Review
Pseudo-NORs: A novel model for studying nucleoli
José-Luis Prieto, Brian McStay
⁎
Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY Scotland, UK
abstractarticle info
Article history:
Received 23 April 2008
Received in revised form 8 July 2008
Accepted 8 July 2008
Available online 18 July 2008
Keywords:
Nucleolus
Nucleolar organiser region
Ribosomal gene
UBF
Pseudo-NOR
Nucleolar organiser regions (NORs) are comprised of tandem arrays of ribosomal gene (rDNA) repeats that
are transcribed by RNA polymerase I (Pol I), ultimately resulting in formation of a nucleolus. Upstream
binding factor (UBF), a DNA binding protein and component of the Pol I transcription machinery, binds
extensively across the rDNA repeat in vivo. Pseudo-NORs are tandem arrays of a heterologous DNA sequence
with high affinity for UBF introduced into human chromosomes. In this review we describe how analysis of
pseudo-NORs has provided important insights into nucleolar formation. Pseudo-NORs mimic endogenous
NORs in a number of important respects. On metaphase chromosomes both appear as secondary
constrictions comprised of undercondensed chromatin. The transcriptional silence of pseudo-NORs provides
a platform for studying the transcription independent recruitment of factors required for nucleolar formation
by this specialised chromatin structure. During interphase, pseudo-NORs appear as distinct and novel sub-
nuclear bodies. Analysis of these bodies and comparison to their endogenous counterpart has provided
insights into nucleolar formation and structure.
© 2008 Elsevier B.V. All rights reserved.
1. Introduction
The eukaryotic nucleus is highly compartmentalised. Partially
decondensed chromosomes occupy discrete chromosome territories
[1–3]. Regulatory proteins are often present in discrete nuclear bodies
[4]. This compartmentalisation has fuelled a large body of research
into how genes interact with nuclear bodies or their constituent
proteins resulting in the formation of transcription factories. The
nucleolus is both the most prominent body and the largest transcrip-
tion factory and represents a paradigm for studying organisation of
gene expression within the nucleus (for recent reviews see [5–7]). The
primary function of nucleoli is ribosome biogenesis. They form around
ribosomal gene (rDNA) repeats and their formation is strictly
dependent on transcription by RNA polymerase I (Pol I). Uniquely,
the genetic loci containing rDNA repeats, termed Nucleolar Organiser
Regions (NORs), can be visualised throughout the cell cycle. During
mitosis when transcription is inactivated and the nucleolus is broken
down, NORs are undercondensed and visible as a secondary constric-
tion on metaphase chromosomes [8]. Pseudo-NORs are novel
artificially constructed arrays that behave in many respects like true
NORs but are transcriptionally silent [9,10]. Here we will review how
analysis of pseudo-NORs has provided important insights into both
secondary constriction and nucleolar formation.
2. Organisation of human rDNA
Human ribosomal gene (rDNA) repeats comprise ∼43 kb [11,12].
Sequences encoding pre-rRNA (13 kb) are separated by long
intergenic spacers (IGSs) of approximately 30 kb. Regulatory
elements, including gene promoters and transcription terminators,
are located in the IGS. The 47S pre-rRNA is processed by a series of
endo and exonucleolytic cleavages to generate one molecule each of
18S, 5.8S and 28S rRNA. In situ hybridisation experiments have
revealed that clusters of rDNA repeats (NORs), are located on the
short arms of the five human acrocentric chromosomes, chromo-
somes 13, 14, 15, 21 and 22 [13]. Pulse-field gel electrophoresis of
genomic DNA digested with enzymes that do not cut human rDNA,
such as EcoRV and Sse83871, revealed a major rDNA band of 3 Mb as
well as several minor bands of 1 and 2 Mb [14]. This implies that most
human NORs are comprised of approximately 70 copies of rDNA
repeats and demonstrates that NORs contain only rDNA. rDNA
repeats are oriented in a telomere to centromere direction [15,16].
More recently this view of rDNA organisation within NORs has proven
to be too simplistic. Single DNA molecule analysis by molecular
combing has revealed that NORs comprise a mosaic of canonical and
non-canonical rDNA repeats [17]. As much as one third of rDNA
repeats are non-canonical, apparently forming palindromic struc-
tures. In human chromosomes rDNA repeats appear to be the only
genes present on acrocentric short arms, thus isolating them from
genes transcribed by Pol II. Isolation of rDNA is further reinforced by
the heterochromatised satellite repeats that comprise much of the
DNA sequence on either side of NORs [18–21].
Biochimica et Biophysica Acta 1783 (2008) 2116–2123
⁎ Corresponding author. Tel.: +353 91 492423; fax: +353 91 525700.
E-mail address: brian.mcstay@nuigalway.ie (B. McStay).
0167-4889/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbamcr.2008.07.004
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