Abstract
In most plants, centromeric DNA contains highly repetitive sequences, including tandem repeats and retrotransposons; however, the roles of these sequences in the structure and function of the centromere are unclear. Here, we found that multiple RNA sequences from centromeric retrotransposons (CRMs) were enriched in maize (Zea mays) centromeres, and back-spliced RNAs were generated from CRM1. We identified 3 types of CRM1-derived circular RNAs with the same back-splicing site based on the back-spliced sequences. These circular RNAs bound to the centromere through R-loops. Two R-loop sites inside a single circular RNA promoted the formation of chromatin loops in CRM1 regions. When RNA interference (RNAi) was used to target the back-splicing site of the circular CRM1 RNAs, the levels of R-loops and chromatin loops formed by these circular RNAs decreased, while the levels of R-loops produced by linear RNAs with similar binding sites increased. Linear RNAs with only one R-loop site could not promote chromatin loop formation. Higher levels of R-loops and lower levels of chromatin loops in the CRM1 regions of RNAi plants led to a reduced localization of the centromeric H3 variant (CENH3). Our work reveals centromeric chromatin organization by circular CRM1 RNAs via R-loops and chromatin loops, which suggested that CRM1 elements might help build a suitable chromatin environment during centromere evolution. These results highlight that R-loops are integral components of centromeric chromatin and proper centromere structure is essential for CENH3 localization.
Highlights
Centromeres are located in the primary constriction of chromosomes and enable the correct separation of chromosomes during mitosis and meiosis
Abundant reads from anti-centromeric H3 variant (CENH3) RNA immunoprecipitation (RIP) sequencing (RIP-seq) data were generated from the centromeric repeats, including CRM1, CRM2, CentA (CentA was the first centromeric retrotransposon (CRM) element identified in maize, and CRM3 is related to the nonautonomous CentA [11]), and CentC (S1B Fig)
We identified a 354-nt back-spliced RNA derived from CRM1, which binds to centromeric regions
Summary
Centromeres are located in the primary constriction of chromosomes and enable the correct separation of chromosomes during mitosis and meiosis. Active centromeres are marked by a centromeric specific histone H3 variant, named CENH3 in plants [1,2] and centromeric protein A (CENP-A) in animals [3,4]. Centromeric DNA contains hierarchical arrays of highly repetitive sequences; in humans, centromeric repeats consist mostly of simple tandem repeats [5,6], whereas in plants, multiple retrotransposons are intermingled with. Back-spliced RNA and chromatin structure in maize centromeres
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