404
Cell Nucleus Biogenesis, Structure and Function
as the SR protein, family, which is de-
Fned by a speciFc serine and arginine rich
motif – and occupies the interchromatin
spaces where the proteins may accumulate
to form large, clustered aggregates. Once
formed, these aggregates – the speckles or
IGCs – are stable in that they occupy a par-
ticular area of nuclear space over many
hours. This spatial stability is seen even
though the components within each site
are in a continual state of flux. However,
the IGCs are also plastic and can change
dramatically under conditions in which
RNA synthesis is inhibited and the need
for splicing is lost; if cells are treated with
the transcriptional inhibitor
α
-amanitin,
the speckles lose their complex surface
texture and collapse locally to form more
dense spherical aggregates. This and the
fact that highly active genes are commonly
found at the periphery of speckles imply
some functional link. Interestingly, cells
that overexpress the serine-arginine (SR)
protein kinase cdc2-like kinase (Clk)/STY
exhibit disrupted speckles. This kinase
hyper-phosphorylates a major IGC com-
ponent, the nuclear SR proteins, which,
as a result move out of the IGCs to dif-
fuse throughout the nucleus. Cells without
IGCs show no obvious defects in short-
term RNA synthesis. However, under the
same conditions RNA ±ISH to a speciFc
transcript shows that splicing, assessed us-
ing a probe to an exon–exon junction,
is severely impaired. This implies that
splicing factors originating from a diffuse
nucleoplasmic pool are not competent to
perform splicing in vivo and that the orga-
nization of the IGC compartment is critical
to this process.
3.5.5
Active Sites – Synthetic Factories
Transcription factories
It is generally ac-
cepted that mammalian nuclei are highly
compartmentalized and that the structure
of different nuclear domains is implicit
to their function. As we have seen, nu-
cleoli provide the outstanding example of
a nuclear compartment that serves a spe-
ciFc function. Nucleoli are dedicated to
the efFcient synthesis of a single primary
RNA transcript and the products generated
from this transcript by RNA processing are
fundamental to ribosome assembly and
function. Within nucleoli, morphologically
distinct compartments deFne the active
centers, where, as many as 500 engaged
polymerase complexes are found within a
typical ±C/D±C complex. In terms of struc-
ture, the nucleolus represents a nuclear
compartment that is dedicated to the syn-
thesis and processing of a speciFc product.
This might lead us to ask if the same
principle applies to transcripts that are
synthesized and processed throughout the
nucleus. Given the relative sizes and com-
plexity of the corresponding sites, it is
not surprising that the architecture of
the nucleoplasmic transcription centers is
less clearly deFned. Classical electron mi-
croscopy techniques have shown that gene
expression generally occurs at the borders
of condensed chromatin in association
with perichromatin Fbrils. The ability to la-
bel the nascent RNA (±ig. 5) using BrUTP
in vitro
or BrU
in vivo
has shown that a pro-
liferative mammalian cell has some 500 to
1000 active sites of RNA polymerase II ac-
tivity for each haploid chromosome set. As
cells have roughly 10 times the number
of active RNA polymerase II holoenzyme
complexes, each active center must repre-
sent a nuclear compartment in which the
transcripts from groups of genes are gen-
erated and processed together. This spatial
coordination of the different steps required
to produce mature mRNAs at speciFc nu-
clear sites forms the basis of the concept of
transcription ‘‘factories’’ where transcripts
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