Human nucleoli comprise multiple constrained territories, tethered to individual chromosomes

In this study, Mangan et al. investigated how ribosomal gene (rDNA) arrays from multiple chromosomal nucleolar organizers (NORs) partition within human nucleoli, which is complicated by the shared DNA sequence composition of five NOR-bearing acrocentric chromosome p-arms. They present a methodology for genetic manipulation of individual NORs and reveal that ribosome biogenesis occurs entirely within constrained territories, tethered to individual NORs inside a larger nucleolus.


gRNA synthesis
Most gRNAs were produced by T7 RNA polymerase transcription of templates prepared by PCR. Transcription templates were prepared in a 2-step PCR process. Firstly, a universal scaffold was prepared using ScaExF and T7gRNArev primers with an stem extension (Ex) and A-U flip (F) and modified gRNA empty vector as template (van Sluis and McStay 2015). T7 promoters and specific gRNA sequences were added by further PCR using the scaffold as template. gRNA templates were in vitro transcribed using the MEGAscript™ T7 Transcription Kit. PCR primer sequences are presented in Supplemental Table S1.

Generation of hTERT-RPE1 Hyg -ve cells
4µg of gRNA targeting immediately downstream of the start codon of the HygR orf in the hTERT expression plasmid pGRN145 was pre-complexed with 10µg Cas9 for 10mins at room temperature. RNP complexes were electroporated into 10 6 hTERT-RPE1 cells using a Nucleofector 2b, the programme T-020 and Nucleofector kit V (Lonza). The clone selected has a 23nt deletion verified (Supplemental Fig. S1). To aid selection during MMCT, cells were subsequently transfected with BSR plasmid pJRC41 and selected in 20µg/ml blastcidin (Melford).

Ribosome and Polysome purification
Purification of cytoplasmic ribosomes was performed from 5 x 15cm dishes of hTERT-RPE1 and derivative cells as described previously (Belin et al. 2010).
Ribosomal RNA was purified using a NucleoSpin RNA II kit (Machery-Nagel).
RNA was eluted in nuclease-free water and stored at -80°C.

Supplemental References
Belin To determine the percentage of nucleolar area occupied by tagged NORs, data from the experiment presented in Fig. 2C were analysed. The area of the tagged NOR, visualised by 28S tag hybridisation signal, was determined as a percentage of the area of the same nucleolus, visualised by 18SE probe hybridisation signal. 25 cells were analysed and the data presented as a box and whisker plot. The outlier is small a nucleolus, presumed to comprise only the tagged NOR (B) As above except that the area of the tagged NOR is expressed as a percentage of the summed area of all the nucleoli in that cell.
Supplemental Fig. S9. Imaging blending of NOR territories during nucleolar stress in live cells. RPE1 15/3 (28SMS2) cells were first transfected with plasmids expressing Nop52-mCherry (red) and mAG-MS2 fusion proteins. Live cells were then treated with AMD and imaged at 5 min intervals thereafter. DNA was visualised in live cells using SiR-DNA (pseudo-coloured in blue). The upper panels show merged images at 5 minute intervals, while those below show only mAG-MS2. Note the progressive spreading of mAG-MS2 throughout the nucleolar volume over the 50 minute imaging period. Scale bars 5 µm.