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(The FASEB Journal. 1999;13:S238-S242.)
© 1999 FASEB

Movement and localization of RNA in the cell nucleus

THORU PEDERSON1

Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, 377 Plantation Street, Worcester, Massachussetts 01605, USA

1Correspondence: Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, 377 Plantation St., Worcester, MA 01605, USA. E-mail: thoru.pederson{at}umassmed.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSION
 REFERENCES
 
The movement of various RNAs from their sites of chromosomal synthesis to their functional locations in the cell is an important step in eukaryotic gene readout, though one less well understood than the transcription, RNA processing, and various functions of RNA. The segregation of the many classes of RNA out into to their appropriate sites in the cell is, from a physical chemical point of view, a remarkable phenomenon. This paper summarizes investigations my colleagues and I have undertaken over the past 7 years to describe the intracellular traffic and localization of RNA in living cells. One approach we have developed is to glass-needle microinject ~0.01 pl of fluorescent RNA solutions into the nucleus or cytoplasm of cultured mammalian cells. This ‘fluorescent RNA cytochemistry’ approach has resolved intranuclear sites (‘speckles’) for which premessenger RNAs (pre-mRNA) have high affinity and has revealed very rapid movements of certain other RNAs from their nucleoplasmic injection sites to the nucleoli. One of these rapidly trafficking nucleolar RNAs is the signal recognition particle (SRP) RNA, and further results indicate that the nucleolus is a site of SRP RNA processing or ribonucleoprotein assembly prior to export to the cytoplasm. In these fluorescent RNA microinjection studies, we have also used mutant RNA molecules to identify specific nucleotide sequences that function as targeting elements for the localization of RNAs at their respective intranuclear sites. In a second approach, we have used fluorescent correlation spectroscopy (FCS), a classical biophysical method for measuring molecular motion in vitro, coupled with confocal fluorescence microscopy to measure the movement of poly(A) RNA in the nucleus, with the interesting finding that these RNAs appear to move about inside the nucleus at rates comparable to diffusion in aqueous solution. Parallel experiments using the method of fluorescence recovery after photobleaching (FRAP) revealed a diffusion coefficient for intranuclear poly(A) RNA close to that measured by FCS. These results bear on the structure of the nucleoplasmic ground substance-an extremely controversial and unsolved problem in cell biology (29) . The methods we have developed and these initial results represent the first major step toward a comprehensive understanding of RNA traffic in the cell nucleus.—Pederson, T. Movement and localization of RNA in the cell nucleus. ;1999>


Key Words:


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSION
 REFERENCES
 
SEVERAL VIGNETTES ABOUT Keith Porter come to mind, but I will only offer a brief one. In 1982, in my capacity as program chair of the American Society for Cell Biology meeting, I reported to Keith that we had chosen Lew Tilney as the first ASCB Keith Porter Lecturer. There was a long pause at the other end of the phone. Then he said, "Well, Thoru, given the selection, I can see why you didn’t need to consult me." That quip had the comedic twist and double layer of meaning that characterized so many of Keith’s remarks—typically clever, often beguiling. When we recall him, we of course primarily think of his pioneering role in creating the field of cell biology. But we also warmly remember his intellectual playfulness.

I would like to present some of the work my colleagues and I have been undertaking over the past ~7 years to understand how RNA molecules get from their DNA birth sites to their correct functional locations throughout the cell. This is a formidable molecular traffic problem from the chemical point of view, and we know (so far) of no Golgi-like sorting apparatus to facilitate the process. Although the proper spatial localization in the cell of the various kinds of RNA is central to gene readout, this aspect of eukaryotic gene expression has, until recently, been much more opaque than our more comprehensive and detailed understanding of transcription, RNA processing, and the various functions of RNA. This state of affairs has been because of, in large part, the lack of ways to study RNA movements inside living cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSION
 REFERENCES
 
Most of the techniques used in our research on RNA movement presented in this symposium paper have been described in considerable detail (1–7).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSION
 REFERENCES
 
Getting started
In 1989–1990, a graduate student in my laboratory, Jin Wang, was doing his thesis research on pre-mRNA splicing from a biochemical perspective (8) . He was a bright student and was making excellent progress. What I did not know was that for a few weeks that winter he had a second ‘secret’ project with a fellow graduate student at the Worcester Foundation, working in the laboratory of Yu-li Wang (no relation to Jin) downstairs from my lab. Jin had been properly impressed by the work of Yu-li Wang, a pioneer in the field of nonmuscle actin dynamics in living cells (9 10 11 12) , and had wondered if fluorescent actin could be microinjected into cells to illuminate pathways of its movement and localization, why couldn’t RNA?

In collaboration with Long-guang Cao, the graduate student in Yu-li Wang’s lab, Jin Wang fluorescently labeled several pre-mRNAs and, with Cao’s collaboration, microinjected them through glass needles into the nuclei of rat NRK fibroblasts. (They did not tell me of these experiments until a few weeks later, when the first suite of them had been successfully done.) What Jin Wang and Long-guang Cao found was that the fluorescent pre-mRNA introduced into the nucleus congressed over a period of 15–30 min into a discrete array of sites peppered throughout the nucleoplasm (1) . These sites were already known to be where certain small nuclear ribonucleoprotein (snRNP) particles are concentrated in the nucleus, (13) and because these snRNPs are cofactors for pre-mRNA splicing, the fluorescent pre-mRNA localization results my student had obtained rang true (1) . As an extension of these studies, he fixed the fluorescent pre-mRNA containing cells and then carried out immunocytochemistry using monoclonal antibodies to known snRNP proteins. This revealed a spatial concordance between the sites at which the pre-mRNA had become localized in the living state and the nuclear regions of concentrated snRNP proteins (1) .

Because the fluorescent pre-mRNAs (including ones for the ß chain of human hemoglobin, a rat neuropeptide pre-mRNA, and an adenovirus pre-mRNA) were transcribed in vitro from cloned DNAs, it was possible to study mutant pre-mRNAs. This revealed that a specific region near the 3' end of the pre-mRNA intron was required for pre-mRNA localization at the discrete nucleoplasmic sites (1) . This was the first demonstration of a sequence-specific localization of exogenously delivered RNA within the cell nucleus.

The ‘new-cleolus’
We then sought to expand this fluorescent RNA cytochemistry approach. A postdoctoral fellow in my laboratory, Marty Jacobson, embarked on studies of the nucleolar localization of two small RNAs using this method and identified nucleolar localization sequences in one of these RNAs (2 , 3) . Marty also elected to study the intranuclear localization of microinjected wild-type and mutant signal recognition particle (SRP) RNA. This study revealed that SRP RNA rapidly shuttles to the nucleolus (within 30–60 s) and that this nucleolar localization involves discrete domains within the SRP RNA molecule (7) . This finding suggests that the biosynthesis of the SRP, a cytoplasmic translational arrest machine, involves a nucleolar station in its intranuclear transit and constitutes part of the evolving evidence that the nucleolus is the site of various RNA processing and ribonucleoprotein assembly events beyond its classically envisioned role in ribosome biosynthesis (14) . Three of the RNAs that have been recently linked to the nucleolus [i.e., SRP RNA (7) , telomerase RNA (14) , and U6 RNA (14) ] are, interestingly, parts of catalytic ribonucleoprotein machines. The SRP is a GTPase, telomerase is a reverse transcriptase, and U6 RNA is an essential part of the catalytic center of the spliceosome. Our fluorescent RNA cytochemistry approach, particularly as applied to SRP RNA (7) , was a key factor in leading me to rethink the nucleolus (14 , 15) , an example of how new techniques can revise one’s conceptual perspective.

Problems and pitfalls
My students and postdocs have always cringed to hear me say after reading their fellowship proposals, "It’s okay, but what about the problems and pitfalls?" It is such a vivid (and accurate) term. When I was a postdoc at Albert Einstein College of Medicine (1968–1971) my mentor was a member of the NIH Cell Biology Study Section and asked me to join him in reading grant applications. I got the sense that the requested section on ‘problems and pitfalls’ was a creative challenge and important opportunity for the applicant, perhaps even the intellectual moment of truth as regards winning the reviewer’s confidence. As a NIH grant applicant soon thereafter and a subsequent Cell Biology Study Section member (1975–1979), I had this notion convincingly confirmed at ‘both ends.’ What are the problems and pitfalls of the fluorescent RNA microinjection approach?

The first and most obvious point to consider is that the microinjected RNAs are tagged by covalently attached fluorescent groups and must therefore be considered derivatized. This issue is formally the same as the one that arises in the use of fluorescent proteins for cell microinjection studies. In our experiments, rhodamine is typically attached to the RNA at an average spacing of 1 molecule every 30–60 nucleotides (1 , 4) . This balances fluorescent signal strength with retaining RNA function as defined by demonstrably correct ribonucleoprotein assembly in vitro (1 2 3) . A second issue is whether the volume of introduced fluorescent RNA (usually 0.01–0.1 pl) or the fluorescent RNA itself impairs cell physiology. The fact that many studies have now shown that nucleus-microinjected RNAs move to the same intranuclear sites as are occupied by their endogenous RNA counterparts (2 3 4 , 6 , 7 , 16 17 18 19) indicates that this aspect of cell function, namely RNA traffic, is not impaired by the presence in the cell of a few thousand fluorescent RNA molecules (4) . We have also observed that cells microinjected with fluorescent RNA divide throughout the subsequent several hours of observation, indicating that the presence of fluorescent RNA does not impair progression through the cell cycle. Thus, fluorescently tagged RNA, as we prepare it (vide supra), appears to behave properly and does not seem to be deleterious to cells. Given the fact that many proteins have been found to behave properly as regards intracellular localization when fused to an ~27,000 molecular weight tag, namely jellyfish green fluorescent protein, the rhodamine labeling of RNA we use is comparatively minor on a molecular mass basis. In certain instances, it may be desirable to keep a certain functional region of a given RNA nonlabeled. This can be accomplished by fluorescently labeling the RNA only at one (or both) of its ends (with an obvious sacrifice of fluorescence intensity per RNA molecule) or by ligating fluorescent and nonfluorescent blocks of RNAs that have the desired nucleotide sequence.

Biophysical and physical chemical aspects of intranuclear RNA motion
The most advanced aspects of this work address the microscale movement of RNA within the nucleus, captured in vivo in real time. Fluorescent correlation spectroscopy (FCS) is a method of fluctuation analysis used to measure molecular transport and chemical kinetics by tracking the number of molecules entering and leaving a very small interrogation volume over successive time intervals (20) . This method of measuring molecular motion has recently been integrated with avalanche photodiode detection systems, having dead times on the order of only 25 ns and advanced confocal optics and algorithms that model the simultaneous diffusion of as many as three components moving at different rates (20 21 22 23) . These advances have led to the advent of FCS as a cell biological tool to study molecular movement in living cells. The Carl Zeiss Company (Jena, Germany) has developed such an instrument, and we were pleased that Zeiss selected the Worcester Foundation as one of the test sites. This fortunate circumstance coincided with our burgeoning interest in more quantitatively measuring RNA movement in cells.

In my laboratory, Joan Politz had previously developed a method, while a postdoctoral fellow in the laboratory of Robert Singer (then at the University of Massachusetts Medical School), to detect poly(A) RNA in cells by an in vivo nucleic acid hybridization procedure (24) . Her method involves incubating rat myoblasts with oligo(dT) and then detecting the intracellular sites at which the oligo(dT) has hybridized to the poly(A) tracts of RNA by fixing the cells and conducting an in situ reverse transcriptase reaction in which the oligo(dT) that is hybridized to poly(A) primes synthesis of a complementary DNA (cDNA). The cDNA products are detected in situ by their incorporation of deoxynucleotides tagged with digoxigenin or other suitable detectors. In collaboration with Elizabeth Browne in the laboratory of David Wolf at the Worcester Foundation, Joan embarked on an investigation of the intranuclear movement of fluorescent oligo(dT) in rat myoblasts using FCS. This study provided a number of very interesting findings (25) . First, a substantial fraction of the intranuclear oligo(dT), as well as most of the control oligonucleotide—oligo(dA), which is not expected to hybridize appreciably to nuclear RNA—was observed to move within the nucleus at mean translation times close to those measured for these oligonucleotides in aqueous solution by the same FCS method (diffusion coefficients = ~4x10-7 cm2/s). Second, a large portion (~45%) of the intranuclear oligo(dT), but not oligo(dA), moved more slowly (diffusion coefficient = ~1x10-7 cm2/s). The amount of this slower-moving oligo(dT) fraction was substantially reduced if the oligo(dT) was prehybridized in solution to oligo(dA) before introduction into the cells, presumably because the oligo(dT) was unable to subsequently hybridize to poly(A) RNA in the cell. Third, this slower-moving oligo(dT), which we interpret as the fraction hybridized to poly(A) RNA, had the same diffusion coefficient as oligo(dT) hybridized to a 7.5-kb polyadenylated RNA in aqueous solution. Fourth, we also detected a small amount of intranuclear oligo(dT), ~15% of the total, that moved considerably more slowly than the poly(A) RNA-hybridized fraction. The average diffusion coefficient of this latter, very slow fraction of oligo(dT), <=1 x 10-8 cm2/s, is consistent with it being hybridized to poly(A) RNA that is itself associated with large macromolecular complexes. The diffusion coefficient of the oligo(dT) fraction that we interpret as hybridized to poly(A) RNA (~1x10-7 cm2/s) is also close to that calculated from Stoke’s law for a 7.5-kb poly(A) RNA molecule in the form of a nuclear hnRNP particle (25) , namely 2.2–9.2 x 10-8 cm2/s, assuming that the ribonucleoprotein particle has a protein:RNA ratio of 4:1 (26 , 27) and treating the particle as either a spherical or ellipsoid structure (28) .

In these studies (25) we also used a different biophysical method to investigate the intranuclear movement of oligo(dT), namely fluorescence recovery after photobleaching (FRAP). Unlike FCS, which is based on the fluorescence intensity fluctuations of molecules darting in and out of a small intracellular (in our case, intranuclear) sampling volume, FRAP measures the rate at which and extent to which a population of fluorescent molecules moves into a region where the existing molecules have been rendered nonfluorescent. Also unlike FCS, FRAP as generally used does not resolve individual kinetic components having characteristically different diffusion coefficients. When we measured the intracellular movement of oligo(dT) by FRAP, the average diffusion coefficient was 1.2 x 10-7 cm2/s (25) . This value is close to the intranuclear diffusion coefficient of oligo(dT) measured by FCS, if the data are treated as a 1-component model, namely ~8.7 x 10-8 cm2/s. This is a remarkable concordance of diffusion coefficients obtained by two independent methods.

The overall conclusion from our FCS and FRAP experiments is that both nonhybridized oligonucleotides and oligo(dT) hybridized to poly(A) RNA move within the nucleus of living mammalian cells with diffusion coefficients similar to those measured for the two respective classes of molecules in aqueous solution. This addresses the physical milieu of the cell nucleus and suggests that the amount of free volume is substantial, irrespective of the existence (or nonexistence) of a nucleoplasmic ground substance or other structural elements that have been proposed to occupy the interchromatin space (29) .

Having used the method at its infancy, my colleagues and I think that FCS is a powerful new tool in the cell biologist’s kit. Its promise has been recently reviewed and some of the potential problems cited (23) have been overcome in our studies (25) . In particular, the level of autofluorescence in the nucleus of the cells we have used is quite low and has not been problematic. In addition, the relatively low irradiance used in our FCS studies, ~100 µJ over the 0.23 µ2 average cross-sectional area in the Zeiss instrument’s confocal volume (25) , is not thermally invasive. Indeed, similar levels of irradiance have been previously used (in non-FCS studies) on cells in Drosophila embryos with no detectable adverse effects (30) .


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSION
REFERENCES
 
Our exploration of RNA movements inside the living cell are at an early stage, reminiscent of the first use of microinjected fluorescent actin and tubulin to study the cytoplasmic microfilament and microtubule systems. We have already learned a great deal about intracellular transport of RNA while developing these new approaches. Our work so far has centered on nuclear RNAs and has revealed specific intranuclear targeting sequences in several (1 2 3 , 7) , indicated new roles for the nucleolus (7 , 14) , and suggested that poly(A) RNAs move within the nucleus by diffusion (25) . It is to be noted that studies of mRNA transport in the cytoplasm of various cells have implicated elements of the cytoskeleton in the localization process. So moving RNA cargo in eukaryotic cells may, perhaps not surprisingly, involve different mechanisms, depending on the RNA and the cell compartment.

Perspective
To cell biologists of my generation (I was born in 1941), Keith Porter carried that all too prosaic label ‘electron microscopist.’ But that was by no means the whole story of his talent. Keith was a passionate advocate of using living cells and indeed was skilled at the nascent art of tissue culture when it was far from the routine practice it is today. He also had a most impressive knowledge of optimal living material among many phyla for revealing important principles of cell biology. (In this respect, Keith was perhaps to the cytoplasm what Joe Gall has been to chromosomes and the nucleus as regards very intelligent selection and adroit exploitation of exceptionally favorable biological material.) I think Keith would not have been at odds with my goal of trying to follow RNA in living cells, though undoubtedly he would have had some penetrating comments. But they would have been delivered with his trademark—a smile—by which he conveyed the shared joy of this endeavor we call cell biology.


   ACKNOWLEDGMENTS
 
My research has been and is supported by NIH grant GM-21595. I thank the National Institute of General Medical Sciences for over 27 years of sustained, enabling support, often for studies that were, at their outset, voyages into the unknown. Portions of this work were also supported by a grant for the study of RNA from the G. Harold and Leila Y. Mathers Foundation, also giving me free rein that is much appreciated. I gratefully acknowledge the three members of my laboratory who have been the major figures in this recent avenue of our research (in chronological order): Jin Wang, Marty Jacobson, and Joan Politz. Joan Politz is supported by NIH NRSA fellowship AR-08361. The initiation of the fluorescent RNA cytochemistry work involved key contributions by Long-guang Cao, and was enormously aided by the expertise of my Worcester Foundation colleague Yu-li Wang, whom I especially thank. I also gratefully acknowledge the collaboration of Elizabeth Browne and David Wolf in the fluorescence correlation spectroscopy experiments, and I thank Ernst Keller of Carl Zeiss for choosing the Worcester Foundation as a developmental test site for the new Zeiss instrument.

Note added in proof: Since this manuscript was submitted, an additional study has confirmed the diffusion-like movement of poly(A) RNA in the nucleus: Politz, J. C., Tuft, R. A., Pederson, T. and Singer, R. H. (1999) Movement of nuclear poly(A) throughout the interchromatin space in living cells. Current Biol. 9, 285–291


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSION
 REFERENCES
 

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