Rio1 downregulates centromeric RNA levels to promote the timely assembly of structurally fit kinetochores.

Kinetochores assemble on centromeres via histone H3 variant CENP-A and low levels of centromere transcripts (cenRNAs). The latter are ensured by the downregulation of RNA polymerase II (RNAPII) activity, and cenRNA turnover by the nuclear exosome. Using S. cerevisiae, we now add protein kinase Rio1 to this scheme. Yeast cenRNAs are produced either as short (median lengths of 231 nt) or long (4458 nt) transcripts, in a 1:1 ratio. Rio1 limits their production by reducing RNAPII accessibility and promotes cenRNA degradation by the 5'-3'exoribonuclease Rat1. Rio1 similarly curtails the concentrations of noncoding pericenRNAs. These exist as short transcripts (225 nt) at levels that are minimally two orders of magnitude higher than the cenRNAs. In yeast depleted of Rio1, cen- and pericenRNAs accumulate, CEN nucleosomes and kinetochores misform, causing chromosome instability. The latter phenotypes are also observed with human cells lacking orthologue RioK1, suggesting that CEN regulation by Rio1/RioK1 is evolutionary conserved.

Centromere and Pericentromere Transcription: Roles and Regulation in Sickness and in Health.

The chromosomal loci known as centromeres (CEN) mediate the equal distribution of the duplicated genome between both daughter cells. Specifically, centromeres recruit a protein complex named the kinetochore, that bi-orients the replicated chromosome pairs to the mitotic or meiotic spindle structure. The paired chromosomes are then separated, and the individual chromosomes segregate in opposite direction along the regressing spindle into each daughter cell. Erroneous kinetochore assembly or activity produces aneuploid cells that contain an abnormal number of chromosomes. Aneuploidy may incite cell death, developmental defects (including genetic syndromes), and cancer (>90% of all cancer cells are aneuploid). While kinetochores and their activities have been preserved through evolution, the CEN DNA sequences have not. Hence, to be recognized as sites for kinetochore assembly, CEN display conserved structural themes. In addition, CEN nucleosomes enclose a CEN-exclusive variant of histone H3, named CENP-A, and carry distinct epigenetic labels on CENP-A and the other CEN histone proteins. Through the cell cycle, CEN are transcribed into non-coding RNAs. After subsequent processing, they become key components of the CEN chromatin by marking the CEN locus and by stably anchoring the CEN-binding kinetochore proteins. CEN transcription is tightly regulated, of low intensity, and essential for differentiation and development. Under- or overexpression of CEN transcripts, as documented for myriad cancers, provoke chromosome missegregation and aneuploidy. CEN are genetically stable and fully competent only when they are insulated from the surrounding, pericentromeric chromatin, which must be silenced. We will review CEN transcription and its contribution to faithful kinetochore function. We will further discuss how pericentromeric chromatin is silenced by RNA processing and transcriptionally repressive chromatin marks. We will report on the transcriptional misregulation of (peri)centromeres during stress, natural aging, and disease and reflect on whether their transcripts can serve as future diagnostic tools and anti-cancer targets in the clinic.

Endocytosis regulates membrane localization and function of the fusogen EFF-1.

Cell fusion is essential for sexual reproduction and formation of muscles, bones, and placenta. Two families of cell fusion proteins (Syncytins and FFs) have been identified in eukaryotes. Syncytins have been shown to form the giant syncytial trophoblasts in the placenta. The FFs are essential to fuse cells in the skin, reproductive, excretory, digestive and nervous systems in nematodes. EFF-1 (Epithelial Fusion Failure 1), a member of the FF family, is a type I membrane glycoprotein that is essential for most cell fusions in C. elegans. The crystal structure of EFF-1 ectodomain reveals striking structural similarity to class II fusion glycoproteins from enveloped viruses (e.g. dengue and rubella) that mediate virus to cell fusion. We found EFF-1 to be present on the plasma membrane and in RAB-5-positive early endosomes, with EFF-1 recycling between these 2 cell compartments. Only when EFF-1 proteins transiently arrive to the surfaces of 2 adjacent cells do they dynamically interact in trans and mediate membrane fusion. EFF-1 is continuously internalized by receptor-mediated endocytosis via the activity of 2 small GTPases: RAB-5 and Dynamin. Here we propose a model that explains how EFF-1 endocytosis together with interactions in trans can control cell-cell fusion. Kontani et al. showed that vacuolar ATPase (vATPase) mutations result in EFF-1-dependent hyperfusion. 1 We propose that vATPase is required for normal degradation of EFF-1. Failure to degrade EFF-1 results in delayed hyperfusion and mislocalization to organelles that appear to be recycling endosomes. EFF-1 is also required to fuse neurons as part of the repair mechanism following injury and to prune dendrites. We speculate that EFF-1 may regulate neuronal tree like structures via endocytosis. Thus, endocytosis of cell-cell fusion proteins functions to prevent merging of cells and to sculpt organs and neurons.

RAB-5- and DYNAMIN-1-Mediated Endocytosis of EFF-1 Fusogen Controls Cell-Cell Fusion.

Cell-cell fusion plays essential roles during fertilization and organogenesis. Previous studies in C. elegans led to the identification of the eukaryotic fusion protein (EFF-1 fusogen), which has structural homology to class II viral fusogens. Transcriptional repression of EFF-1 ensures correct fusion fates, and overexpression of EFF-1 results in embryonic lethality. EFF-1 must be expressed on the surface of both fusing cells; however, little is known regarding how cells regulate EFF-1 surface exposure. Here, we report that EFF-1 is actively removed from the plasma membrane of epidermal cells by dynamin- and RAB-5-dependent endocytosis and accumulates in early endosomes. EFF-1 was transiently localized to apical domains of fusion-competent cells. Effective cell-cell fusion occurred only between pairs of cell membranes in which EFF-1 localized. Downregulation of dynamin or RAB-5 caused EFF-1 mislocalization to all apical membrane domains and excessive fusion. Thus, internalization of EFF-1 is a safety mechanism preventing excessive cell fusion.