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While many features of the detailed biochemistry of nuclear metabolism are well defined, the organiz- ation or compartinentalization of these processes is, in general, poorly understood. Understanding nu- clear organization is particularly important for at least two reasons. First, evidence already available clearly indicates that the nucleus and cytoplasm compartmentalize some metabolic processes very dif- ferently. Thus, stu,]ies of nuclei may yield new prin- ciples or methods of biological organization. Second, contemporary nuclear organization is likely to con- tain important molecular 'fossils' of organization of metabolism in the late stages of pre-cellular evol- ution. For example, nuclei might reveal how metabolic processes were compartmentalized before the development of extensive lipid bilayer structures.

Technical progress during the past few years - especially in high-resolution light microscopy, in situ detection and the application of new genetic and cytogenetic strategies - has produced a new gener- ation of studies of this problem. These and earlier studies provide clear evidence for organization; how- ever, the nature, extent and function of these sub- nuclear structures remain substantially unclear. Our purpose here is to review critically selected features of this body of evidence in light of recent work suggesting that much of the compartmentalization of nuclear pre-mRNA metabolism may, in fact, have a relatively simple basis.

A simple view of nuclear compartmentalization Two recent studies place components of pre-mRNA

splicing machinery and the relatively decondensed chromatin making up actively transcribing genes in a small subnuclear compartment~,L This compart- ment consists of a network of fine channels appar- ently defined by exclusion from condensed strut. tures, Including condensed chromatin (such as diploid chromosome axes), nucleoll, the nuclear sur- face and possibly other structures. IThls compartment was Independently abbreviated ECN ~ and ICD 2. We suggest for general use the hybrid term ICN (for Inter- chromosomal channel network) to Indicate this com- partment specifically as decribed in the first sentence of this paragraph.] This organization appears to exist in both diploid and polytene nuclei in Drosophila t and in diploid mammalian nuclei z. Various earlier analy- ses of nuclear organization, while providing substan- tially less topological detail than these recent studies, are also consistent with this picture (for example, see Refs 3, ~, and 5 and references therein).

Furthermore, in one of these studies = we con- structed a novel experimental system that allowed, for the first time, visualization (at steady state) of a specific pre-mRNA moving away from its gene to the nuclear surface. This pre-mRNA behaved precisely as expected if movement occurs by diffusion through the ICN. Specifically, this pre-mRNA is distributed throughout the ICN, indicating movement away from the gene in a spatially uniform (isotropic) fashion. Moreover, the rates of this isotropic move- ment are consistent with diffusion. Lastly, bulk nuclear mature mRNA [as assessed by hybridization to poly(A)! colocalizes with this specific pre-mRNA

Nuclear pre-mRNA metabolism: channels and

tracks

We review new evidence suggesti~g that metazoan nuclear pre-

mRNA metabolism occurs in a small subnuclear compartment

consisting of a network of channels defined by exclusion from

various condensed stntctures. Nuclear components, including

mRNA en route from the gene to the nuclear surface, apparently

move through these channels by conventional diffusion.

throughout the ICN, indicating that most or all mRNAs likely move from their genes to the nuclear surface by diffusion.

These observations suggest a simple picture in which pre-mRNA metabolism goes on in the ICN, Moreover, movement of actors in tills process within the ICN likely occurs by conventional diffusion. We wlU refer to this as the 'dlffuslonal ICN' model. While Important details, Including means of maintaining 'open' channels between condense:l nuclear struc- tures t,2, remain to be elucidated, this model is suf- ficiently well defined to warrant evaluation of its potential generality.

This mode of compartmentalization is expected to provide large advantages by co-concentrating el- ements of metabolic machinery and thereby acceler- ating their rates of interaction. For example, the rates at which splicing factors find new pre-mRNAs, and mature mRNAs find nuclear pore complexes for export, would be substantially increased relative to the corresponding rates if the entire nuclear volunie were diffusionaUy accessible.

While this model is attractively simple, a number of earlier observations have been interpreted to indi. care more complex subnuclear organization. In the following sections we interpret (in some cases, re- interpret) these observations on the diffusional ICN model. We will argue that all available evidence is ............................................................ consistent with this model. The authors are at

Nuclear organization of pre.mRNAs A large number of studies indicate a high density of

nascent transcripts forming 'Christmas trees' organ- ized around the template DNA of very, actively tran- scribing genes (Ref. 6 and references therein).

the Department of Biochemistry and Cell Biology, University of New York, Stony Brook, NY 11794, USA.

TRENDS IN CELL BIOLOGY VOL. 4 FEBRUARY 1994 © 1994 Elsevier Science Lid 0962.8924/94/$07.00 3S

C('!:) ~l)]]]] i : : ~ 11/- FORUM

Acknowledgements

We apologize to our many

colleagues whose important work

could not be directly cited

because of limits on the number of references, We are

qrateful to many colleagues for

important discussions,

including Ann Beyer, Stan Fakan, Paul Fisher,

David Ish.Horowicz, Edwin Smith,

David Spector and Deborah Spikes. Work from this

laboratory is supported by NIH grant GM32003.

Moreover, the slow rate of transcription 7 relative to expected rates of pre-mRNP diffusion I predicts that the steady-state levels of nascent pre-mRNAs should be substantially higher than those of mature mRNAs in transit from the gene to the nuc!ear surface. Given the limited sensitivity of current in situ hybridization techniques, the diffusional ICN model predicts that nascent transcripts from actively transcribed genes should be detected in diploid nuclei but the mature mRNAs in transit to the nuclear surface should not 1.

Images obtained from in situ hybridization exper- iments with diploid nuclei are uniformly consistent with these predictions. Individual dots or short tracks of RNAs intimately associated with the correspond- ing genes (presumably nascent t/anscript 'Christmas trees') are seen but little or no additional nuclear RNA is detected (see, for example, Refs 8, 9 and 10 and ref- erences therein). We will refer to this interpretation as the 'tracks-as-trees' model.

An alternative to this interpretation is that pre- mRNA 'tracks' represent some novel structure associ- ated with post-transcriptional processing and/or transport of nuclear pre-mRNAs (reviewed in Ref. 10; referred to below as the 'post-transcriptional-struc- ture' model for tracks). Which of these alternative interpretations is correct remains to be unambiguously established. However, we believe the tracks-as-trees model is substantially more plausible for the follow. ing reasons.

First, Lawrence and colleagues have suggested that nuclear pre-mRNA 'tracks' are too long to represent nascent transcript 'Christmas trees' and, thus, likely represent some other structure (reviewed In Ref. 10). However, in our view, there is no conclusive evidence for tracks longer than those predicted by the tracks- as.trees model. Specifically, only very actively tran- scribing genes produce sufficient steady-state nuclear RNA levels to permit in situ detection (see, for example, Refs 1 and 10). Thg chromatin of this highly selected subset of DNA segments is expected to show extensive local decondensatlon (Ref. 6 and references therein). This is In contrast to the large malority of geaomic sequences which, at any moment in time, are not being heavily transcribed and are expected to be highly condensed. Assuming a mean level of chromatin condensation (relative to fully extended, naked DNA) of approximately five. to tenfold for these genes, all the observed tracks of which we are aware have lengths consistent with the expected dimensions of the corresponding nascent transcript 'Christmas trees' (see Ref. 1 for more details). Moreover, in no case of which we are aware has hybridization with multiple probes for unique sequences within the transcription units in question been carried out to show directly whether template DNA and pre-mRNA do or do not colocalize in these tracks in diploid nuclei.

Second, the tracks-as-trees model is consistent with the expected kinetics of pre-mRNA metabolism, while the post-transcriptional.structure model for tracks apparently is not.-Specifically, the highest steady-state concentrations of nuclear pre-mRNA are expected to occur at/on structures where the slowest steps in the proce,s occur. Thus, the post-transcrip.

tional-structure model requires the existence of some post-transcriptional step occurring in 'track' struc- tures that takes substantially longer than the very slow process of transcription itself. No such lengthy step is known or expected.

This difficulty with the post-transcriptional-struc- ture model can be appreciated by considering the fol- lowing specific example. It takes about 40 minutes to synthesize the full-length transcript from the large fibronectin gone 7,1o. We estimate conservatively that at least 90% of the fibronectin RNA localized to the observed 'track' by in situ hybridization would be in the proposed post-transcriptional structure (rather than in the nascent transcript array) on this model. If so, the average RNA molecule would spend about six hours in this post-transcriptional structure. A gen- erally occurring step of this duration is not plausible. For example, less than six hours is required for the entire sequence of events in which one gene family is activated by maternal information to produce pro- tein products that activate a second gone family that, in turn, produces protein products regulating a third set of genes during the patterning of the Drosophila embryo (reviewed in Ref. 11).

In conclusion, in our view, there is currently no conclusive evidence for residence of pre-mRNAs or pre-mRNA components in specific nuclear structures other than nascent transcript trees and the ICN.

Organization of splicing components A large body of evidence from work over the last

ten years using Drosophila polytene nuclei and amphibian lampbrush chromosomes (Refs 12, 13 and references therein) indicates that components of the splicing machinery concentrate in the immediate vicinity of actively transcribing genes. Recent work Indicates that the same is true in mammalian nuclei (for example see Ref. 14). These observations are readily Interpretable on the dlffuslonal ICN model if diffusing splicing components are captured by binding to nascent transcripts.

In addition to concentration by binding to nascent transcripts, some concentration of splicing compo- nents in punctate structures ('speckles') not directly associated with nascent transcripts is also widely observed (see, for example, Refs 4, 13 and 15-18). The most extensive studies indicate that these compo- nents are not exclusively concentrated In punctate structures but rather some are also more widely distributed t9 - apparently throughout the ICN I`

These punctate structures can be interpreted to indicate some elaborate subnuclear organization. However, they ate also simply interpretable on the dlffuslonal ICN model. Specifically, crystal-like pre- cipitates of splicing components (speckles) in equi- librium with components dissolved in the aqueous ICN would provide a highly efficient mechanism for homeostatic control of the concentrations of soluble components in the face of fluctuating rates of tran- scription. Consistent with this view of these struc- tures, short.term metabolic labelling experiments indicate that these punctate structures are not sites of active pre-mRNA metabolism (reviewed in Refs 8 and 17).

36 TRENDS IN CELL BIOLOGY VOL. 4 FEBRUARY 1994

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Concluding remarks The diffusional ICN hypothesis is consistent with

a large body of evidence and has a useful simplicity. Whether this model proves to be correct in its essen- tial features (as we expect) or not, it will provide an important counterpoint to more-complex models as analysis of subnuclear organization proceeds. Further progress here, in turn, should provide important new insights into both contemporary function and pre- cellular evolution.

References 1 ZACHAR, Z., KRAMER, I., MIMS, I. P. and BINGHAM, P. M.

(1993) I. Cell Biol. 121,729-742 2 ZIRBEL, R. M., MATHIEU, U. R., KRUZ, A., CREMER, T. and

LICHTER, P. (1993) Chromosome Res. 1, 93-106 3 FAKAN, S. and PUVION, E. (1980) Int. Rev. CytoL 65, 255-299 4 SPECTOR, D. (1990) Proc. NatlAcad. Sci. USA 87, 147-151 5 HUTCHtSON, N. and WEINTRAUB, H. (1985) Ce1143, 471-482 6 BEYER, A. ~nd OSHEIN, Y. (1988) Genes Dev. 2, 754-765 7 THUMMEL C. S. (1992) Science 255, 39-40

8 HUANG, S. and SPECTOR, D. L. (1991) Genes Dev. 5, 2288-2302

9 KOPCZYNSKI, C. C. and MUSKAVITCH, M. A. T. (1992)/. Cell Biol. 119, 503-512

10 XING, Y. and LAWRENCE, ]. B. (1993) Trends CellBiol. 3, 346-353

11 AKAM, M. (1987) Development 101,1-22 12 PII~IOL-ROMA, S. and DREYFUSS, G. (1993) Trends Cell Biol. 3,

151-155 13 WU, A., MURPHY, C., CALLAN, H. G. and GALL ]. G. (1991)

J. Cell Biol. 113, 465-483 14 IIMENEZ-GARCIA, L F. and SPECTOR, D. L. (1993) Cell 73,

47-60 15 LERNER, E. A., LERNER, M. R., ]ANEWAY, C. A. and STEITZ, I. A.

(1981) Proc. Notl Acad. Sci. USA 78, 2737-2741 16 FU, X-D. and MANIATIS, T. (1990) Nature 343, 437-441 17 LAMOND, A. I. and CARMA-FONSECA, M. (1993) Trends Cell

Biol. 3, 198-204 18 LI, H. and BINGHAM, P. M. (1991) Cell67, 335-342 19 MATERA, A. G. and WARD, D. C. (1993)/. CellBioL 121,

715-728

Apoptosis (Greek for 'drop-out') is a process by which cells can be eliminated in response to a wide range of physiological and toxicological signals (see Refs 1-3 for reviews). It occurs during embryogenesis (e.g. in the formation of digits from a limb bud), the negative selection of lmmunocompetent T cells, hormone- Induced tissue atrophy and tumour regression, and in many tissues to provide a stable balance of cellular mass. The morphological eve~Rs ot apoptosls differ from those of necrosis, in which cells die by plasma membrane lnlury. During apoptosis, the formation of protuberances on the cytoplasmic membrane Is accompanied by chromatln condensation and the formation of t:rescent.shaped deposits along the nuclear envelope (Fig. 1).

DNA Iragmentatlon during apoptosls One of the hallmarks of apoptosls is the enzymatic

internucleosomal degradation of chromatin, which can be seen as the formation of the so.called DNA lad- der (multiples of about 180 bp) after electrophoretic separation of the extracted DNA on agarose gels (Fig. l). This DNA fragmentation is due to multiple single- strand nicks preferentially induced in the linker region between nucleosomes 4 and follows the gener- ation of large fragments of 300 and/or 50 kbp s indica- tive of the initial release of chromatin loop domains. Wyllie was the first to place nuclease activation and DNA fragmentation into the general context of apop- tosis almor ~, ~3 ~ a~', ~,~6 but the characteristic lad- der had previously been shown by Hewish and Burgoyne 7 when they demonstrated the Ca 2÷- and MgZ÷.dependent autodigestion of DNA in isolated liver cell nuclei. More recently, these studies were extended to isolated nuclei of thymocytes and other cells (mostly of haematopoietic origin) that show similar autodigestion when exposed to divalent cations.

The apoptosis endonucleases:

cleaning up after cell death?

~, : ~ ~,~,"

The tern1 apoptosis describes the predictable stntctural dlanges

associated with many loons of programmed cell death. One of the

first visible events of apoptosis is the collapse of the nucleus.

Nuclear degradation is manifested by digestion of chromatin into

nucleosome-sized fragments or multiples of these. This digestion of

DNA is enzymatic, and several attempts have been made to

characterize apoptosis-specific endodeoxyribomwleases. Although

there are strong candidates for such enzymes, direct evidence for

their role in apoptosis is yet to be provided.

Since endonuclease activation is one of the earliest changes denoting irreversible commitment to cell death, it is generally thought to be involved in the triggering of cell death rather than being the result of it. However, based on several studies of culture cell

TRENDS IN CELL BIOLOGY VOL. 4 FEBRUARY 1994 © 1994 Elsevier Science Ltd 0962-8924/94/$07 O0 3 7