Back-spliced RNA from Retrotransposon Binds to Centromere and Regulates Centromeric Chromatin Loops in Maize

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 retrotransposon-derived back-spliced RNA can bind to the centromere through R-loops and regulate the formation of centromeric chromatin loops. Multiple RNA sequences from centromeric retrotransposons (CRMs) were enriched in maize (Zea mays) centromeres and back spliced RNA from CRM1 were detected. We identified three types of circular CRM1 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 RNAi was used to target the back-spliced 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 that centromeric chromatin organization by circular CRM1 RNAs via R-loops and chromatin loops. R-loops are integral components of centromeric chromatin. Proper centromere structure is essential for CENH3 localization. CRM1 elements may have helped to build a suitable chromatin environment during centromere evolution. Young leaves were cross-linked in 0.4 M sucrose, 10 mM Tris-HCl (pH = 8), 1 mM EDTA, 1 mM PMSF and 1% formaldehyde in a vacuum for 30 min, after which the reaction was terminated by adding 2 M glycine. The leaves were washed three times with RNase-free water, and Kimwipes (Kimberly-Clark Professional) were used to remove water drops from the leaves. The leaves were then transferred to liquid nitrogen and ground into a fine powder. The following experiment was carried out using the native ChIP protocol as previously described with slight modification. The chromatin was digested into single nucleosomes using 0.02 U/μl DNase I (RNase-free) at 37°C. Dynabeads Protein A (ThermoFisher, Cat#:10001D) was used for binding the anti-CENH3 antibody. After washing the beads incubated with the antibody and chromatin fragments, the beads were eluted twice at 65°C for 15 min using elution buffer containing 50 mM Tris-HCl (pH = 7.5), 10 mM EDTA, 200 mM NaCl and 1% SDS. The eluted fractions were combined, and Proteinase K (Invitrogen, Cat#: 25530-49) was added to relieve cross-linking at 37°C overnight after which the RNA was extracted using TRIzol reagent, and the residual DNA was removed using RNase-free DNase I (NEB, Cat#: M0303S). The cDNA libraries of RIP samples were prepared using the standard manual provided in the NEB Next Ultra RNA Library Prep Kit for Illumina (NEB, Cat#: E7530S). The samples were applied to the Illumina HiSeq 2500 sequencing system, generating ~37 million 125-bp paired-end reads.