Mechanism of mRNA transport in the nucleus

Abstract

The mechanism of transport of mRNA-protein (mRNP) complexes from transcription sites to nuclear pores has been the subject of many studies. Using molecular beacons to track single mRNA molecules in living cells, we have characterized the diffusion of mRNP complexes in the nucleus. The mRNP complexes move freely by Brownian diffusion at a rate that assures their dispersion throughout the nucleus before they exit into the cytoplasm, even when the transcription site is located near the nuclear periphery. The diffusion of mRNP complexes is restricted to the extranucleolar, interchromatin spaces. When mRNP complexes wander into dense chromatin, they tend to become stalled. Although the movement of mRNP complexes occurs without the expenditure of metabolic energy, ATP is required for the complexes to resume their motion after they become stalled. This finding provides an explanation for a number of observations in which mRNA transport appeared to be an enzymatically facilitated process.gene expression ͉ live cell imaging ͉ mRNA export ͉ nuclear viscosity A fter mRNAs are synthesized, processed, and become associated with a number of different proteins at the transcription site, they are released into the nucleoplasm (1). The mechanism by which these large mRNA-protein (mRNP) complexes then move through dense nucleoplasm to reach the nuclear pores has been the subject of intense study and speculation (2, 3). Early workers proposed that mRNP complexes are transferred along a chain of receptors until they reach a nuclear pore, expending metabolic energy in the process (4). This solid-state transport model is supported by observations made in fixed nuclei that show some transcripts distributed along tracks that originate from the locus of the parent gene (5, 6). A second theory, called the ''gene-gating'' hypothesis, proposes that active genes are situated near the nuclear periphery and that mRNAs exit the nucleus through the nearest pores (7). This idea is supported by observations that certain mRNAs exit from one side of the nucleus (8) and that, in yeast, many transcriptionally active gene loci are located near the nuclear periphery (9). By contrast, a number of other studies have found that mRNP complexes move quite freely within the nucleus (10-16). This view is supported by studies of the distribution of newly synthesized Balbiani ring RNA in the salivary gland cells of insects (11), fluorescence recovery after photobleaching and fluorescence correlation spectroscopy studies of probes that bind to the poly(A) tails of mRNAs (12-15), and from single-particle analysis of mRNP complexes bound to GFP-linked proteins (16).Although the latter studies found that mRNP complexes are able to diffuse within the nuclear matrix, there was a paradoxical active transport component to their motility, because both a reduction in temperature and ATP depletion curtailed the mobility of the complexes (14-16). To better understand the nature of mRNP mobility, we have developed a system of fluorogenic probes and mRNA constructs that allo...

Discontinuous movement of mRNP particles in nucleoplasmic regions devoid of chromatin

“…The DNA-oligonucleotide-labeled particles displayed a considerably higher mobility than recorded in previous single-particle experiments (6,7,14). To rule out the possibility that the fast motion was caused by fragmentation of the particles by RNase H, we performed microinjection experiments using fluorescently labeled 2Ј-O-methyl-RNA oligonucleotides complementary to BR2 RNA.…”

“…However, the mobility can be affected by energy depletion, which suggests that ATP-dependent processes also play a role (5). Recently, it became feasible to track individual mRNP particles in the nucleoplasm (6,7). It could then be confirmed that the particles move by free diffusion, but the diffusion is often spatially constrained.…”

“…mRNPs travel in interchromatin channels (8,9), and the movement of mRNPs is probably hindered in tight channels and even stalled in cavities formed by chromatin. In the single-particle-tracking experiments, ATP depletion showed no effect on the individual, still-mobile mRNPs but further constrained the movement of the particles, probably because of a more condensed organization of the chromatin (6,7). To further study the movement of mRNPs, we have now chosen an experimental system that allows us to follow the movement of individual mRNPs in large nucleoplasmic regions lacking chromatin, and thus we avoid the complex influence of chromatin.…”

“…Messenger RNPs can be imaged, because they are large and can be brightly labeled. Two recent studies used synthetic genes to produce highly fluorescently labeled mRNA in vivo and followed the pathways of these artificial mRNA molecules by single-particle-tracking microscopy (6,7).…”

Rapid, Diffusional Shuttling of Poly(A) RNA between Nuclear Speckles and the Nucleoplasm

“…However, reductions of this magnitude are not easily explained by rate decreases in enzymatic reactions because, as explained above, one would expect to see a much larger effect upon a 15°C temperature shift (see also Shav-Tal et al, 2004 for a discussion of this point). In addition, a recent study of mRNA movement in the nucleus of mammalian cells has somewhat clarified the situation by revealing that ATP is required for the resumption of movement when RNA becomes corralled within tight confinements, rather than the nucleotide being involved in the movement itself (Vargas et al, 2005). Molenaar et al (2004) also reported that several poly(A) binding proteins moved in and out of speckles at rates similar to those reported here for both bound SC35 and poly(A) RNA, and by others for RNA-binding proteins (Phair and Misteli, 2000;Misteli, 2001;Calapez et al, 2002), but they reported a 5-to 10-fold slower diffusion coefficient for poly(A) RNA itself.…”