interference (RNAi) has revolutionized the fields of genetics and molecular biology and continues to inspire groundbreaking research across the scientific community. The presence of double-stranded RNA molecules triggers the RNAi mechanism to recognize and direct degradation of complementary messenger RNAs or prevent their translation into protein. Originally a defense mechanism against invasive nucleic acids -exogenous or endogenous- it has evolved into a complex regulatory phenomenon that not only protects the genome but also controls gene expression. RNAi functionality relies on short RNA molecules (sRNAs) to guide catalytic complexes to their targets by RNA base pairing. This mechanism is conserved in every major eukaryotic lineage, though there are diverse biogenetic pathways that result in different types of sRNAs. The fungal kingdom is an excellent example of this diversity because it encompasses many RNAi pathways involved in controlling the vegetative and sexual stages of their lifecycles. Here in this work, we propose the early-diverging fungus Mucor circinelloides as a model to study the role of RNAi in controlling essential biological processes involved in chromosome function and pathogenesis. M. circinelloides harbors canonical and non-canonical RNAi pathways that are intertwined to regulate genome expression, stability, and transmission, having an impact on its singular ecology. A ubiquitous inhabitant of the soil, M. circinelloides displays a saprophytic lifestyle, but can become an opportunistic animal pathogen and cause an often-lethal infectious disease known as mucormycosis. We identified M. circinelloides conserved homologs of the kinetochore complex, a protein bridge that binds the centromeres to the microtubules during cell division. Surprisingly, M. circinelloides and all of the Mucorales lack the essential centromeric histone H3 variant CENP-A that binds directly to the centromeric chromatin but retain most of the remaining kinetochore proteins. A functional analysis and ChIP-seq assay of conserved kinetochore proteins discovered nine centromeres that anchor kinetochores throughout the cell cycle in a monocentric arrangement. M. circinelloides mosaic centromeres bear features of the genetically-defined point centromeres, like their short length and a highly conserved DNA motif; while also exhibiting regional centromere determining characteristics, mainly their large pericentric regions colonized by a retrotransposable element. This retrotransposon is similar to the human LINE1 and conserved in all species belonging to the subphylum Mucoromycotina that lack CENP-A. Thus, we named it genomic retroelement of Mucoromycotina LINE1-like, or Grem-LINE1, and proposed its involvement in centromere identity in the absence of CENP-A. Grem-LINE1 sequences are being actively silenced by canonical sRNAs, indicating that RNAi is involved in maintaining genome stability and determining centromere identity in M. circinelloides. Intriguingly, there is an increased number of canonical antisense sRNAs targeting Grem-LINE1 sequences in mutants lacking the non-canonical RNAi pathway (NCRIP). Hence, our data support previous observations of an antagonistic interaction of the non-canonical over the canonical RNAi pathway in M. circinelloides. This coordinated RNAi regulation plays a critical role in the interaction with the host innate immune defenses. A comparison between two pathotypes with opposite virulence potentials revealed the genetic response involved in survival and germination during macrophage phagocytosis. The hostile phagosomal environment triggers this response by inducing two basic leucine-zipper activating transcription factors (Atf), Atf1 and Atf2, and remodeling a vast gene network that includes genes encoding an aquaporin aqp1, and two putative membrane-bound or secreted effectors chi1 and pps1. Most of the principal components of this Atf-mediated germination pathway are also induced during in vivo interaction with peritoneal mouse macrophages and are needed for a full virulence in a murine infection model. NCRIP exerts a fine control of this response by repressing it during non-stressful or saprophytic conditions and releasing it upon stressful challenges like those encountered during phagocyte interaction. Our data suggest that NCRIP activity is restrained during macrophage phagocytosis, correlating with an increased expression of the canonical RNAi genes. This increased expression is also observed in mutants lacking NCRIP activity. As a result, NCRIP mutants are unable to repress the response to phagocytosis and develop a constitutive pre-exposure adaptation to stressful conditions that protects them from oxidative damage. This is manifested and explained at a transcriptional level because the transcriptomic profile of these NCRIP mutants cultured under non-stressful conditions mimics most of the wild-type response to macrophage phagocytosis. Surprisingly, the NCRIP mutants are significatively less virulent than a wild-type strain, suggesting that the genetic deregulation provoked by the lack of NCRIP activity affects other fungal processes required for virulence. Overall, our results offer a detailed analysis of how two interacting RNAi pathways in M. circinelloides contribute to essential biological functions and provide insights into its virulence traits, which could lead to therapeutic breakthroughs against the difficult-to-treat mucormycosis.