Construction of synthetic nucleoli and what it tells us about propagation of sub-nuclear domains through cell division

Abstract

The cell nucleus is functionally compartmentalized into numerous membraneless and dynamic, yet defined, bodies. The cell cycle inheritance of these nuclear bodies (NBs) is poorly understood at the molecular level. In higher eukaryotes, their propagation is challenged by cell division through an "open" mitosis, where the nuclear envelope disassembles along with most NBs. A deeper understanding of the mechanisms involved can be achieved using the engineering principles of synthetic biology to construct artificial NBs. Successful biogenesis of such synthetic NBs demonstrates knowledge of the basic mechanisms involved. Application of this approach to the nucleolus, a paradigm of nuclear organization, has highlighted a key role for mitotic bookmarking in the cell cycle propagation of NBs.

Keywords: 1°, primary; 2°, secondary; CBs, Cajal bodies; CDK, cyclin-dependent kinase; DFC, dense fibrillar component; DJ, distal junction; FCs, fibrillar centers; GC, granular component; HLBs, histone locus bodies; HMG, high mobility group; IGS, intergenic spacers; NBs, nuclear bodies; NORs, nucleolar organizer regions; Nucleolar Organizer Region (NOR); PJ, proximal junction; PML, promyelocytic leukemia; PNBs, pre-nucleolar bodies; TFs, transcription factors; UBF; UBF, Upstream binding factor; XEn, Xenopus enhancer; cell cycle; mitotic bookmarking; neo-NOR; neonucleoli; nuclear bodies; nucleolus; pol, RNA polymerase; pre-rRNA, precursor rRNA; pseudo-NOR; rDNA, ribosomal genes; rRNA, ribosomal RNA; RNP, ribonucleoprotein; synthetic biology; t-UTPs, transcription U 3 proteins.

Figure 1.

Schematic representation of nucleolar compartmentalization. Top left diagram represents a nucleolus within a DAPI-stained nucleus (blue). Nucleoli are subdivided into FC (green), DFC (yellow) and GC (red). FCs constitute the core of nucleoli and are surrounded by DFCs. These compartments are embedded within the GC that is typically surrounded by perinucleolar heterochromatin (dark blue). rDNA transcription by the pol I transcription machinery (UBF, SL1, Rrn3 and pol I) occurs at the interface between FCs and DFCs (white box). The resulting 47 S pre-rRNA transcripts undergo a highly coordinated series of cleavages (green arrows) and modifications to generate the mature 18 S, 5.8 S and 28 S rRNA. Early processing events occur within the DFC (yellow box), while later processing events and assembly of ribosomal subunits occur within the GC (red box). Note that pseudo-NORs support the view that FCs are the interphase counterparts of the mitotic 2° constrictions and that they contain non-transcribed rDNA sequences and unengaged pol I machinery recruited in a UBF-dependent manner (green box).

Figure 2.

UBF depletion results in loss of NOR competency. The top cartoon indicates the features of human NORs, including an array of rDNA repeats (red), surrounded by a conserved distal junction (DJ, green) and proximal junction (PJ, white). Each rDNA repeat is comprised of a transcription unit and an intergenic spacer (IGS). The DJ and PJ were named in regard to their positions relative to the centromere. Competent NORs appear as 2° constrictions, gaps in DAPI staining (blue), on metaphase chromosome (middle left) and participate in nucleolar formation in interphase (bottom left). Interestingly, DJ sequences appear as foci localized within perinucleolar heterochromatin. DJ foci identify individual NORs and indicate that mature nucleoli are comprised of multiple NORs. Note, the association of neo-NORs with endogenous human NORs has revealed the existence of NOR territories, indicated by a dashed white line (bottom left), within mature nucleoli. Upon UBF depletion, a subset of competent NORs loses UBF and become silenced. Consequently, they fail to form 2° constrictions during mitosis (middle right) and fail to participate in nucleolar formation in interphase (bottom right).

Figure 3.

Synthetic NORs’ contribution to the understanding of the nucleolar cycle. The left panel summarizes the findings that resulted from the construction of synthetic NORs. Pseudo-NORs, UBF-binding site arrays, demonstrate that UBF seeds the formation of 2° constrictions during mitosis and FCs in interphase (top). Neo-NORs, arrays of UBF-binding sites interspersed with rDNA transcription units, indicate that pre-rRNAs are the only architectural requirement for DFC and GC formation (bottom). While UBF depletion has revealed that mitotic bookmarking is necessary for nucleolar formation, pseudo-NORs establish that it is not sufficient. Thus, the cell cycle inheritance of nucleoli is a staged process (represented in the right panel). Endogenous NORs that were active in the previous interphase are bookmarked by UBF, forming mitotic 2° constrictions that retain most of the FC components. This UBF-dependent bookmarking ensures the reactivation of rDNA transcription in late telophase, with pre-rRNAs seeding the recruitment of DFC and GC factors. Hence, staging establishes a temporal order to nucleolar compartmentalization and ensures the rapid re-formation of nucleoli in early G1. For diagrammatic simplicity, individual chromosomes are merged.