Meiotic DNA Repair in the Nucleolus Employs a Nonhomologous End-Joining Mechanism

Abstract

Ribosomal RNA genes are arranged in large arrays with hundreds of rDNA units in tandem. These highly repetitive DNA elements pose a risk to genome stability since they can undergo nonallelic exchanges. During meiosis, DNA double-strand breaks (DSBs) are induced as part of the regular program to generate gametes. Meiotic DSBs initiate homologous recombination (HR), which subsequently ensures genetic exchange and chromosome disjunction. In Arabidopsis (Arabidopsis thaliana), we demonstrate that all 45S rDNA arrays become transcriptionally active and are recruited into the nucleolus early in meiosis. This shields the rDNA from acquiring canonical meiotic chromatin modifications and meiotic cohesin and allows only very limited meiosis-specific DSB formation. DNA lesions within the rDNA arrays are repaired in an RAD51-independent but LIG4-dependent manner, establishing that nonhomologous end-joining maintains rDNA integrity during meiosis. Utilizing ectopically integrated rDNA repeats, we validate our findings and demonstrate that the rDNA constitutes an HR-refractory genome environment.

Sequencing and analysis ofArabidopsis thalianaNOR2 reveal its distinct organization and tissue-specific expression of rRNA ribosomal variants

“…It is also interesting to note that we could never detect rRNAs bearing the CAT insertion in any of our and previously published [22][23][24][25] total RNA sequencing datasets. This indicates that even though both NORs are transcriptionally active in young seedlings 29 and in inflorescences 19 , not all repeats of NOR2 are transcribed. This is in contrast to what was previously hypothesized since NOR2 is not transcriptionally silent in adult somatic tissues.…”

“…Utilizing an LNA probe specifically directed against the CAT insertion rDNA unit type we detected signals only on NOR2. In addition, the probe allowed us to visualize rDNA expansion and re-arrangement events in the hda6-6, nuc2-2 and rtel1-1 mutant lines which are known to have unstable rDNA copy numbers 19,20 . The expansions were further corroborated by qPCR experiments.…”

“…We designed an LNA probe (CAT+) that specifically detects the CAT insertion located within NOR2. We performed a FISH experiment on spreads of pollen mother cells (PMCs) also employing an LNA probe directed against a universal rDNA sequence (SalI probe), detecting all 45S rDNA in Arabidopsis 19 ( Fig. 4a).…”

“…rtel1-1, nuc2-2 and hda6-6 mutants [19][20][21] . In the rtel1-1, nuc2-2 and hda6-6 mutants we detected a different localization of the CAT+ probe.…”

“…This set of experiments confirms that the rRNA gene variants we detect in the rDNA are expressed in a tissue-specific manner and present in translating ribosomes. Furthermore, the presence of only 25S rRNA variants suggests that NOR4 is mainly contributing to the pool of ribosomes even within tissues that have both NOR2 and NOR4 transcriptionally active (Young Leaves and Inflorescences) 6,19 .…”